Therapeutic effects of Lentinan in gastrointestinal inflammation and carcinogenesis – A scoping review of evidence from preclinical and clinical studies

Abstract

Lentinan, a β-(1,3)-glucan derived from the mushroom Lentinus edodes, is known for its health-beneficial including anti-oxidant, anti-inflammatory, and anti-tumor effects particularly in the gastrointestinal tract, and even regarded as adjuvant in cancer treatment in Asia. There is, however, a knowledge gap regarding lentinan's clinical application and the underlying mechanisms of its pleiotropic effects. This prompted us to perform a scoping review summarizing current knowledge of lentinan's health-promoting and disease-alleviating properties in the gastrointestinal tract and beyond. Preclinical and clinical studies revealed protective and therapeutic effects of the compound in distinct gastrointestinal pathologies, including colitis, chemotherapy- and immunosuppression-induced intestinal injury, infection models as well as in colorectal carcinogenesis by modulating intestinal epithelial barrier integrity, immune signaling, and microbiota composition. Lentinan was reported to exert its anti-tumor effects by interfering with apoptosis, autophagy, stemness, and angiogenesis. Notably, lentinan enhanced the effectiveness of chemotherapy and chimeric antigen receptor T-cell (CAR-T) therapy, reduced treatment-related adverse events, improved survival and quality of life. The observed effects were dependent on lentinan's molecular weight, dose, and route of administration. In conclusion, lentinan application constitutes a promising intervention strategy in gastrointestinal and extra-intestinal including systemic morbidities that should be further investigated in preclinical and clinical studies.

1. Introduction

1.1. The gastrointestinal barrier

The gastrointestinal tract is a highly regulated and coordinated system that fulfills a wide array of vital functions [1, 2]. Its expansive surface area allows direct contact with external substances, thereby making nutrient digestion and absorption possible [1, 2]. But this constant exposure also demands strict barrier integrity to prevent translocation of pathogens, toxins, and dietary antigens beyond the intestinal lumen [2–4]. To meet these demands, the gut consists of specialized populations of epithelial cells that are connected by several types of cell-cell junctions and supported by protective mucus layers, commensal microorganisms, and a well-orchestrated mucosal immune system [1, 3]. Together, these components create a tight barrier with selective permeability [2–4]. Housing the body's largest population of immune cells, the digestive tract maintains constant immunological surveillance while sustaining tolerance to commensal microorganisms and harmless antigens [5, 6].

1.2. Barrier disruption and disease progression

Distinct events such as inflammation, microbial infections, or iatrogenic injury can disrupt the physical and chemical barriers of the gut, resulting in increased intestinal permeability also termed “leaky gut” [7–9]. When barrier function is compromised, microbes and microbial products can translocate into the circulation and distant body sites, triggering immune responses both locally and systemically [10]. Such immunopathology is often accompanied by gut dysbiosis, that alters the microbial ecosystem and its metabolic output, further affecting epithelial integrity and triggering inflammatory signaling. Together, these processes are now recognized as central contributors to a broad range of diseases, including inflammatory bowel disease (IBD), colorectal cancer, and extra-intestinal disorders mediated through gut-organ axes [7].

IBD, encompassing Crohn's disease and ulcerative colitis, displays the consequences of chronic intestinal inflammation and barrier dysfunction [11]. The prevalence of IBD is rising steadily, imposing a significant burden on patients' quality of life (QOL) and healthcare systems around the world [12, 13]. Although recent advances in biologics and small-molecule therapies have improved disease management, many patients fail to achieve sustained remission or experience substantial side effects. Consequently, there is an increasing need for complementary therapeutic strategies that restore intestinal homeostasis through multi-targeted mechanisms [14, 15].

Most importantly, patients with long-standing IBD have a two-to three-fold increased chance of developing colorectal cancer [16], with a risk rising with disease duration and extent [17]. Unlike sporadic colorectal cancer, which typically follows the adenoma-carcinoma sequence initiated by adenomatous polyposis coli mutations, colitis-associated colorectal cancer often features early tumor suppressor protein p53 mutations that occur as “field changes” in chronically inflamed mucosa prior to histologic dysplasia [17]. Multiple mechanisms lead to the progression from normal mucosa to carcinoma. Research positioned oxidative DNA damage and pro-inflammatory cytokine signaling as inductors of epigenetic changes and activators of transcriptional regulators such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT)3 [18]. Dysbiotic microbial structures further promote tumorigenesis via the production of genotoxins, modulation of immune responses, and metabolic reprogramming [19].

Apart from colitis-associated colorectal cancer, gastrointestinal neoplasms represent a critical clinical burden due to their rising incidence, substantial prevalence, and poor long-term outcomes in advanced disease [20–23]. Though there have been a number of advances in screening, surgery, chemotherapy, and immunotherapy, challenges such as intrinsic and acquired resistance, systemic toxicity, and deterioration in QOL still frequently limit treatment efficacy [24–28]. Moreover, a multitude of evidence indicates that intestinal barrier dysfunction, immune dysregulation, and microbiota alterations influence the progression of colorectal cancer. Therefore, the integrity of the gastrointestinal system should be viewed as a critical determinant for both, prevention and treatment of gastrointestinal malignancies [29, 30].

1.3. Iatrogenic injury and treatment limitations

Iatrogenic intestinal injury poses a major clinical challenge in both oncological and immunological therapies. A large proportion of patients undergoing cytotoxic therapies develop chemotherapy-induced gastrointestinal toxicities like nausea, mucositis, and diarrhea [31, 32]. Such side effects frequently require dose reductions or premature discontinuation of treatment, thereby compromising treatment efficacy and negatively affecting patient outcomes [31, 33]. Similarly, immunosuppressive treatments, commonly used in oncology and autoimmune disease management, significantly disrupt gut microbial communities and mucosal immune defenses, enhancing the vulnerability to infection or inflammation [34–36]. Despite this, effective strategies to preserve intestinal barrier integrity and immune competence without affecting the therapeutic efficacy of anti-cancer or immunosuppressive agents remain limited [37–39].

1.4. Gut-liver axis in systemic disease

Growing evidence displays the gut-liver axis as a critical system of bi-directional communication [40]. Disruption of this axis allows microbial components, viable bacteria, and inflammatory mediators to translocate from the intestine to the liver via the portal circulation. This process activates hepatic innate immune pathways, including Toll-like receptor (TLR) signaling, inflammasome activation, and fibrogenic responses [40–44]. At the same time, disease-associated changes in gut microbial composition and metabolic activity, such as impaired short-chain fatty acid production and dysregulated bile acid metabolism, further exacerbate liver injury [41, 45]. Collectively, these disturbances have been implicated in the development and progression of liver diseases, including metabolic dysfunction-associated steatotic liver disease, nonalcoholic steatohepatitis, cholangiopathies or even hepatocellular carcinoma [40, 41, 44, 46, 47].

Extending beyond hepatic pathologies, increasing evidence has linked the systemic dissemination of microbial products to extra-intestinal manifestations such as metabolic disorders, autoimmune diseases like rheumatoid arthritis, and neurodegenerative morbidities, including Parkinson's and Alzheimer's diseases. Increased gut epithelial permeability and bacterial translocation have further been associated with hypertension and with extensive endotoxemia and systemic inflammation including multi-organ dysfunction in sepsis [48–50]. Collectively, these findings highlight the repair of the compromised intestinal mucosal barrier and microbial homeostasis as a uniting therapeutic strategy in intestinal, hepatic, and systemic inflammatory including metabolic diseases [1, 7, 48, 51].

1.5. Therapeutic gaps and phytomedicinal rationale

The rising global prevalence of gastrointestinal and systemic inflammatory diseases, together with the limitations of current pharmacological interventions, underscore the need for new therapeutic approaches. In this context, phytomedicine has gained increasing attention as a complementary intervention strategy [52, 53]. While conventional single-target therapies, such as biologics directed against tumor necrosis factor alpha (TNF-α), have transformed disease management, they often fail to address the complex, multi-layered disruption of intestinal homeostasis that underlies many chronic inflammatory conditions [15, 54–56]. In addition, the high costs of these treatments, their association with adverse effects including predisposing to secondary infections, and the frequent loss of response over time represent a critical unmet clinical need [15, 57].

Consequently, research efforts have increasingly turned towards bioactive natural compounds, which often exert pleiotropic effects across multiple biological pathways [58, 59]. Polyphenolic compounds such as curcumin and silymarin among others have been widely studied for their immunomodulatory, anti-oxidant, hepatoprotective, and prebiotic properties, for instance [58, 60, 61]. Among these natural products, also fungal polysaccharides, particularly β-glucans, have emerged as exceptional biological response modifiers [62, 63]. These complex carbohydrates have demonstrated the ability to enhance host defense mechanisms and maintain tissue integrity without inducing the systemic toxicity frequently seen with conventional chemo-preventive agents [62, 63]. Within this group one particular molecule is especially notable out due to its well-defined structure and decades of clinical use in oncology.

1.6. Source, structure, and pharmacological profile of lentinan

The polysaccharide lentinan stems from the fruiting body and mycelium of Lentinus edodes [64], one of the most popular edible mushrooms worldwide and better known as shiitake [65]. The polysaccharide consists of a linear β-(1,3)-D-glucan backbone with periodic β-(1,6) side chains that construct a distinctive triple-helix conformation [66]. This unique structure makes lentinan a water soluble, thermally stable molecule that exerts characteristic receptor-binding specificity [66]. Lentinan's immunomodulatory, anti-inflammatory, anti-neoplastic, and anti-oxidant properties have been mentioned in the literature, explaining its central position across multiple research contexts regarding its utility as a pharmaceutical agent [66]. Lentinan draws special attention in Eastern Asia, where it has a long history of use in traditional medicine and has been approved as an adjunctive therapy for cancer in China and Japan since the 1980s [65].

1.7. Rationale and scope of investigating lentinan in gastrointestinal diseases

The rationale for consolidating current knowledge on lentinan's effects within the gastrointestinal tract arises from several considerations. Gastrointestinal diseases are inherently multi-factorial, involving overlapping processes such as chronic inflammation, immune dysregulation, and disturbances of the gut microbial ecosystem [65, 67]. Effective therapeutic strategies therefore need to act across multiple biological pathways, an attribute that lentinan appears to possess based on preliminary studies [65, 66]. In parallel, growing recognition of the bi-directional gut-liver axis and the contribution of intestinal dysfunction to systemic disease [40] emphasizes the importance of evaluating lentinan's effects beyond the intestine. What is more, the increasing global incidence of gastrointestinal malignancies, particularly colorectal cancer, underscores the urgent need for safe and effective preventive and adjunctive therapies [20–23]. Early studies suggested anti-neoplastic activity of lentinan, together with its capacity to enhance anti-tumor immune responses, make it a promising candidate to address this unmet clinical demand [65, 68]. In oncological settings, chemotherapy and immunosuppressive agents frequently disrupt both mucosal and systemic immunity, thereby increasing infection risk and weakening host defense mechanisms [69–71]. Interventions that can restore immune competence without compromising antitumor efficacy are therefore highly desirable.

Taken together, lentinan's combined immunomodulatory and cytotoxic properties support its potential role as an adjuvant to conventional cancer therapies [65, 68].

1.8. Aim

Our scoping review aims to provide a structured and comprehensive overview of lentinan's health-promoting and disease-alleviating effects in the gastrointestinal tract. We summarize mechanisms by which lentinan modulates intestinal epithelial barrier integrity, immune signaling, and intestinal microbiota composition. On this basis, we also explore its protective and therapeutic effects in preclinical models of gastrointestinal pathologies, including colitis, chemotherapy- and immunosuppression-induced intestinal injury, as well as in colitis-associated and sporadic colorectal carcinogenesis. Furthermore, we assessed lentinan's clinical utility and therapeutic efficacy as an adjunctive agent for gastrointestinal cancers.

2. Methods

2.1. Literature search strategy

From 21 November 2025 to 20 December 2025, we conducted a structured literature search using the public database “PubMed”. The following search queries.

#1 (“lentinan”[Title/Abstract]) AND (“gastrointestinal tract”[MeSH Terms] OR “gastrointestinal”[Title/Abstract] OR “gut”[Title/Abstract] OR “intestine”[Title/Abstract] OR “intestinal”[Title/Abstract]) AND ((English[Filter] OR German[Filter]) AND (2005:2025[pdat]))

#2 (“lentinan”[Title/Abstract] OR “lentinus edodes”[Title/Abstract]) AND (“gastrointestinal neoplasms”[MeSH] OR “gastrointestinal tumor” OR “oesophageal cancer” OR “esophageal cancer” OR “gastric cancer” OR “stomach cancer” OR “colorectal cancer” OR “colon cancer”) AND ((English[Filter] OR German[Filter]) AND (2005:2025[pdat])),

were combined using the Boolean operator “OR” to form the search strategy (#1 OR #2).

We screened the resulting titles and abstracts for eligibility according to the predefined criteria listed in the following section. Subsequently, the remaining articles' full texts were assessed, excluding any non-relevant reports. The complete process of study selection, which ultimately led to the inclusion of a total of 43 studies, is displayed in Fig. 1.

Fig. 1.

PRISMA 2020 flow diagram of the literature selection process

2.2. Inclusion and exclusion criteria

We included articles if they were published between 1 January 2005 and 20 December 2025 and were freely available as an English or German full text. Only original articles, explicitly using lentinan as a primary intervention in research questions regarding gastrointestinal disease, were included in this review. Therefore, studies were excluded if they evaluated lentinan only as part of multi-component preparations, used any extracts from Lentinus edodes, or examined lentinan only as a positive drug control without isolating the its specific effects. Furthermore, studies that did not report on gastrointestinal outcomes or any non-original articles were disregarded in the construction of this paper.

3. Results

3.1. Pharmacokinetics of oral lentinan administration

To unravel pharmacokinetic features of lentinan in vivo, Wang et al. orally administered cyanine 5.5-labeled lentinan (50 mg kg⁻¹) to mice [72]. Near-infrared imaging visibly illustrated the prolonged retention of cyanine 5.5 in the animals' gastrointestinal tracts compared with free dye controls. Lentinan appeared to be preferentially absorbed in the small intestine and transiently accumulated in Peyer's patches. The molecule was further observed in liver and kidneys, indicating systemic distribution and clearance. Ex vivo imaging confirmed that Peyer's patch targeting was absent in free dye controls. Ligated jejunal loop and cleared-tissue confocal microscopy demonstrated a colocalization of lentinan with follicle-associated epithelial M cells. The authors further showed a time-dependent transepithelial transport of lentinan from apical uptake to basolateral translocation over two hours. In an inverted human colorectal adenocarcinoma Caco-2 cell/human B lymphoblastoid Raji B-cell co-culture M-cell model, lentinan exposure upregulated Dectin-1 and clusterin gene expression, microvillus effacement, as well as cumulative lentinan transmembrane transport which exceeded that observed in Caco-2 monocultures. Dectin-1 antagonist laminarin, spleen tyrosine kinase (Syk) inhibitor piceatannol, and NF-κB inhibitor Bay 11-7085 decreased M-cell apparent permeability to a significant degree, supporting pathway involvement. Flow cytometric analysis of Peyer's patch immune cells revealed that CD11c⁺ mononuclear phagocytes were the major uptake population (41%) for lentinan, with Dectin-1, Syk, and NF-κB inhibitors markedly inhibiting absorption. Bone marrow-derived dendritic cells (BMDCs) and murine macrophage-like RAW264.7 cells exhibited similar trends. In multiple Cell Counting Kit-8 (CCK-8) assays, lentinan dose-dependently increased the proliferation of Peyer's patch cells, BMDCs and RAW264.7 macrophages. At the transcriptional level, lentinan administration increased TNF-α, interleukin (IL)-1β, IL-6, IL-12, and interferon-gamma (IFN-γ) mRNA expression in Peyer's patches but reduced expression of both anti-inflammatory IL-4 and IL-10 levels. In BMDCs, elevated TNF-α, IL-6, IL-12, and IFN-γ mRNA expression was subsequently followed by an increase of dendritic cell activation and maturation, as indicated by increased CD86 and major histocompatibility complex class II (MHCII) expression. In RAW264.7 macrophages, lentinan induced increased expression of IL-1β, IL-6, IL-12, and IFN-γ mRNA, in accordance with classically activated macrophages, i.e. M1 macrophage polarization. In summary, orally administered lentinan was preferentially absorbed in the small intestine through M-cell-mediated delivery to Peyer's patches, where it was mainly taken up by mononuclear phagocytes. Subsequent dendritic cell maturation and macrophage M1-like pro-inflammatory activation was mediated by Dectin-1/Syk/NF-κB signaling [72].

Zheng et al. studied the pharmacokinetics of intestinal absorption and cellular uptake of lentinan in rats and Caco-2 monolayers [73]. Technetium-99m-lentinan administration to rats showed gastrointestinal retention, with peak uptakes in the duodenum (1.11% ID/g at 1 h) and colon (4.43% ID/g at 2–4 h). A partial bioavailability persisted up to 8 h and lentinan appeared to be predominantly excreted via the feces (63.41%) rather than urine (12.92%). Ussing chamber experiments further demonstrated time-dependent absorption, with the ileum being the primary site of uptake. The viability of Caco-2 cells was not significantly affected by exposure to fluorescent lentinan (0–800 μg mL⁻¹) for 8 h. Cellular uptake was both time- and concentration-dependent and confocal microscopy revealed homogeneous cytoplasmic distribution following a 4-h incubation with 500 μg mL⁻¹ lentinan. In polarized Caco-2 monolayers, the uptake of lentinan was energy-dependent and predominantly mediated by clathrin-mediated endocytosis and actin-dependent macropinocytosis, with no involvement of caveolae-mediated endocytosis. Lysosomal acidification regulated lentinan endocytosis, whereas endoplasmatic reticulum and microtubule function contributed to endocytosis and exocytosis, respectively. Stable transepithelial electrical resistance in Caco-2 monolayers exposed to apical fluorescent lentinan suggested that epithelial tight junctions were not affected. The cumulative basolateral transport of lentinan reached 2.65 ± 0.62 μg cm⁻², with an apparent permeability coefficient of 3.18 ± 0.72 × 10⁻⁶ cm s⁻¹, supporting time-dependent transcellular transport of lentinan. Inhibition experiments revealed that dynamin- and phosphoinositide 3-kinase (PI3K)-dependent clathrin endocytosis, as well as actin-mediated macropinocytosis, were involved in lentinan internalization. Together, these results demonstrated lentinan's oral bioavailability, predominant gastrointestinal retention, and fecal excretion, while preserving integrity of the epithelial barrier. Intestinal absorption was mainly observed in the ileum and mechanistically dependent on active, energy-dependent clathrin-mediated endocytosis and macropinocytosis. These results support that the transcytosis of lentinan occurred in epithelial cells without affecting tight junction integrity, suggesting its relevance for oral gastrointestinal applications [73].

3.2. Acute intestinal inflammatory injury models

Liu et al. examined whether orally administered lentinan (5, 10 or 20 mg kg⁻¹) attenuated acute dextran sulfate sodium (DSS)- or 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in mice, under therapeutic or prophylactic regimens, in comparison to salicylazosulfapyridine (SASP) as a positive control [74]. Lentinan proved to be more effective than SASP in reducing the disease activity index (DAI) scores at medium and high doses. High-dose lentinan preserved body weight and colon length in both, DSS and TNBS colitis. In DSS-treated mice, colons showed luminal mucus or purulent secretions, while TNBS administration also induced severe pathology, including thickened colon walls, ulcerations, necrosis, and in extreme cases, bowel perforation or megacolon. Of note, lentinan reduced macroscopic colon damage scores in a dose-dependent manner in both models. Medium- and high-dose lentinan significantly outperformed SASP in TNBS-induced colitis, whereas only high-dose lentinan exceeded SASP efficacy in DSS-treated mice. Histological analysis revealed that DSS mice showed epithelial lesions, crypt erosions, and neutrophil infiltration, whereas TNBS mice exhibited granuloma formation. In both models, lentinan dose-dependently decreased erosion area and microscopic damage scores, with high-dose lentinan showing the greatest efficacy and outperforming the positive control SASP. Lentinan substantially restored the commensal intestinal microbiota structure in mice suffering from acute colitis, increasing the proportion of Firmicutes while reducing Proteobacteria, Eubacterium, and Parabacteroides. Pretreatment with 20 mg kg⁻¹ of lentinan prior to DSS exposure resulted in lower DAI scores, weight loss, extent of colon shortening, and histological damage scores. Lentinan significantly preserved epithelial integrity and led to less neutrophilic infiltration compared with untreated DSS mice, effectively preventing colitis development. In vitro, lentinan dose-dependently inhibited TLR-4 signaling in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. This effect involved suppression of calcium influx and subsequent calcium/calmodulin-dependent protein kinase II (CaMKII) activation. Treatment with lentinan reduced the expression of downstream signaling proteins such as CaMKII, interleukin-1 receptor-associated kinase (IRAK)4, myeloid differentiation primary response 88 (MyD88), inhibitor of nuclear factor kappa-B kinase subunit beta (IKBKB) and TNF-receptor-associated factor 6 (TRAF6) in a dose-dependent manner. Moreover, lentinan reduced NF‐κB activity, translocation of the NF‐κB p65 subunit to the nucleus as well as the expression of several types of NF‐κB-regulated downstream inflammatory cytokines, including IL-13 and CD30L, in RAW264.7 macrophages. Similar effects were observed in LPS-stimulated Caco-2 cells, where lentinan attenuated NF-κB-p65 nuclear translocation in a dose-dependent fashion. Overall, the study demonstrates that high-dose lentinan showed significant effects against acute colitis across therapeutic and prophylactic regimens. The polysaccharide's effects might be attributed to its modulation of the gut microbiota and inhibition of the TLR-4/CaMKII/NF-κB pathway [74].

You et al. studied the effects of lentinan and probiotics, administered individually or in combination, in murine DSS-induced ulcerative colitis [75]. Treatment group animals received intraperitoneal injections of lentinan at doses of 5, 10 or 20 mg kg⁻¹. The authors also exposed HT-29 and human Caco-2 cells that had been stimulated with 2% DSS to lentinan (2 mg mL⁻¹) alone or in combination with probiotics. Although the study also discussed probiotic and combination treatment groups, this assessment will only focus on the effects of sole lentinan treatment. Lentinan treatment attenuated clinical disease parameters such as body weight loss, colon shortening, and DAI scores in a dose-dependent manner. Colonic pro-inflammatory cytokine levels, specifically TNF-α, IL-6, and IL-1β, were significantly suppressed in a dose-dependent manner and immunoblotting data further demonstrated a concentration-dependent reduction in levels of phosphorylated NF-κB p65 and phosphorylated inhibitor of κB alpha (IκBα) compared with DSS-treated control animals. Histopathological examination revealed that lentinan-treated mice showed significantly smaller erosion areas, less lymphocyte infiltration, and lower histological damage scores. In vitro experiments demonstrated that lentinan treatment enhanced HT-29 cell proliferation and reduced apoptosis of these cells. Western blot analyses indicated a significant decrease in levels of phosphorylated NF-κB p65 and IκBα in lentinan-treated cells. Taken together, these findings show that lentinan dose-dependently improved DSS-induced colitis in both, in vitro and in vivo models. Lentinan's protective effects were associated with suppression of the NF-κB pathway and improved epithelial cell survival [75].

Mizuno et al. investigated the effects of lentinan and fucoidan, a fiber from seaweed, on intestinal inflammation using a transwell co-culture model [76]. The experimental design consisted of Caco-2 cells cultured on the apical side and RAW264.7 macrophages on the basolateral side. Treatment cultures received lentinan or fucoidan at a concentration of 500 μg mL⁻¹. Budesonide was added as a positive control. Considering this review's aims, only findings regarding lentinan are discussed. Basolateral LPS-stimulation upregulated TNF-α expression in the macrophages and IL-8 in Caco-2 cells. In contrast, apical lentinan decreased IL-8 mRNA levels but less strongly than budesonide did. In contrast to budesonide, lentinan did not significantly suppress TNF-α production in macrophages. Moreover, lentinan was not detected in the basolateral compartment of the co-culture by anti-lentinan ELISA, indicating that lentinan did not cross the Caco-2 monolayer, which is consistent with epithelial signaling rather than transepithelial transport [76].

Zhang et al. studied the effects of lentinan treatment on DSS-induced colitis in mice [77]. Lentinan treatment groups consisted of one that was orally gavaged with 100 mg kg⁻¹ and one that received lentinan capsules (100 mg kg⁻¹). A positive control group was administered 5-aminosalicylic acid (5-ASA). In vivo, both lentinan formulations significantly reduced weight loss, DAI scores, and colon shortening compared with DSS controls. Lentinan limited bacterial invasion of the colonic lumen, with only slight penetration into the mucus layers, in contrast to subepithelial crypt penetration observed in untreated DSS mice. Lentinan application improved crypt architecture, reduced goblet cell loss, and lowered histopathological scores, whereas the tight junction proteins occludin and claudin-1, as well as mucin 2 expression and distribution, were significantly increased. Lentinan also reduced serum levels of 4-kDa fluorescein isothiocyanate (FITC)-dextran and LPS, with effects comparable to those seen upon 5-ASA application. Lentinan further increased the expression of intestinal stem cell markers leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) and olfactomedin 4, antimicrobial peptide regenerating islet-derived protein 3 gamma (REG3γ), fucosyltransferase 2 (Fut2), and overall epithelial fucosylation in colitis-induced mice, as evidenced by immunofluorescence and flow cytometry. Lentinan treatment significantly increased anti-inflammatory IL-10 production in the colon and reduced concentrations of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β. Furthermore, lentinan expanded the murine CD45⁺CD11b⁺ macrophage marker and F4/80^hi MHCII⁺ macrophages in the colonic lamina propria but decreased infiltrating F4/80^int macrophages and neutrophil populations in DSS-induced colitic mice. The polysaccharide increased colonic retinoic acid receptor-related orphan receptor gamma t (RORγt)-positive type 3 innate lymphoid cell (ILC3) and IL-22⁺ frequencies within lamina propria lymphocytes, elevated colonic IL-22 secretion, and upregulated CD163 and Dectin-1 expression in DSS-treated mice. Colonic ligature experiments showed that fluorescent-labeled lentinan penetration was restricted under physiological conditions, whereas colitis allowed mucosal breach and contact with epithelial and lamina propria immune cells. Notably, IL-22 blockade abolished the protective effects on body weight, DAI scores, colon length, mucin 2 expression, Fut2 expression, and epithelial fucosylation. In vitro, lentinan activated Dectin-1 signaling in RAW264.7 macrophages and enhanced ILC3-dependent secretion of IL-1β, as small interfering RNA validated. In a co-culture of RAW264.7 macrophages and ILC3s, lentinan indirectly enhanced RORγt⁺ ILC3 expansion, function, and IL-22 production via macrophage-mediated effects. In summary, lentinan mitigated DSS-induced colitis by reinforcing mucosal barrier integrity, limiting bacterial translocation, and promoting epithelial regeneration. These effects were mediated through macrophage-dependent activation of ILC3s and IL-22 production via Dectin-1 signaling. These findings identify Dectin-1/IL-22 signaling as a central mechanism in lentinan's anti-colitic impacts [77].

Nishitani et al. evaluated the anti-inflammatory activity of intra-gastric lentinan application at 50, 100, or 200 µg/mouse, starting 7 days before DSS and continuing through the experiment [78]. Lentinan significantly attenuated DSS-induced weight loss and colon shortening at the intermediate dose. Intermediate and high doses of lentinan also significantly decreased histological damage scores. In the colon, lentinan significantly reduced IFN-γ mRNA expression at all doses, as well as IL-1β expression at the highest dose, compared with DSS controls. In vitro, using a co-culture system of apical Caco-2 cells and basolateral RAW264.7 macrophages, apical lentinan suppressed IL-8 mRNA induction in Caco-2 cells without reducing LPS-induced TNF-α production by RAW264.7 cells. Additional analyses revealed that lentinan decreased tumor necrosis factor receptor 1 (TNFR1) mRNA expression, protein levels, surface TNFR1 expression, and basolateral localization in Caco-2 cells. These effects on mRNA expression were confirmed in vivo using an intestinal ligated loop assay in mice. Mechanistically, these effects were clathrin-dependent, as they were inhibited by the clathrin endocytosis inhibitor monodansylcadaverine and partially attenuated by the presence of anti-lentinan antibodies, independently of effects on macrophage TNF-α production. Overall, oral lentinan was effective in preventing DSS-induced colitis and reducing inflammatory response. This effect was consistent with clathrin-dependent TNFR1 endocytosis and down-regulation rather than macrophage suppression [78].

Minato et al. examined whether oral or rectal pretreatment with lentinan (100 μg daily) could even more effectively protect mice from DSS-induced colitis [79]. Both routes suppressed DSS-associated weight loss, but oral administration was significantly more effective. In DSS-treated mice, oral, but not rectal, lentinan was associated with increased ileal Ifng mRNA expression. In healthy mice, oral lentinan upregulated ileal expression of T-box transcription factor (Tbx21) and Il12b as opposed to rectal lentinan decreasing the latter, whereas neither route of administration altered colonic Il12b expression. Further analyses demonstrated that oral lentinan increased proliferation of ileal lymphocytes and their migration to the inflamed colon, particularly of CD4⁺ T helper cells and regulatory T cells. In vitro experiments using a Caco-2/BMDC co-culture model showed that apical exposure to lentinan reduced Tnfr1 expression in Caco-2 cells. In contrast, direct lentinan exposure of BMDCs increased Il12b mRNA, but did not affect this expression by apical administration of lentinan to Caco-2 cells. These findings show that lentinan's anti-inflammatory effects were route-dependent, as oral, but not rectal, lentinan pretreatment protected against colitis. Lentinan promoted dendritic cell activation and epithelial TNFR1 modulation, consistent with a mechanism involving small-intestinal immune stimulation and epithelial responsiveness [79].

Wang et al. examined the effects of dietary lentinan in a piglet model of intestinal barrier injury and microbial dysbiosis induced by intraperitoneal LPS injection [80]. During LPS injury, lentinan supplementation maintained ileal claudin-1 protein expression; and even in the absence of LPS challenge, lentinan increased claudin-1 expression, jejunal villus height-to-crypt depth ratio, and ileal villus height and crypt depth. Regardless of LPS challenge, a diet supplemented with lentinan significantly decreased TNF-α levels in both jejunal and ileal mucosa, while IL-6 and IL-1β levels were specifically reduced in the ileum. Jejunal pro-inflammatory IL-1β, IL-6, and TNF-α mRNA expression as well as ileal TNF-α and IL-1β mRNA levels were decreased by lentinan supplementation. In addition, lentinan attenuated IL-6 expression in LPS treated piglets. Compared with control diet piglets, lentinan decreased mRNA expression of jejunal TLR-4, lipopolysaccharide-binding protein (LBP), myeloid differentiation factor 2 (MD2), MyD88, IRAK1, TRAF6, nucleotide-binding oligomerization domain-containing protein 1 (NOD1), receptor-interacting serine/threonine-protein kinase 2 (RIP2) and NF-κB, as well as ileal TRAF6 and NOD1, independent of the challenge. LPS increased jejunal and ileal heat shock protein 70 (HSP70) mRNA and protein levels, while lentinan further elevated HSP70 mRNA expression above LPS-induced levels. Irrespective of LPS challenge, lentinan enhanced short-chain fatty acids in cecal digesta, including propionate, butyrate, isobutyrate, and isovalerate. Bacterial taxa such as Prevotella_9 and unclassified Lachnospiraceae were positively correlated with these metabolites. Taken together, dietary lentinan preserved intestinal barrier structure, suppressed inflammatory signaling, and enhanced short-chain fatty acid production in a piglet model independent of LPS challenge. These effects were also associated with the induction of stress-protective HSP70 responses [80].

Ren et al. tested the effects of lentinan supplementation on intestinal inflammation in young taimen that had been induced by intraperitoneal injection of LPS [81]. Lentinan treatment groups were added 5 or 10 g kg⁻¹ lentinan to the fish's basal diet. Low-dose lentinan outperformed the high dose in preserving mid-intestinal claudin-d expression, maintaining mucosal integrity with intact microvilli and goblet cells, and reducing inflammatory cell infiltration in the lamina propria compared with LPS controls. Lentinan administration significantly reduced mRNA expression of transforming growth factor beta (TGF-β), TNF-α, IL-1β, IL-6, and IL-8, while raising IκBα mRNA in LPS-challenged fish, with the low dose showing superior effects. With lentinan intervention, the activities of the anti-oxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH Px), and catalase were 41.88%, 41.96%, and 93.13% higher at low doses and 14.93%, 16.27%, and 20.31% at high doses, respectively. Malondialdehyde (MDA) levels were significantly reduced in both groups, with low-dose lentinan showing better efficacy. These results were supported by partial restoration of SOD and catalase mRNA expression in LPS-challenged fish, with more pronounced effects in the low-dose group. Dietary lentinan also modified the intestinal microbial community. Low-dose lentinan samples clustered with LPS-treated controls, whereas high-dose samples clustered closer to the naïve control group. Specifically, lentinan application was accompanied by decreased Proteobacteria, Bacteroidetes, and Fusobacteria but elevated Firmicutes, Cyanobacteria, and Actinobacteria numbers. At lower taxonomic levels, lentinan increased beneficial Lactobacillaceae, Lachnospiraceae, and Ruminococcaceae populations, while it decreased pathogenic Enterobacteriaceae and Fusobacteriaceae if compared to the LPS control group. Overall, low-dose dietary lentinan restored intestinal barrier function, consistent with lower NF-κB–associated inflammatory signaling, enhanced anti-oxidant capacity, and favorably remodeled microbiota in LPS-challenged fish. The better efficacy of lower dosing suggests a non-linear dose-response relationship exerted by lentinan [81].

3.3. Effects of lentinan in chronic colitis and colon cancer

3.3.1. Lentinan and large intestinal carcinogenesis

Liu et al. addressed whether orally administered lentinan (5, 10, and 20 mg kg⁻¹ daily) alleviated chronic DSS-induced colitis in mice, using SASP at 200 mg kg⁻¹ as a reference control. In addition, the authors examined the protective effects of oral lentinan (20 mg kg⁻¹ daily) in an azoxymethane (AOM)/DSS-induced murine model of colitis-associated colorectal cancer. Lentinan was administered from the beginning of the third DSS cycle.

In the chronic DSS model, lentinan administration significantly reduced macroscopic colon damage scores compared with untreated controls, with the highest dose of lentinan even outperforming SASP. Consistently, lentinan significantly ameliorated microscopic damage scores in a dose-dependent manner, reducing epithelial lesions, crypt erosions and lymphocytic infiltration.

In AOM/DSS-treated mice, lentinan administration mitigated body weight loss and led to fewer and smaller tumors. Histological evaluations revealed reduced lymphocyte infiltration, less atypical hyperplasia, and fewer nuclear abnormalities in comparison with the diseased placebo control group. Furthermore, lentinan down-regulated the expression of IL-13, CD30L, as well as the colorectal cancer-related markers carcinoembryonic antigen (CEA), cytokeratin 8, and cytokeratin 18 and reduced p53 immunostaining. Collectively, oral lentinan application significantly ameliorated chronic DSS-induced colitis and also reduced the tumor burden in colitis-associated colorectal cancer. The anti-tumor effects of lentinan were accompanied by suppression of colorectal cancer markers [74].

3.3.2. Anti-tumor effects of lentinan depending on molecular weight fractions

Liu et al. evaluated the anti-tumor efficacy of molecular weight-fractionated lentinan polysaccharides (ultrasonic degradation-derived fractions UD-0 - highest molecular-weight fraction, UD-5, UD-20, and UD-60 - lowest molecular-weight fraction) in an AOM/DSS-induced murine colorectal carcinoma model, with 5-fluorouracil (5-FU) as a reference treatment regimen [82]. Oral administration of lentinan polysaccharide fractions significantly reduced occult fecal blood in a molecular weight-dependent manner, prevented body weight loss, restored colon length, and attenuated tumor burden in AOM/DSS-challenged mice. The therapeutic efficacy of UD-60 was similar to that of the positive control 5-FU, but with even fewer toxic effects. The results of histological analyses indicated that major organs were not damaged by oral administration of lentinan polysaccharide fractions, as opposed to 5-FU-induced lung toxicity, suggesting a favorable safety profile of the former. In vivo fluorescent imaging demonstrated that the ingested polysaccharide fractions mainly accumulated in the intestine and were gradually metabolized. Assessment of intestinal barrier integrity revealed that lentinan polysaccharide treatment preserved crypt architecture, reduced inflammatory cell infiltration, and enhanced epithelial ultrastructural integrity in AOM/DSS-induced colorectal carcinoma mice. Lentinan fractions also restored the expression of tight junction proteins zonula occludens-1 (ZO-1; encoded by TJP1) and claudin-1. Lentinan polysaccharides also increased goblet cell numbers, enhanced mucus secretion, and elevated fecal secretory immunoglobulin A (sIgA) levels in colorectal cancer mice, with lower-molecular-weight fractions performing better than factions of higher molecular weight. Treatment notably decreased proliferation antigen kiel 67 (Ki67)-positive proliferating cell numbers and increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cell counts, with anti-tumor effects similar to those seen upon 5-FU application. Serum concentrations of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β, were significantly decreased in AOM/DSS-challenged mice after administration of lentinan polysaccharides. In vitro, lentinan also inhibited the production of nitric oxide in LPS-stimulated RAW264.7 macrophages. Intestinal microbiota analyses revealed that the addition of lentinan polysaccharide fractions effectively restored microbial community structure in a molecular weight-specific manner. At the phylum level, an increase in beneficial Bacteroidetes, a decrease in potentially pathogenic Proteobacteria and a normalization of the Firmicutes/Bacteroidetes ratio was noted due to lentinan treatment. At the genus level, lentinan polysaccharides increased the abundance of Duncaniella and Muribaculum while reducing Helicobacter. In addition, lentinan polysaccharide treatment reversed AOM/DSS-induced reductions in short-chain fatty acid contents, significantly increasing propionate, butyrate, and valerate levels, with UD-60 achieving the highest concentrations. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses also suggested that the polysaccharide fractions could influence microbial phosphonate and phosphinate metabolism. In vitro experiments further demonstrated that UD-0, UD-5, UD-20, and particularly UD-60 suppressed colorectal cancer cell growth by inhibiting proliferation and promoting apoptosis in a molecular weight-dependent manner. All tested fractions reduced the viability of both HT-29 and SW480 human colorectal cancer cells in a dose- and molecular weight-dependent fashion, reaching maximum inhibition at 400 μg mL⁻¹. UD-60 reduced cell viability to 57% in HT-29 cells and to 60% in SW480 cells. No cytotoxicity was detected in normal L929 murine fibroblasts or RAW264.7 macrophages. Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) flow cytometry showed molecular weight-dependence in the induction of early apoptosis, as UD-60 induced early apoptosis in 22.6% of HT-29 cells and in 29.7% of SW480 cells. Collectively, molecular weight-fractionated lentinan polysaccharides exhibited significant anti-tumor effects in colitis-associated colorectal cancer, with lower molecular weight fractions demonstrating the best efficacy. These effects were caused by the preservation of intestinal barrier integrity, suppression of inflammation, induction of tumor cell apoptosis, and restoration of microbiota composition and short-chain fatty acid production. Among the fractions, UD-60 exhibited anti-tumor activity that was comparable to the positive control 5-FU while maintaining a favorable safety profile [82].

3.4. Direct anti-tumor mechanisms of lentinan

3.4.1. Lentinan's effect on apoptosis

Wang et al. investigated the anti-tumor effects of water-extracted soluble lentinan (SLNT) at concentrations of 0.2, 1.0, and 5.0 mg kg⁻¹ on HT-29 cells and corresponding xenograft tumors [83]. SLNT reduced volume and weight of HT-29 xenografts in nude mice by 17.88, 48.87, and 57.90%, respectively, close to the effect achieved by 5-FU (67.23%). Histopathological examination showed apoptosis-associated morphological alterations such as karyorrhexis and irregular karyomorphism in SLNT-treated tumors. In vitro, SLNT inhibited HT-29 cell proliferation in a concentration-dependent manner. At 1,600 μg mL⁻¹, cell viability reached only 35.57% based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The polysaccharide also dose-dependently induced apoptosis, producing classical apoptotic morphology such as DNA condensation. Significant activation of cysteine-dependent aspartate-specific protease (caspase)-3 was detected both in vitro and in vivo. Pharmacological inhibition of caspase-3 using a caspase-3 inhibitor I (namely, Ac-DEVD-CHO), however, reduced apoptosis from 32.91 to 15.88% in vitro. However, apoptosis levels remained above baseline, suggesting that apoptosis was at least in part caspase-independent. SLNT treatment further upregulated the activity of cleaved caspase-9 and elevated levels of cytosolic cytochrome c and Bcl-2-associated X protein (Bax). On the other hand, SLNT decreased the expression of B-cell lymphoma 2 (Bcl-2), consequently resulting in a 3-fold higher Bax/Bcl-2 ratio compared with control HT-29 cells. Comparable effects were observed with SLNT-treated HT-29 tumor xenografts. In addition, SLNT induced mitochondrial membrane potential depolarization in HT-29 cells. Intracellular reactive oxygen species (ROS) levels were significantly increased following SLNT exposure, with the strongest effects observed at 1,600 μg mL⁻¹ in vitro. Consistently, serum ROS levels were significantly elevated in mice treated with SLNT at doses of 1 and 5 mg kg⁻¹. Apoptosis was reduced by pretreatment with N-acetylcysteine (NAC) from 32.91 to 21.49%, accompanied by reduced cytochrome c release and normalization of the Bax/Bcl-2 ratio. However, about 20% of apoptotic cells still remained, indicating incomplete ROS dependency. In addition, SLNT activated caspase-8 and significantly reduced NF-κB-p65 nuclear translocation both in vitro and in vivo, while increasing TNF-α levels in serum and cell culture could be measured. Together, SLNT significantly suppressed colorectal tumor growth by inducing apoptosis via both intrinsic and extrinsic pathways, that involved ROS-dependent mitochondrial dysfunction, caspase activation, cytochrome c release, and inhibition of NF-κB signaling. Partial resistance to caspase and ROS inhibition indicated the contribution of parallel apoptotic mechanisms to lentinan-mediated tumor suppression [83].

3.4.2. Lentinan and stemness

Liu et al. investigated whether intravenous SLNT exerted direct effects on tumor growth and on stemness constituting a fundamental molecular and functional capacity of a cell to self-renew, persist over time, and differentiate into multiple specialized cell lineages [84]. To address this, the authors applied CD133⁺ colorectal cancer cells and murine xenografts models. In vivo, technetium-labeled SLNT rapidly accumulated at tumor sites, indicating preferential tumor localization. In a HCT116 human colorectal cancer xenograft model applying nude mice, SLNT significantly reduced tumor volume, weight, and Ki67 expression, without causing liver or kidney toxicity or body weight loss. In vitro, SLNT decreased the viability and clonogenic capacity of colorectal cancer cells. It also decreased the number and size of spheres formed by HCT116 and SW620 human colorectal cancer cells, as well as CD133 expression within these spheres. Gain- and loss-of-function studies using CD133 knockdown in high CD133-expressing HCT116 cells and CD133 overexpression in low CD133-expressing SW480 human colorectal adenocarcinoma cells demonstrated that CD133 promoted colorectal cancer proliferation, stemness, and tumorigenesis both in vitro and in vivo. In vivo, knockdown of CD133 reversed the tumor-suppressive function of SLNT in nude mouse xenografts, whereas overexpression of CD133 increased its efficacy. SLNT administration resulted in a significant suppression of CD133 protein levels in HCT116 cells, their CD133⁺ subpopulations, and established HCT116 xenograft tumors, as well as CD133 mRNA levels in HCT116 cells. SLNT also down-regulated the PI3K/protein kinase B (Akt) signaling pathway. Dose-dependent reductions in PI3K regulatory subunit p85, phosphorylated tyrosine kinase Src (p-Src), and phosphorylated protein kinase B (p-Akt) levels were observed in HCT116 cells as well as xenograft tumors following SLNT treatment. Collectively, SLNT directly targeted colorectal cancer stem-like cells by binding to CD133 and suppressing tumor growth and stemness both in vitro and in vivo. This effect was mediated through inhibition of the PI3K/Akt signaling pathway and selective cytotoxicity toward CD133⁺ tumor cells [84].

3.4.3. Lentinan and autophagy

Zhang et al. investigated the immune-independent anti-tumor effects of SLNT in HT-29 xenografts [85]. Intravenous SLNT application in nude mice significantly reduced tumor volume and weight in a dose-dependent manner. In vitro, SLNT induced autophagy in HT-29 cells, as indicated by increased levels of lipidated microtubule-associated protein 1 light chain 3 (LC3-II) and Beclin-1, enhanced autophagic flux, reduced sequestosome-1 (p62) levels, and by the accumulation of autophagosomes and autolysosomes. Pharmacological inhibition of autophagy using chloroquine or 3-methyladenine significantly attenuated SLNT-induced tumor cell death in vitro and reduced anti-tumor efficacy in vivo. SLNT treatment induced increases of endoplasmatic reticulum stress markers, including binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP) both in HT-29 cells and tumor tissues. SLNT activated unfolded protein response pathways via protein kinase R-like endoplasmic reticulum kinase (PERK) and serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1 alpha (IRE1α). Collectively, these findings demonstrate that SLNT induced immune-independent colorectal tumor suppression through endoplasmic reticulum stress–mediated autophagic and apoptotic cell death. This process was driven by IP₃R-dependent calcium release and activation of PERK and IRE1α unfolded protein response pathways [85].

3.4.4. Anti-angiogenic effects of lentinan

Deng et al. investigated the anti-cancer activity of lentinan in CT26 mouse colon carcinoma cell line tumor-bearing mice [86]. Lentinan exhibited potent anti-tumor effects, with an inverted U-shaped dose-response that showed maximum tumor suppression at 1.0 mg kg⁻¹ rather than at higher doses of 4.0 to 5.0 mg kg⁻¹. Treatment significantly suppressed tumor growth and reduced vascular perfusion within tumors, while preserving the vasculature of normal colon tissue, indicating tumor-specific vascular modulation. Mechanistically, lentinan impaired tumor vascular function without decreasing vessel density. Transcriptome and enzyme-linked immunosorbent assay (ELISA) analyses revealed an upregulated expression of angiostatic factors, including IFN-γ, TNF-α, C-X-C motif chemokine ligand (CXCL)9, tissue inhibitor of metalloproteinases (TIMP)1, and thrombospondin 1 (TSP1) in lentinan-treated tumors. Lentinan also promoted infiltration of CD4⁺ and CD8⁺ T cells, NK1.1⁺ natural killer cells, and myeloid cells, while suppressing tumor-associated macrophages. Neutralizing IFN-γ partially restored the inhibitory effects of lentinan on tumor growth and vascular function. Tumor suppression was largely maintained, and vascular alterations persisted in T cell-deficient nude mice, suggesting a T cell-independent IFN-γ axis likely supported by myeloid cells. Together, these findings demonstrate that lentinan inhibited colorectal tumor growth by inducing IFN-γ-dependent angiostasis and immune activation without disrupting normal vascular architecture. Tumor suppression was associated with impaired tumor perfusion, with upregulation of angiostatic factors and immune cell infiltration, and with myeloid cells contributing to IFN-γ signaling [86].

3.5. Lentinan as chemotherapy adjuvant - preclinical studies

3.5.1. Immunotherapeutic synergy

Mushiake et al. addressed whether the addition of intraperitoneal lentinan could augment anti-tumor efficacy of tegafur/gimeracil/oteracil (S-1) in Colon-26 colorectal tumor-bearing mice [87]. Compared with S-1 alone, combined treatment with S-1 and lentinan significantly improved survival, whereas body weight, adipose tissue mass, serum calcium and cytokine concentrations were not further affected. Within tumors, the combination therapy selectively increased the percentage of CD86⁺ cells, while total CD11c⁺ dendritic cell numbers were not affected. Dendritic cells isolated from tumor-bearing mice receiving lentinan plus S-1 immunotherapy showed significantly greater antigen-presenting capacity, as evidenced by increased T cell proliferation. In addition, effector immune cells from lentinan co-treated mice showed more pronounced cytotoxic activity against Colon-26 tumor cells compared to those derived from S-1 treated mice. This cytotoxicity was tumor specific, while no activity was observed against unrelated syngeneic or natural killer cell-sensitive targets. Importantly, the survival benefit of combined lentinan and S-1 treatment was abrogated in nude mice, even though efficacy of monotherapy with S-1 remained. No apparent adverse reactions were noted during long-term chemoimmunotherapy, supporting a favorable safety profile. Collectively, lentinan potentiated S-1 anti-tumor activity by promoting dendritic cell maturation and tumor-specific T cell-mediated immunity. The survival benefit required an intact adaptive immune system and was associated with increased antigen presentation and cytotoxic effector function [87].

Niu et al. examined whether lentinan enhanced chimeric antigen receptor T-cell (CAR-T) efficacy against epidermal growth factor receptor variant III (EGFRvIII)-positive and human CD19 (hCD19)-positive MC38 mouse colorectal carcinoma tumors [88]. Lentinan potentiated CAR-T-induced tumor growth inhibition in both EGFRvIII⁺ and hCD19⁺ MC38 immunocompetent mouse models. Compared with CAR-T or lentinan monotherapy, combination treatment significantly reduced tumor burden and significantly prolonged survival. Combined therapy increased intra-tumoral frequencies of CD8⁺CD107a⁺ cytotoxic T cells and central memory T cells, along with enhanced IFN-γ and IL-2 production in the tumors. Furthermore, lentinan diminished the expression of inhibitory receptors programmed cell death protein 1 (PD-1), T cell immunoglobulin and mucin domain-containing protein (TIM)-3, and lymphocyte activation gene-3 (LAG-3) on CAR-T cells in vivo. Transcriptomic tumor profiling revealed that lentinan had no effect on alternatively activated M2 macrophage polarization but significantly upregulated the expression of M1 macrophage-associated genes, including nitric oxide synthase 2 (Nos2), Cd86, and Il1b. In vitro, lentinan significantly enhanced the cytolytic activity of EGFRvIII-targeted CAR-T cells against MC38 colon cancer cells, as evidenced by increased CD107a⁺ degranulation and elevated IL-2 and IFN-γ secretion, without exerting direct cytotoxicity. CAR-T-mediated tumor toxicity was significantly enhanced at lentinan concentrations of 10 ng mL⁻¹, with further improvement up to 10 μg mL⁻¹, even though the effect did not strictly correlate with dose. Lentinan promoted CAR-T differentiation toward central memory (CD44⁺ CD62L⁺) and stem-like memory (CD44⁺ CD62L⁺) phenotypes without affecting naïve, effector memory, effector T cell differentiation, Ki67⁺ proliferation or granzyme B expression. At the transcriptional level, lentinan upregulated Tcf7 and Foxo1 (forkhead box O1) mRNA expression, while leaving PR domain zinc finger protein 1 (Prdm1) and Tbx21 mRNA expression unchanged. In addition, lentinan reduced CAR-T cell exhaustion, characterized by decreased TIM-3 and CD317 expression in vitro. Collectively, lentinan enhanced CAR-T cell cytotoxicity, promoted memory differentiation through the TCF1/FOXO1 axis, partially alleviated exhaustion phenotypes, and shifted the macrophage transcriptional profiles toward an M1-like phenotype [88].

3.5.2. Chemotherapy sensitization

Wang et al. explored whether lentinan was capable of promoting the chemosensitivity of the folinic acid, fluorouracil, irinotecan (FOLFIRI) regimen in human colon carcinoma cell lines (CBS, Moser, FET, and SW480) [89]. Lentinan co-treatment more than doubled FOLFIRI-induced cytotoxicity and remarkably inhibited tumor cell invasion and clonogenic capacity compared with FOLFIRI alone. Moreover, lentinan stimulated the P1 and P2 promoters of the calcium sensing receptor (CaSR) via vitamin D response elements, increasing the expression level of CaSR similarly to those induced by calcitriol and calcipotriol. Knockdown of CaSR abolished lentinan-induced chemosensitization, thus verifying receptor dependency. In CaSR-competent cells, lentinan inhibited the expression and promoter activity of thymidylate synthase and survivin. Furthermore, lentinan suppressed canonical Wingless-related integration site (Wnt)/β-catenin signaling through enhanced E-cadherin/β-catenin membrane complex formation and inhibition of transcription factor TCF4-dependent nuclear transcription. These effects were abolished in CaSR-silenced cells. Notably, lentinan induced CaSR expression to a degree comparable with vitamin D analogs without related side effects such as hypercalcemia. Collectively, lentinan potentiated FOLFIRI efficacy through CaSR mediated transcriptional regulation of thymidylate synthase, survivin, and Wnt/β-catenin signaling while maintaining a favorable safety profile [89].

Zhao et al. studied the effects of lentinan, either alone or in combination with docetaxel and cisplatin, on BGC823 human gastric cancer cells [90]. The authors reported that individual lentinan treatment at doses ranging from 1.56 to 12.5 μg mL⁻¹ significantly inhibited the proliferation of BGC823 cells in a dose-dependent manner compared with control cells. Combined treatment with docetaxel and cisplatin effectively suppressed cell proliferation. The triple combination of lentinan, docetaxel, and cisplatin further enhanced anti-proliferative effects relative to docetaxel/cisplatin treatment alone, exhibiting an additive interaction at 6.25 + 2.5 + 50 μg mL⁻¹. Flow cytometry analysis indicated that independent treatment with lentinan (6.25 μg mL⁻¹) led to apoptosis in 19.84% of cells, whereas the combination group of docetaxel plus cisplatin resulted in apoptosis in 50.22% of cells and the triple combination increased apoptosis to 72.06%. Collectively, these findings demonstrate that lentinan exerted direct anti-proliferative effects and significantly enhanced docetaxel- and cisplatin-induced anti-proliferative and pro-apoptotic effects in gastric cancer cells in vitro. The combination therapy of lentinan, docetaxel, and cisplatin increased apoptosis beyond that achieved by chemotherapy alone, indicating additive anti-tumor activity [90].

3.6. Lentinan as chemotherapy adjuvant - clinical trials

3.6.1. Phase III randomized controlled trial

Yoshino et al. conducted a phase III randomized controlled trial in order to evaluate the clinical benefits of adding lentinan to S-1 chemotherapy compared with S-1 monotherapy in patients suffering from advanced gastric cancer [91]. A total of 295 patients with unresectable or recurrent gastric cancer were randomly assigned to receive oral S-1 (40–60 mg twice daily, for 4 weeks followed by a 2-week rest) alone or in combination with weekly intravenous lentinan (2 mg) until disease progression or intolerable toxicity. Median overall survival was 9.9 months in the lentinan plus S-1 group compared with 13.8 months in the S-1 monotherapy group, and time-to-treatment failure was shorter in patients treated with lentinan (2.6 vs 4.3 months). Objective response rates were 22.3% with S-1 alone vs 18.7% with S-1 plus lentinan, although the difference between groups was not significant. Safety profiles were also alike between groups, with comparable rates of hematologic and non-hematologic adverse events, treatment discontinuation due to toxicity (13.1% vs 19.9%) or treatment-related mortality (one treatment related death in the lentinan group). QOL, assessed by Functional Assessment of Cancer Therapy - Biological Response Modifier (FACT-BRM), remained stable through 12 weeks in both treatment arms. Flow cytometric analysis revealed wide inter-individual variability in the fraction of CD14⁺ monocytes that bound lentinan, ranging from 0.03 to 38.7% in a total of 309 patients and remaining stable over the treatment period. Lentinan was associated with significantly prolonged survival in high binding capacity (≥2.0%) patients who received at least two courses of chemotherapy (3-year survival rate: 24.1 vs 3.1%), whereas no survival benefit was observed in low-binding patients. Hence, the addition of lentinan to S-1 chemotherapy did not improve overall survival in the overall gastric cancer population but demonstrated significant benefit in patients with high monocyte lentinan-binding capacity. Of note, lentinan showed no additional toxicity, supporting its safety in clinical use [91].

3.6.2. Colorectal cancer trials

Hazama et al. assessed the tolerability and QOL impact of orally administered superfine dispersed lentinan (SDL; 15 mg day⁻¹ for 12 weeks) in patients with unresectable colorectal cancer [92]. Among 71 eligible patients (median age 65 years), only few SDL-related adverse events were reported (n = 4; 5%) that were limited to mild grade 2 diarrhea, rashes, or constipation, resolving during treatment. Chemotherapy-related toxicities were infrequently observed in this cohort, given that only one event of grade 4 neutropenia after treatment with 5-FU/levofolinate occurred while the rest were grade 1–2, implying high level of tolerability relative to standard chemotherapy. QOL, assessed in 48 patients, remained stable overall but showed significant improvement in patients with low baseline QOL. 65% of this subgroup experienced a measurable improvement. Flow cytometric analyses demonstrated that enhanced lentinan-binding capacity (≥5%) of peripheral CD14⁺ monocytes in patients was significantly associated with greater QOL improvement compared with patients exhibiting lower binding capacity. The authors hypothesized that the clinical benefit of SDL could depend on host immunoreactivity and interactions between monocytes and lentinan. Oral SDL was well tolerated and maintained overall QOL in patients with unresectable colorectal cancer. Patients with low baseline QOL and high monocyte lentinan-binding capacity showed the greatest benefit [92].

Dong et al. investigated the treatment efficacy and safety of intravenous lentinan in combination with capecitabine/oxaliplatin (XELOX) chemotherapy in patients with digestive tract malignancies in comparison with XELOX alone [93]. Sixty patients with gastric, colon or rectal cancer were divided by order of admission and were subsequently treated with XELOX chemotherapy (oxaliplatin 130 mg m⁻² on day 1; capecitabine 1,000 mg m⁻² on days 1–14; four 28-day cycles), with the experimental group (n = 30) additionally receiving intravenous lentinan (2 mL, twice weekly). Following treatment, the lentinan group had significantly higher hemoglobin, serum albumin, CD3⁺ T cell, and CD4⁺ T cell numbers, but lower CD8⁺ T cell counts compared with controls, despite reductions from baseline in both groups. Lentinan significantly reduced chemotherapy-associated toxicity, with the total incidence of adverse events declining to 3.33% compared with 20.0% in controls. This decrease included fewer cases of leukopenia, liver and kidney dysfunction, and gastrointestinal side effects. Short-term efficacy assessed by Response Evaluation Criteria In Solid Tumors (RECIST) criteria showed superior outcomes in the lentinan group, with clinical benefit rates of 96.67% vs 76.67% and objective response rates of 86.67% vs 46.67% if compared to controls. Hence, lentinan co-administration with XELOX chemotherapy improved short-term treatment efficacy, immune parameters, and hematologic tolerance in patients with digestive tract malignancies. The combination significantly reduced chemotherapy-associated toxicity while enhancing clinical response rates [93].

3.6.3. Gastric cancer trials

Ina and colleagues conducted a retrospective chart review to evaluate the efficacy of lentinan (2 mg intravenously every two weeks) combined with S-1-based chemotherapy in gastric cancer patients with liver metastases [94]. Among 138 metastatic gastric cancer patients who underwent treatment between 2010 and 2015, a total of 12 patients (8 men, 4 women; median age: 67 years) received lentinan in combination with S-1 monotherapy, S-1/cisplatin or paclitaxel/S-1/cisplatin (PSC) triple combination therapy. Trastuzumab was added in human epidermal growth factor receptor 2 (HER-2)-positive cases. The overall response rate was 42% (one complete remission, four partial remissions), with a disease control rate of 83%. Median overall survival amounted to 407 days and objective responders (n = 5) showed significantly longer overall survival than patients with stable or progressive disease (n = 7). One complete response was seen in a 65-year-old male patient that achieved sustained disappearance of multiple liver metastases following PSC/lentinan therapy, with remission maintained for 33 months [94].

In their study, Ina and coworkers administered intravenous lentinan (2 mg, every 2–3 weeks) as an adjuvant to S-1-based regimens, including S-1 alone, S-1/paclitaxel, S-1/cisplatin and PSC triple therapy, in patient with inoperable gastric cancer [95]. Sixty-eight eligible patients (chemotherapy alone, n = 37; lentinan plus S-1, n = 31) were analyzed. The authors observed significantly longer median overall survival in the lentinan group compared with chemotherapy alone (689 vs 565 days). One-, two-, and five-year survival rates were also higher in the lentinan group (91.3, 45.7, and 10.0%, respectively) than in controls (59.4, 32.7, and 0%). No significant differences were observed between groups in the occurrence of grade 3–4 toxicities, including neutropenia or mucositis. While granulocyte-to-lymphocyte ratios showed no differences at baseline or after three months, patients receiving lentinan tended to maintain lower ratios (i.e., around or below 2) at one-year post-treatment and one month prior to death compared with patients that only received chemotherapy. Lentinan adjuvant therapy significantly prolonged overall survival and improved long-term survival rates in patients with inoperable gastric cancer without increasing severe toxicity. Sustained immune balance, reflected by lower granulocyte-to-lymphocyte ratios, was associated with improved outcomes [95].

Higashi et al. conducted a clinical study involving 39 patients with unresectable gastric cancer. Nineteen patients received intravenous lentinan (2 mg, on days 1 and 8) in addition to S-1/paclitaxel, while 20 were treated with S-1/paclitaxel alone [96]. Baseline characteristics were not different between treatment groups. The authors did not report a significant improvement in overall survival by administration of lentinan compared with chemotherapy alone. Although the median treatment duration in the lentinan group was 79 days longer than that in the control group, this difference did not reach statistical significance. The overall incidence of adverse events was 61.5%, and lentinan co-administration was associated with fewer cases of leukopenia, nausea, peripheral neuropathy, stomatitis, and dysgeusia. Together, lentinan co-administration did not significantly improve overall survival but chemotherapy-associated adverse events tended to be less frequent and treatment duration was prolonged. These findings suggest a supportive rather than survival-prolonging role for lentinan in this treatment setting [96].

3.6.4. QOL-focused studies

Kataoka and coworkers evaluated whether lentinan (2 mg/week) improved QOL during S-1/paclitaxel chemotherapy in patient with advanced gastric cancer [97]. Twenty patients were treated with S-1 (80 mg m⁻² on days 1–14) and paclitaxel (50 mg m⁻² on days 1 and 8) on a 3-week cycle for 12 weeks combined with lentinan initiated from either the first cycle (12-week group, n = 10) or the third cycle (6-week group, n = 10). The proportion of CD14⁺ monocytes binding lentinan ranged from 0.16 to 11.95% and did not change during treatment. The overall response rate was 57.9% (70% in the 12-week arm versus 44% in the 6-week arm, but the difference was not significant) and the median survival time amounted to 314 days. Grade ≥3 hematologic toxicities included anemia, neutropenia, and leukopenia, while grade ≥3 non-hematologic toxicities involved diarrhea, anorexia, and allergic reactions. One grade 5 pulmonary fibrosis was observed in the 6-week cohort. QOL, assessed by the Quality of Life Questionnaire for Cancer Patients Treated with Anti-Cancer Drugs (QOL-ACD), improved from baseline to week 11 in the 12-week group but not in the 6-week treatment arm. Collectively, early and prolonged lentinan administration enhanced QOL during combination chemotherapy for advanced gastric cancer. QOL benefits were time-dependent and more pronounced with earlier initiation of treatment [97].

Yoshino and colleagues tested whether oral SDL (15 mg day⁻¹ for 12 weeks) provided QOL and survival benefits in patients with unresectable or recurrent gastric cancer [98]. Twenty-six eligible patients (median age 66 years; 88% with stage IV disease) received SDL in combination with standard chemotherapy for 12 weeks. All 18 reported adverse events were determined to be due to chemotherapy or disease progression, given that no SDL-related adverse events were observed. The median survival time was 17.1 months, with 1- and 2-year rates of survival of 47.8 and 31.9%, respectively, with six patients (23%) surviving for more than three years. QOL analyses showed strong Spearman correlations between survival and QOL-ACD scores at week 12, including the overall score as well as activity, physical, psychological, and social domains. Survivors displayed significantly higher QOL-ACD scores than non-survivors and patients with higher QOL-ACD scores exhibited superior survival compared with those with lower scores. Overall, oral SDL was well tolerated, and QOL-ACD scores at week 12 were strongly associated with overall survival, supporting QOL as a clinically meaningful endpoint and potential prognostic indicator in this cohort [98].

3.6.5. Esophageal cancer trial

Another study by Wang et al. examined whether the addition of lentinan to tegafur chemotherapy improved clinical outcomes in esophageal cancer patients compared with tegafur monotherapy [99]. Fifty patients, that were divided according to their order of hospitalization, completed two 21-day treatment cycles consisting of intravenous tegafur (1,000 mg day⁻¹ for 5 days), while the experimental group (n = 25) additionally received intravenous lentinan (1 mg every other day). Both the lentinan and the control groups showed significant post-treatment improvement in the Karnofsky performance status, Eastern Cooperative Oncology Group (Zubrod-ECOG-WHO) performance status, and QOL scores. However, the QOL scores were more profoundly improved in the lentinan group. Assessment based on World Health Organization criteria for solid tumors indicated higher proportions of partial responses, improvement, and disease stabilization after two courses of treatment, whereas no significant differences were observed after a single cycle. There was a T helper type 1 (Th1) shift of serum cytokine profiles in both groups, with increased serum concentrations of pro-inflammatory IL-2, IL-6, and IL-12 and decreased levels of anti-inflammatory IL-4, IL-5, and IL-10 relative to baseline, with more pronounced effects seen in the lentinan group. Hence, lentinan enhanced QOL and clinical response when combined with tegafur chemotherapy in esophageal cancer patients. The treatment promoted a Th1-shifted cytokine profile and improved disease control over repeated cycles [99].

3.7. Epithelial-targeted injury models

3.7.1. Chemotherapy-induced mucositis

Zhang et al. examined the impact of lentinan on intestinal mucositis in mice induced by intraperitoneal administration of 5-FU [100]. Mice from the treatment group were orally gavaged with lentinan at doses of 5 mg kg⁻¹ or 50 mg kg⁻¹, while an additional group was treated with tablets containing viable probiotic bacteria such as bifidobacteria and lactobacilli. Given this review's focus, only effects of lentinan are discussed. High-dose lentinan significantly attenuated 5-FU-induced reductions in body weight, food intake, and colon length. Immunohistochemical analysis and alcian blue-periodic acid-Schiff (PAS) staining revealed that high-dose lentinan effectively preserved mucin 2 expression and goblet cell numbers compared with untreated mucositis mice. Consistently, immunofluorescence analysis showed that high-dose lentinan significantly restored colonic ZO-1 and claudin-1 fluorescence intensity in inflamed tissues, indicating improved epithelial barrier integrity. Histological examination showed that lentinan ameliorated epithelial necrosis, crypt loss, glandular cavitation, and inflammatory cell infiltration in a dose-dependent manner. Compared with untreated 5-FU controls, lentinan-treated mice exhibited significantly increased villus height and reduced crypt depth. Lentinan additionally modulated systemic immune responses and gut microbial composition. ELISAs revealed decreased serum concentrations of TNF-α, IL-1β, and IL-6 and increased IL-10 concentrations in the high-dose lentinan group relative to 5-FU controls, whereas fecal microbiota analysis showed that β-diversity and microbial taxa in lentinan-treated groups clustered more closely with healthy controls than the 5-FU group. Lentinan application was accompanied by reduced Proteobacteria abundance and increased Lachnospiraceae, Muribaculaceae, and Prevotellaceae families in fecal samples compared with 5-FU-induced mucositis mice. Thus, high-dose oral lentinan effectively mitigated chemotherapy-induced intestinal mucositis by preserving epithelial barrier integrity, goblet cell function, and tight junction expression. These protective effects were accompanied by suppression of systemic inflammation and restoration of gut microbiota composition [100].

In their study Suga and colleagues applied a murine colon cancer model by subcutaneous inoculation of C26 colon carcinoma cells [101]. In a long-term experimental protocol, mice received oral S-1 chemotherapy five days per week and were additionally administered lentinan intravenously at 0.1 mg twice weekly. A short-term protocol was also employed, in which mice received three intraperitoneal injections of lentinan (0.1 mg per mouse), followed by oral S-1 administration after the final injection. Lentinan co-administration prevented loose stools and normalized ileal crypt apoptosis by day 3 without affecting body weight compared with S-1 treatment alone. Single-dose experiments confirmed dose-dependent increases in ileal apoptotic cell bodies following S-1 at doses from 10 to 25 mg kg⁻¹, which were significantly suppressed by lentinan pretreatment. Immunofluorescence analysis revealed expansion of CD11b⁺TIM-4⁺ cells within ileal crypts, whereas this effect was abolished by depletion with liposomal clodronate. Elimination of these cells abrogated lentinan-mediated protection, demonstrating that CD11b⁺TIM-4⁺ phagocytic cells are required for lentinan-mediated protection. Further immunofluorescence studies also showed that CD11b⁺ cells were closely associated with apoptotic debris, as nuclear fragments were detected inside the phagocytes. These findings provide evidence that lentinan prevented S-1-induced gastrointestinal toxicity by stimulating local CD11b⁺TIM 4⁺ phagocytic cells that clear apoptotic enterocytes [101].

3.7.2. Lentinan in antibiotic- and microbiota-targeted injury

Ji et al. examined lentinan's effects on intestinal inflammation and dysbiosis following oral administration of four antibiotics, namely ampicillin, vancomycin, metronidazole, and neomycin in mice [102]. Animals in the treatment group were orally gavaged with lentinan at 200 mg kg⁻¹ per day. Following antibiotic withdrawal, oral lentinan accelerated body weight recovery and normalized cecal size. Furthermore, lentinan treatment enhanced intestinal barrier integrity, as real time quantitative polymerase chain reaction (RT-qPCR) demonstrated the restoration of antibiotic-induced reductions in colonic ZO-1 and occludin mRNA expression. Histological examination of murine intestinal ex vivo biopsies showed reduced villus damage and inflammatory cell infiltration, along with improved tissue architecture if compared to untreated antibiotic-exposed mice. Analysis of mucosal immune parameters showed lower colonic TNF-α and IL-6 levels and reduced NF-κB signaling activity in lentinan-treated mice relative to antibiotic controls. The authors further evaluated whether oral lentinan application altered the gut microbial community structure and metabolic output. Unique fraction metric principal coordinates analyses confirmed that antibiotic treatment disrupted microbial β-diversity, whereas lentinan recovered those alterations. At the phylum level, lentinan increased Firmicutes abundance, normalized the Firmicutes/Bacteroidetes ratio, and restricted Proteobacteria expansion. At lower taxonomic levels, the relative abundance of beneficial bacteria such as Ruminococcus, Allobaculum, and Lactobacillus in lentinan-treated mice was considerably higher, whereas potentially pathogenic genera like Parabacteroides and Klebsiella showed a decline. Moreover, lentinan treatment increased fecal total short-chain fatty acid concentrations as well as acetate, propionate, and butyrate levels if compared to antibiotics-challenged control mice. Overall, lentinan restored intestinal homeostasis following broad-spectrum antibiotic–induced injury by enhancing epithelial barrier integrity, suppressing mucosal inflammation, and normalizing gut microbiota composition. These effects were accompanied by enrichment of potentially health-beneficial commensal bacterial taxa and increased short-chain fatty acid production in the intestinal tract [102].

Jin et al. infected mice with the parasite Trichinella spiralis and subsequently administered lentinan orally either alone or in combination with a STAT6 inhibitor in order to block type-2 signaling or with broad-spectrum antibiotics to disrupt the complex intestinal microbiota [103]. Lentinan significantly reduced Trichinella spiralis burdens and remained effective even when STAT6 was inhibited. The authors reported that infection induced an upregulation of mucin 2 that was even further enhanced by lentinan treatment. Antibiotic-mediated microbiota depletion suppressed both infection-driven and lentinan-induced mucin 2 expression, indicating that lentinan's effects were dependent on an uncompromised complex gut microbiota composition. Notably, microbial profiling revealed that lentinan even significantly increased α-diversity indices, whereas phylum-level shifts included increased Bacteroidetes and reduced Proteobacteria abundances if compared with infected mock control mice. 16S ribosomal RNA analysis further showed restoration of Clostridiales, Bacteroidales, Muribaculae, and Lachnospiraceae NK4A136 families, whereas the fecal metabolites butyrate and isobutyrate were much higher as compared to infected control mice. The authors further examined whether lentinan's effects could be attributed to fecal butyrate concentrations. Therefore, sodium butyrate or Clostridium tyrobutyricum were independently administered to the mice. Both interventions recapitulated lentinan's protective effects and reduced helminth burden, increased goblet cell numbers, enhanced mucus secretion, and elevated mucin 2 expression. KEGG functional prediction suggested enrichment of microbial pathways related to glycan biosynthesis and metabolism. Collectively, these findings demonstrate that oral lentinan enhanced host resistance to Trichinella spiralis infection through microbiota-dependent mechanisms that promoted butyrate production and mucin 2-mediated mucus responses independently of STAT6-driven type-2 immunity [103].

3.7.3. Lentinan in viral infection

Fan and colleagues studied whether dietary lentinan could prevent damage caused by rotavirus infection in both pigs and porcine intestinal epithelial IPEC-J2 cells [104]. Treatment groups were supplemented with 84 mg kg⁻¹ lentinan in addition to their basal diet. In vivo, lentinan supplementation exerted significant main effects on average daily gain, feed conversion, and serum urea nitrogen concentrations, with supplemented pigs showing higher daily gain and reduced feed conversion and serum urea nitrogen than control diet pigs under both healthy and rotavirus-challenged conditions. Lentinan delayed diarrhea onset and reduced diarrhea duration and severity in rotavirus-infected pigs. Significant main effects of both rotavirus infection and lentinan supplementation were observed for immunological parameters including IgG, IgM, sIgA, IFN-β, and rotavirus-specific antibodies. While rotavirus challenge increased serum immunoglobulin levels, lentinan further enhanced these responses. Whereas serum IgG was significantly influenced by diet, this was not the case upon rotavirus infection. In jejunal mucosa samples, rotavirus challenge suppressed sIgA levels, whereas lentinan supplementation restored and enhanced mucosal immunity in both healthy and infected pigs. A significant interaction was observed for rotavirus-specific serum antibodies, whereby lentinan selectively increased those levels in infected pigs. Lentinan also exerted a significant main effect on jejunal rotavirus-specific antibody levels across both conditions and consistently upregulated IFN-β expression, with the highest levels observed in lentinan-supplemented, rotavirus-infected pigs. Furthermore, rotavirus challenge suppressed the expression of porcine beta-defensin (pBD)1, pBD2, and pBD3, while lentinan supplementation upregulated these peptides in both healthy and infected pigs. Lentinan also enhanced expression of antiviral signaling molecules, including TLR-3, retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated protein 5 (MDA5), IFN-β, and interferon-stimulated gene 15 (ISG-15), relative to controls. A significant interaction was also observed for mitochondrial antiviral signaling protein (MAVS), since lentinan increased its expression in infected pigs and particularly in healthy animals. Lentinan further reduced jejunal rotavirus nonstructural protein 4 (NSP4) levels, especially in rotavirus-infected pigs. In vitro, lentinan improved viability of rotavirus-challenged IPEC-J2 cells, reduced NSP4 levels in culture media, and upregulated pBD2, pBD3, RIG-I, MDA5, MAVS, IFN-β, and ISG-15 mRNA expression. Collectively, dietary lentinan increased anti-viral mucosal immunity and attenuated rotavirus-induced intestinal injury by enhancing immunoglobulin responses, antimicrobial peptide expression, and IFN-mediated anti-viral signaling [104].

Mao et al. also used a diet-based lentinan intervention strategy in weaned piglets, followed by oral rotavirus infection resulting in intestinal injury [105]. Lentinan-supplemented diet contained both the basal components and 84 mg kg⁻¹ of lentinan product, added by replacing the same amount of corn starch. Lentinan supplementation reduced diarrhea severity and jejunal NSP4 levels in rotavirus-infected pigs and significantly lowered serum diamine oxidase activity in both healthy and infected animals compared with the control diet group. A significant main effect of lentinan diet was observed for jejunal mucin 1, mucin 2, ZO-1, and occludin expression, with higher levels in lentinan-supplemented pigs across conditions. Significant interactions between lentinan diet and rotavirus infection were detected microscopically for villus height and crypt depth, with lentinan increasing villus height and reducing crypt depth in healthy pigs, but specifically in infected animals. Lentinan diet was also accompanied by elevated villus height-to-crypt depth ratio in both healthy and challenged animals. Moreover, lentinan exerted significant effects on oxidative stress and apoptosis-related parameters, increasing total anti-oxidant capacity and reducing MDA, Bax, and caspase-3 levels in both conditions. A significant interaction was further observed for Bcl-2 expression, which was increased by lentinan in infected pigs and specifically elevated in healthy animals. Lentinan application increased the fecal abundances of bifidobacteria in both healthy and infected pigs, whereas lactobacilli and total bacterial counts were elevated in lentinan-challenged infected pigs. Lentinan supplementation also increased cecal the short-chain fatty acids acetate and propionate as well as total volatile fatty acid levels, with acetate and total volatile fatty acid levels being elevated in both conditions and propionate increased particularly in rotavirus-infected pigs. Collectively, these findings demonstrate that lentinan provides comprehensive intestinal protection against rotavirus-induced intestinal injury by enhancing barrier function, anti-oxidant capacity, suppressing apoptosis, and promoting health-beneficial gut microbiota and short-chain fatty acid production during rotavirus challenge [105].

Ren and colleagues investigated the protective effects of crude lentinan (CLNT) in rainbow trout challenged with infectious hematopoietic necrosis virus (IHNV). Fish were fed diets supplemented with CLNT at 1.0 or 2.0% prior to intraperitoneal IHNV infection. IHNV infection resulted in a mortality rate as high as 93.3% in the infection group, whereas CLNT supplementation significantly improved survival. With 23.3%, the low-dose group showed a higher survival rate than the high-dose group with 17.7%. Dietary addition of CLNT significantly reduced viral burden in the intestine as indicated by reduced mRNA expression of IHNV nucleoprotein (IHNV-N) and polymerase (IHNV-L) genes compared with infected controls. RT-qPCR experiments revealed that CLNT enhanced the intestinal gene expression of tight junction proteins claudin-d, ZO-1, and occludin. Further histological analyses demonstrated reduced goblet cell hypertrophy, intraepithelial leukocytosis, and epithelial vacuolation when compared with infected controls. Interestingly, these effects were even more pronounced in low-dose versus high-dose CLNT supplementation. RT-qPCR displayed that CLNT significantly suppressed TNF-α, IL-1β, IL-6, and TGF-β mRNA expression. Again, low-dose CLNT was more effective than the higher dose in restoring TNF-α, IL-6, and TGF-β expression toward baseline levels. Moreover, CLNT supplementation markedly enhanced the total intestinal anti-oxidant capacity as indicated by increased SOD, but reduced MDA levels, with greater effects observed with the lower dose. Finally, CLNT's impact on intestinal microbiota composition and metabolic activity were assessed. Gut microbial community analyses indicated partial restoration of microbial homeostasis, characterized by increased relative abundances of Proteobacteria and Firmicutes and reduced levels of Myxococcota, Nannocystis, and Mycobacterium compared with infected controls. The administration of CLNT also increased acetic acid concentrations and altered predicted microbial functional pathways, given down-regulation of amino acid biosynthesis, carbon metabolism, and purine metabolism relative to IHNV infection models. Collectively, dietary lentinan improved survival following viral infection by attenuating intestinal inflammation, restoring epithelial barrier function, enhancing anti-oxidant defenses, and normalizing gut microbial homeostasis. The superior efficacy of lower versus higher doses indicates a dose-dependent immunomodulatory effect on viral intestinal injury [106].

3.8. Lentinan in immunosuppression models

Jin et al. investigated whether oral gavage of lentinan (200 mg/kg/day) for 10 days alleviated immunosuppression and intestinal injury in mice following cyclophosphamide (CP) challenge [107]. Lentinan attenuated CP-induced body weight loss and improved spleen and thymus immune indices. RT-qPCR analysis revealed a significant increase of ileal claudin-1 mRNA expression due to lentinan application. Mucin 2 and G-protein coupled receptor (GPR) 43 transcripts were elevated across experimental groups rather than uniquely induced by lentinan. Histological analyses revealed reduced villus injury and epithelial disruption in the terminal ileum of CP-challenged mice receiving lentinan. Lentinan also showed significant impacts on immune parameters reversing CP-induced suppression of serum IL-2, IL-6, and IFN-γ concentrations, as well as ileal IL-1β and IFN-γ mRNA expression levels. Furthermore, fecal microbiota analysis demonstrated significant increases in α- and β-diversity indices in lentinan-treated mice. The intervention significantly raised Bacteroidetes numbers, but decreased Firmicutes and Proteobacteria phyla, and restored distinct genera such as Lactobacillus, Bifidobacterium, and Roseburia that had been depleted by CP. In summary, lentinan reversed CP-induced immunosuppression by restoring intestinal barrier integrity, cytokine levels, and gut microbiota composition [107].

Jiang et al. applied a CP-induced immunosuppression mouse model, where treatment group animals were challenged with oral lentinan (800 mg/kg/day) for 7 days [108]. Lentinan partially restored spleen and thymus immune indices and significantly increased intestinal sIgA levels compared with mock treated diseased counterparts. Concanavalin A (ConA)/pokeweed mitogen-stimulated lymphocytes from both Peyer's patches and the spleen showed enhanced proliferation. Histological examination of intestinal tissues revealed that lentinan partially restored Peyer's patch size, lymphocyte density, and ulex europaeus agglutinin I (UEA-1)-positive M cell numbers in the follicle-associated epithelia. Lentinan-treated samples exhibited higher CD19⁺ B-cell proportion and elevated activation-associated T cell subsets, including CD3⁺CD25⁺ and CD3⁺CD69⁺ compared with diseased placebo controls. On the other hand, the intervention decreased the CP-induced elevation of CD3⁺ T cell proportion toward baseline. Furthermore, the authors generated an M cell-like in vitro model by co-culturing Caco-2 cells with Peyer's patch lymphocytes. The M cell-like differentiation demonstrated increased transcytosis of fluorescent microspheres and less microvilli formation compared with Caco-2 monocultures. ConA stimulated Peyer's patch lymphocytes that were isolated from CP-treated mice failed to induce M cell differentiation in Caco-2 cells, as evidenced by reduced microsphere transcytosis compared with controls. In contrast, co-cultures that used ConA-stimulated lymphocytes from lentinan-treated mice promoted Caco-2 differentiation toward an M cell-like phenotype and significantly increased transcytosis. In accordance, electron microscopical histological examination showed a clearer loss of microvilli and more pronounced M cell-like appearance of Caco-2 cells co-cultured with lymphocytes isolated from lentinan-treated mice. Collectively, lentinan restored mucosal immunity in immunosuppressed mice by promoting Peyer's patch recovery, lymphocyte activation, and M cell differentiation through lymphocyte-mediated epithelial reprogramming [108].

In their study, Jiao et al. applied a murine dexamethasone-induced immunosuppression mouse model. Lentinan was administered to mice by oral gavage every other day (6, 12, or 18 mg/mouse per dose) [109]. Mice were immunized with ovalbumin (OVA) via primary and secondary injection while receiving dexamethasone. Medium- and high-dose lentinan significantly restored duodenal sIgA levels, with high-dose lentinan additionally increasing ileal goblet cell counts and PAS⁺ mucus area if compared with mock-treated diseased control mice. The highest dose of lentinan increased CD11c⁺ dendritic cells as well as CD11b⁺F4/80⁺CD80⁺ macrophages in Peyer's patches after primary immunization. After the secondary OVA vaccination, the medium- and high-doses of lentinan treatment significantly elevated CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell populations in Peyer's patches, while these changes were not observed in inguinal or popliteal lymph nodes. The authors further reported restored OVA-specific serum IgG levels. Fecal microbiota analysis showed that lentinan administration was accompanied by a shifted microbiota composition and altered bacterial genera, characterized by increased potential health-beneficial Parabacteroides and decreased Allobaculum in the high-dose lentinan group. Furthermore, the overall short-chain fatty acid levels increased upon lentinan application, with acetate and butyrate being elevated at all doses, while propionate increased only in the high-dose group. Functional prediction suggested enrichment of pathways including streptomycin biosynthesis in the commensal gut microbiota. In vitro, lentinan exhibited no toxicity across concentrations ranging from 39 to 625 μg mL⁻¹ in splenic T cells. The addition of lentinan (300 or 600 μg mL⁻¹) increased splenic T cell proliferation and differentiation and further upregulated IFN-γ, IL-2, and IL-12mRNA expressions. Together, these findings indicate that lentinan partially restored mucosal immune function by enhancing goblet cell function, T cell activation, antigen-specific antibody responses, and microbiota-derived short-chain fatty acids. These effects collectively restored intestinal immune competence during glucocorticoid-induced immunosuppression in mice [109].

3.9. Lentinan in systemic injury and inflammation models

3.9.1. Lentinan in sepsis

Kuang et al. surgically induced gut-origin sepsis in mice and assessed the therapeutic effects of lentinan on small intestine, liver, and lung injury [110]. Therefore, treatment group mice received intraperitoneal lentinan at doses of 5, 10, or 20 mg kg⁻¹ starting 6 h after surgery that was subsequently administered once daily for 7 days; intraperitoneal ulinastatin served as a positive control compound. Histological analyses revealed that lentinan exerted a similar effect like ulinastatin as indicated by attenuated inflammatory cell infiltration, villus damage, and mucosal atrophy, as well as restored organization of epithelial cells and goblet cells. Lentinan also exhibited apoptosis-protective effects in a dose-dependent fashion, as evidenced by lower Bax and higher Bcl-2 mRNA and protein expression compared with mock-treated sepsis control mice. Caspase-3 expression was also significantly decreased at medium and high lentinan doses. Furthermore, lentinan dose-dependently reduced serum concentrations of TNF-α, IL-1β, IL-6, and high-mobility group box 1 (HMGB1), while systemic anti-inflammatory IL-10 secretion was enhanced, whereas the lowest dose of lentinan did neither affect HMGB1 nor IL-10. Furthermore, lentinan inhibited sepsis-induced upregulation of NF-κB and TLR-4 at both the mRNA and protein levels in a dose-dependent manner. Lentinan exerted its protective effects even beyond the intestinal tract given that sepsis-associated lung and liver injuries were alleviated, whereas lentinan also dampened bacterial translocation from the gut to mesenteric lymph nodes, liver, and lung in a dose-dependent manner. In addition, lentinan decreased myeloperoxidase activity and MDA levels and restored SOD, effects that were comparable to ulinastatin application. Together, these results demonstrate that lentinan protected against sepsis-induced multi-organ injury by reducing intestinal TLR-4 and NF-κB expression, attenuating oxidative stress, suppressing apoptosis, and limiting systemic inflammation [110].

3.9.2. Lentinan in hypertension

Zhao et al. investigated the effects of lentinan in murine hypertension. To address this, hypertension was induced by subcutaneous infusion of angiotensin II (Ang II), and mice belonging to the treatment groups received oral lentinan at doses of 5 or 20 mg kg⁻¹ body weight, starting one day prior to Ang II administration [111]. Captopril was used as a positive treatment control. In a separate trial, the authors injected adeno-associated virus serotype 9 expressing either scrambled control or short hairpin RNA targeting angiotensin II type 1 receptor-associated protein (ATRAP) into mouse tail veins, that was followed by Ang II infusion and lentinan (20 mg kg⁻¹) treatment. The results revealed that lentinan dose-dependently attenuated Ang II-induced increases in systolic blood pressure, aortic wall thickening, fibrosis, and infiltration of galectin-3 positive macrophages. Lentinan treatment also restored acetylcholine-induced and endothelium-dependent vasodilation. At the molecular level, lentinan significantly down-regulated mRNA expression of multiple aortic inflammatory and fibrotic genes in hypertensive mice. Another notable observation was the reduction of aortic superoxide accumulation as well as the suppression of genes involved in oxidative stress regulation. In the intestine, lentinan dose-dependently mitigated Ang II-induced fibrosis, muscularis thickening, goblet cell loss, and villus shortening. Lentinan significantly restored intestinal cell proliferation and, further, reduced apoptosis. The macromolecule preserved small intestinal ZO-1 immunofluorescence and prevented Ang II-induced declines in tight junction protein mRNA expression. Lentinan also suppressed intestinal Il1b, Il6, and Tnf genes and reduced macrophage Cd68 expression relative to hypertensive mock controls. Mechanistically, lentinan dose-dependently reduced chymotrypsin-like proteasome activity in the serum and intestine, intestinal proteasome subunit beta type 8 (PSMB8) expression, prevented ATRAP degradation, and lowered protein levels of PSMB8, phosphorylated NF-κB p65 protein subunit, TGF-β, and phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). ATRAP knockdown abolished lentinan's protective effects and exacerbated Ang II-induced vascular and intestinal pathology. In summary, these findings show that lentinan attenuated murine Ang II-induced hypertension and vascular remodeling while simultaneously preserving intestinal barrier integrity and suppressing gut inflammation. These effects depended in the small intestine on limiting PSMB8-mediated ATRAP degradation and inhibiting downstream NF-κB/ERK1/2/TGF-β signaling [111].

3.10. Lentinan and the gut-liver axis

3.10.1. Lentinan in high fat diet-induced steatohepatitis

Yang et al. used a high-fat diet-induced steatohepatitis mouse model, with lentinan incorporated into the diet at 500 mg kg⁻¹ in diet (approximately 60 mg/kg/day) [112]. Significant in vivo effects of lentinan supplementation were observed including prevention of body weight gain and liver weight increase. Lentinan administration significantly reduced hepatocyte swelling and lipid droplet accumulation seen in high-fat diet-fed mice. RT-qPCR and western blot analyses of jejunal barrier integrity showed that, compared with high-fat mock controls, lentinan significantly upregulated both mRNA and protein expression of occludin and ZO-1. Notably, lentinan also decreased serum LPS concentrations. Analyses of jejunal anti-oxidant signaling revealed activation of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase quinone 1 (NQO1), and glutamate-cysteine ligase catalytic subunit (Gclc) with simultaneous reduction of jejunal inducible nitric oxide synthase (iNOS) protein in lentinan-treated mice. Quantification of hepatic immune mediators showed significant down-regulation of hepatic TNF-α, IL-1β, IL-6, and monocyte chemoattractant protein 1 (MCP-1) mRNA, alongside upregulated IL-10 and arginase 1 mRNA expression levels in liver samples derived from lentinan-supplemented mice compared with untreated counterparts. Lentinan treatment normalized hepatic LBP and TLR-4 mRNA expression, decreased F4/80⁺ macrophage infiltration, and altered macrophage polarization, characterized by the reduction of CD68 and CD11c expression and an enhancement of CD206 expression. In addition, lentinan increased hepatic insulin receptor expression and attenuated blood glucose levels at later time points (i.e., 60 to 120 min). Transcriptomic analyses showed a pronounced modulation of hepatic immune and metabolic pathways, including PI3K-Akt signaling, cytokine-cytokine receptor interactions, and arachidonic acid metabolism, identifying protein tyrosine phosphatase 1B (PTP1B)-Akt as a central regulatory molecule that linked inflammation and insulin signaling. Gut microbiota analyses revealed that lentinan significantly recovered α-diversity abundance-based coverage estimator (ACE) and Chao richness estimator (Chao1) indices. Furthermore, lentinan suppressed the expansion of Proteobacteria and Epsilonbacteraeota and enhanced the abundances of Actinobacteria and Firmicutes. At lower taxonomic levels, the potentially pro-inflammatory taxa Deltaproteobacteria, Desulfovibrionales, Desulfovibrionaceae, Campylobacteria, Campylobacterales, Helicobacteraceae, and Helicobacter were reduced by lentinan, while some beneficial Actinobacteria taxa including Bifidobacteriales, Bifidobacteriaceae, and Bifidobacterium were enriched. Correlation analyses indicated that Proteobacteria and Epsilonbacteraeota were positively correlated with LPS-TLR-4 signaling and hepatic inflammatory cytokines, while Actinobacteria was negatively correlated with TLR-4 and positively correlated with IL-10. KEGG pathway analysis showed that lentinan supplementation modified metabolic pathways in the digestive tract of the high-fat diet group, including carbohydrate metabolism, nucleotide metabolism, glycan biosynthesis, and metabolism related to amino acids. Together, these findings show that lentinan prevented steatohepatitis by reversing gut barrier dysfunction, reshaping microbiota composition, suppressing hepatic inflammation, and restoring insulin PTP1B-Akt signaling [112].

3.10.2. Lentinan in acute liver injury

Zhao et al. applied a mouse model of acute alcoholic liver injury to test lentinan for its hepatoprotective effects and associated modulation of the gut microbiome [113]. Prior induction of acute liver injury by ethanol application, mice were perorally subjected to lentinan at doses of 100, 200, and 400 mg kg⁻¹ body weight, using silymarin as a positive treatment control. Regarding clinical parameters, low and intermediate doses of lentinan prevented weight loss comparable to silymarin, whereas all three doses effectively decreased liver index and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities compared with ethanol-treated controls. Lentinan treatment did not only dose-dependently improve histopathological changes due to ethanol-induced liver injury, but also enhanced hepatic anti-oxidant capacities and alleviated hepatic secretion of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6. Gut microbiota analyses demonstrated restoration of Shannon and Simpson α-diversity indices and β-diversity profiles, with the medium dose of lentinan showing the strongest effect. Lentinan significantly decreased Akkermansia and Escherichia-Shigella, but increased commensal members of Lactobacillus, Muribaculum, Parabacteroides, Ruminococcus, Bifidobacterium, and Faecalibacterium as compared to untreated alcohol-induced mice. Correlation analysis showed that Morganellaceae, Akkermansiaceae, and Oscillospiraceae were positively associated with liver injury markers, oxidative stress, and inflammatory cytokines, but negatively correlated with anti-oxidant enzymes. Lactobacillaceae, Ruminococcaceae, and Bifidobacteriaceae, however, were positively correlated with anti-oxidant activity and inversely associated with hepatic and inflammatory indices. Taken together, lentinan showed dose-dependent hepatoprotective effects in murine ethanol-induced liver injury by enhancing anti-oxidant defenses, suppressing inflammatory cytokines, and enriching beneficial gut microbiota [113].

Li et al. assessed the protective effects of lentinan in surgically induced hepatic ischemia-reperfusion injury (HIRI) in mice and applied oral or intraperitoneal lentinan doses of 100 mg kg⁻¹ once daily for 7 days prior surgery [114]. The authors found that oral, but not intraperitoneal lentinan significantly improved HIRI-induced liver damage, reduced histopathological scores and apoptotic cell responses in the liver, which also held true for serum ALT and AST levels if compared to diseased mock controls. Remarkably, these effects could not be observed when the gut microbiota was depleted by combined application of vancomycin, neomycin, metronidazole, and ampicillin, but could be restored by fecal microbiota transplantation from lentinan-pretreated donors. Similarly, oral lentinan significantly suppressed hepatic pro-inflammatory genes such as Tnf-α, Il-1β, Il-6, and Cxcl-2 in mice suffering from hepatic ischemia. Again, the disease-preventive effects of intraperitoneal or oral lentinan administration were abolished in microbiota-depleted mice but could be restored upon reconstitution of the complex murine gut microbiota. Lentinan application per se had also an effect on fecal microbial communities as indicated by increased abundances of Allobaculum, Erysipelotrichaceae, and Erysipelotrichales upon prophylactic application in HIRI mice. Further analyses demonstrated that these gut microbial changes were associated with alterations in fecal metabolites given that isoursodeoxycholic acid (IsoUDCA) levels were almost doubling in both, plasma and stool. Again, this effect was abolished by antibiotic treatment but could be replenished by reintroducing the gut microbial ecosystem upon fecal microbiota transplantation. Notably, a single-dose IsoUDCA pretreatment was sufficient to significantly reduce HIRI severity, as evidenced by improved liver histology scores, lower serum ALT and AST levels, and down-regulated hepatic TNF-α, IL-1β, IL-6, CXCL-1, and CXCL-2 mRNA. Macrophage depletion significantly reduced liver injury compared with diseased mock controls, whereas IsoUDCA did not provide any additional benefit in the absence of macrophages. Collectively, lentinan pretreatment protected against ischemia-reperfusion liver injury through a microbiota-dependent mechanism involving increased IsoUDCA production and suppression of macrophage-driven inflammation [114].

4. Discussion

4.1. Principal findings

In our review we summarized studies from two decades of research on lentinan which collectively portrayed lentinan not as a generic immunostimulant, but rather as a macromolecule with distinct disease-alleviating and even protective and health-beneficial effects on intestinal, extra-intestinal, and systemic morbidities of different etiologies. The reviewed studies showed potent anti-inflammatory effects of lentinan in acute experimental colitis [74, 75, 77–79], but also in chronic colitis and colitis-induced colorectal cancer [74, 82]. The anti-tumor effects of the compound were reported to be due to interference of lentinan with apoptosis [83], stemness [84], autophagy [85], and angiogenesis [86]. Preclinical studies provided evidence for immunotherapeutic synergies of lentinan with other anti-tumor agents including CAR-T in the combat of colorectal cancer [87, 88], but the compound could also promote chemosensitivity of established chemotherapeutic agents when tested against colonic [89] and gastric cancer cells [90]. Clinical trials showed potent synergistic effects of lentinan as adjuvant given in combination with distinct anti-neoplastic regimen for treating gastric, esophageal, and colorectal cancer [91–99]. Moreover, anti-inflammatory effects were observed when lentinan was applied in chemotherapy-induced mucositis [100, 101], in antibiotic-induced dysbiosis and intestinal mucosal damage [102], and in parasitic [103] and viral [104–106] infection models. Lentinan could also counteract intestinal tissue injury induced by immunosuppressive agents [107–109]. Strikingly, lentinan was proven effective to alleviate systemic inflammatory morbidities such as sepsis [110], but could also reduce murine hypertension [111]. Furthermore, lentinan interacted within the gut-liver axis and alleviated distinct hepatic injuries such as high-fat diet induced steatohepatitis [112], acute alcohol-induced liver injury [113], and hepatic induced ischemia [114].

The mushroom compound exerted its action via a variety of mechanisms, combining preservation of the epithelial barrier, regulation of mucosal immunity, and alterations of microbiota-dependent metabolic signaling, for instance. The literature further indicated that the therapeutic efficacy of lentinan was, in fact, dependent on its structure, molecular weight, and route of administration [72, 78, 82].

4.2. Structure-activity relationships of lentinan

Lentinan's structure allowed for its pronounced bioactivity by enabling the polysaccharide to bind with high affinity to pattern recognition receptors, particularly Dectin-1, and the maintenance of its structural integrity substantially affected its therapeutic potency [72]. Lower molecular weight fractions, such as UD-60, demonstrated more pronounced anti-tumor and epithelial-protective effects, while lower molecular weight derivatives, including SDL or SLNT, showed better tissue infiltration and accumulation at tumor sites [82, 84]. Hence, fractionation based on the molecular weight should be an important consideration for future research on lentinan and its derivatives.

4.3. Route-dependent efficacy and precision dosing of lentinan

That lentinan's activity differed depending on its route of administration was consistently shown across preclinical and clinical studies. Oral administration of lentinan, for instance, acted predominantly on the intestinal surface, where it stabilized intestinal barriers and modulated microbial communities [72, 74, 77].

The relatively high molecular weight of lentinan is often considered as a limitation for effective pharmacological application. It could be shown, however, that even at its high molecular size lentinan could to be transported across intestinal epithelia via Peyer's patch M cell-mediated transport, thus bypassing standard epithelial uptake, and directly interact with the lymphatic system [72]. This process explains how orally administered lentinan was able to exert systemic immunologic and metabolic responses despite minimal plasma bioavailability [72, 73]. In contrast, intravenous or intraperitoneally administrated lentinan could directly contact circulating and tissue-resident immune cells [85, 101, 107, 115]. These dynamics explain the reported impacts of oral lentinan application in colitis [74, 77], dysbiosis [102, 103], mucositis [100], and metabolic disease models [112] and further account for why systemic delivery was chosen for studies examining anti-tumor and chemotherapy-synergy effects [83–86].

Importantly, lentinan showed non-linear dose-response relationships, with lower doses often exhibiting better effects in immune-mediated contexts, suggesting receptor saturation or feedback inhibition at higher doses [81, 86, 106]. Future research should therefore focus on lentinan dosing fitting the individual disease context rather than keeping the standard escalation approach.

4.4. Mechanistic insights into lentinan's interaction with immune-dependent and -independent signaling pathways

The preclinical evidence of lentinan demonstrates its action through both immune-dependent and immune-independent mechanisms. Immune-dependent actions of lentinan involved activation of mucosal mononuclear phagocytes via Dectin-1/Syk/NF-κB signaling pathways, maturation of dendritic cells as well as macrophage polarization towards M1 phenotype [72, 77]. Furthermore, lentinan promoted epithelial repair via IL-22-STAT3 signaling pathways, enhancement of antimicrobial peptides, restoration of goblet cell counts, and enhancement of FUT2-mediated fucosylation [77, 82]. Moreover, lentinan exerted immune-independent mechanism involving direct action on the epithelium, displayed by clathrin-mediated down-regulation of TNFR1, inhibition of TLR-4/CaMKII/NF-κB signaling [74, 76, 78], preservation of tight junctional complexes [77, 82, 111], and intestinal stem cells. [77] Lentinan could also be shown to modulate the intestinal microbiota composition by increasing short-chain fatty acid-producing bacteria [80, 81]. These metabolites activate G-protein-coupled receptors to coordinate immune responses with epithelial cell metabolism [107]. There was also evidence of direct anti-tumor effects of lentinan. In immune-deficient models, for instance, the polysaccharide directly induced ROS-mediated mitochondrial apoptosis, endoplasmatic reticulum stress/autophagy-mediated cell death, PI3K/Akt suppression in tumor stem-like cells, and IFN-γ-dependent angiostasis [83–86]. Furthermore, the activation of CaSR led to enhanced FOLFIRI efficacy, suppressed invasion and clonogenic growth independent of T cell activity [89]. This mechanistic flexibility enables lentinan to function in a variety of contexts, including in patients with immunologically “cold” tumors (i.e., tumors with low immune cell particularly T cell infiltration that do not trigger a strong immune response and often resist immunotherapy) or in subjects with compromised immune function [86, 107, 109].

4.5. Extra-intestinal effects of lentinan: interaction with the gut-liver axis and vascular crosstalk

Lentinan exerts its effects beyond the intestinal tract by interacting with the gut-liver axis and crosstalk with the gut-vascular barrier [111, 112]. The molecule was shown to consistently attenuate hepatic inflammation and metabolic injury in models of alcohol-induced damage, high-fat diet-induced steatohepatitis, and gut-origin sepsis, for instance [110, 112, 113]. These potent protective effects were largely attributed to the restoration of tight junction integrity and the reduction of endotoxin and pathogen leakage into the portal circulation [110, 112, 113]. Accordingly, the microbiota-mediated alteration of bile acid concentrations such as IsoUDCA exhibited additional hepatoprotective effects by reducing hepatocyte apoptosis and inhibiting Kupffer cell-mediated inflammatory immune responses [114]. Lentinan further successfully mitigated Ang II-induced intestinal and vascular injury through modulation of the PSMB8-ATRAP axis. Hence, these findings correlate an intact intestinal barrier with vascular homeostasis [111].

4.6. Microbiota-mediated epithelial protection by lentinan

Besides its direct immunomodulatory and epithelial barrier tightening properties, lentinan was shown to also indirectly support intestinal epithelial integrity through ins interaction with the complex gut microbial ecosystem [100, 102]. Several studies reported that the lentinan administration was accompanied by increased intestinal short-chain fatty acid concentrations due to increased abundances of, for instance, butyrate-producing bacterial commensal taxa such as Lachnospiraceae and Bacteroidales [74, 80, 81]. Furthermore, effects of lentinan on microbial diversity and changes of the intraluminal milieu resulted in distinct disease-alleviating and even protective actions in inflammatory including parasitic and viral infection models, by enforcing host defenses, facilitating pathogen expulsion, and limiting bacterial translocation [77, 103, 106].

4.7. Translational outlook

Evidence from clinical trials suggest that the capacity of monocytes to bind to lentinan correlated with its clinical responsiveness [92, 115]. These findings may provide a way to identify likely responders before the initiation of treatment. Most importantly, apart from direct toxicity and enhancement of chemotherapy efficacy, the capacity of lentinan to reduce iatrogenic collateral injuries which often limit therapeutic outcomes, represents a significant clinical advantage that should not be disregarded [93, 100, 101]. The clinical efficacy of lentinan will largely depend on the development of molecular weight-standardized formulations, quality control metrics, and biomarker-driven patient stratification [82, 92, 115]. Accordingly, future clinical trials on the reduction of toxicity as well as the improvement of QOL and long-term treatment resilience, especially in combination with chemotherapy and CAR-T cell therapy, are likely to exhibit the greatest translational impact [88, 92].

4.8. Limitations

There are several limitations of our study. Scientific interest in lentinan has increased over the past two decades. But still, the complex interactions between immune modulation, epithelial signaling, and microbiota remodeling have not entirely been understood. Importantly, several of the proposed modes of actions exerted by lentinan were based on data derived from single studies and await further replication in independent investigations. Moreover, the available human studies differed significantly regarding the experimental design and parameters like formulation, dosing, and selection of patients, which considerably impairs the comparability across trials. While acute intestinal inflammation and oncological settings are comparatively well represented, there were only a handful of studies on the effects of lentinan in liver disease, systemic inflammatory conditions, immunosuppression-induced pathology, or gut-liver axis-mediated disease processes, for instance. This imbalance highlights key knowledge gaps in clinically relevant disease settings.

Furthermore, although some studies suggested intestinal uptake via M cell-mediated transcytosis or clathrin-dependent endocytosis, comprehensive research on the pharmacokinetics of lentinan, including its absorption, bioavailability, distribution or metabolism have not yet been performed, particularly not in human subjects. The absence of such data is a major obstacle to clinical translation. One also needs to take into consideration that the therapeutic efficacy and safety of lentinan can only be interpreted with caution, and the results of this review do not support definitive clinical recommendations.

This review also exhibits methodological limitations. The literature search was conducted using the online database PubMed only, which may exclude studies and leads to publication bias. No formal, standardized assessment of bias or study quality was performed, which limits the methodical evaluation of the included studies. Furthermore, additional synonyms and supplementary approaches such as reference list screening could have been used in the search strategy. Although more than 40 studies were included, it is very well possible that not all literature that meets the defined inclusion and exclusion criteria was identified. Hence, despite our careful approach and precise work, mistakes cannot be ruled out.

4.9. Conclusion

The summarized studies provide evidence that lentinan constitutes a promising intervention strategy in gastrointestinal and extra-intestinal including systemic morbidities due to its pleiotropic effects. Future studies need to redirect from viewing lentinan as a simple, general supplement and rather view it as a precise agent that demands molecular weight-standardized formulations and biomarker-driven clinical strategies. Furthermore, more studies should perform broader and more systematic research, include multiple databases, integrate more pharmacokinetic analyses, and prioritize human studies to improve translational relevance and clinical applicability.

List of abbreviations

5-ASA

5-aminosalicylic acid

5-FU

5-fluorouracil

Akt

protein kinase B

ALT

alanine aminotransferase

Ang II

angiotensin II

AOM

azoxymethane

AST

aspartate aminotransferase

ATRAP

angiotensin II type 1 receptor-associated protein

Bax

Bcl-2-associated X protein

Bcl-2

B-cell lymphoma 2

BMDC

bone marrow-derived dendritic cell

CaMKII

calcium/calmodulin-dependent protein kinase II

CAR-T

chimeric antigen receptor T cell

CaSR

calcium-sensing receptor

Caspase

cysteine-dependent aspartate-specific protease

CLNT

crude lentinan

ConA

concanavalin A

CP

cyclophosphamide

Cxcl

C-X-C motif chemokine ligand

DAI

Disease Activity Index

DSS

dextran sulfate sodium

EGFRvIII

epidermal growth factor receptor variant III

ELISA

enzyme-linked immunosorbent assay

FOLFIRI

folinic acid, fluorouracil, irinotecan chemotherapy regimen

FOXO1

forkhead box O1

Fut2

fucosyltransferase 2

GSH-Px

glutathione peroxidase

HIRI

hepatic ischemia-reperfusion injury

HMGB1

high-mobility group box 1

HSP70

heat shock protein 70

IBD

inflammatory bowel disease

IFN

interferon

IHNV

Infectious Hematopoietic Necrosis Virus

IκBα

inhibitor of κB alpha

IL

interleukin

ILC3

Type 3 innate lymphoid cell

IRAK

Interleukin-1 receptor-associated kinase

IRE1α

serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1 alpha

ISG-15

Interferon-stimulated gene 15

IsoUDCA

isoursodeoxycholic acid

KEGG

Kyoto Encyclopedia of Genes and Genomes

Ki67

Antigen kiel 67

LBP

lipopolysaccharide-binding protein

LPS

lipopolysaccharide

M1

classically activated macrophage

M2

alternatively activated macrophage

MAVS

mitochondrial antiviral signaling protein

MD2

myeloid differentiation factor 2

MDA

malondialdehyde

MDA5

melanoma differentiation-associated protein 5

MHCII

major histocompatibility complex class II

MyD88

myeloid differentiation primary response 88 (MyD88)

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

NOD1

nucleotide-binding oligomerization domain-containing protein 1

NSP4

rotavirus nonstructural protein 4

OVA

ovalbumin

p-Akt

phosphorylated protein kinase B

p65

NF-κB p65 protein subunit

PAS

Periodic acid-Schiff

PERK

protein kinase R-like endoplasmic reticulum kinase

PI3K

phosphoinositide 3-kinase

pBD

porcine beta-defensin

PSC

paclitaxel/S-1/cisplatin

PSMB8

proteasome subunit beta type 8

PTP1B

protein tyrosine phosphatase 1B

p-Src

phosphorylated tyrosine kinase Src

QOL

quality of life

QOL-ACD

Quality of Life Questionnaire for Cancer Patients Treated with Anti-Cancer Drugs

RIG-I

retinoic acid-inducible gene I

RORγt

retinoic acid receptor-related orphan receptor gamma t

ROS

reactive oxygen species

RT-qPCR

real-time quantitative polymerase chain reaction

S-1

tegafur/gimeracil/oteracil

SASP

salicylazosulfapyridine

SDL

superfine dispersed lentinan

sIgA

secretory immunoglobulin A

SLNT

soluble lentinan

SOD

superoxide dismutase

STAT

signal transducer and activator of transcription

Syk

spleen tyrosine kinase

Tbx21

T-box transcription factor

TGF-β

transforming growth factor beta

Th1

T helper type 1

TIM

T cell immunoglobulin and mucin domain-containing protein

TLR

Toll-like receptor

TNBS

2,4,6-trinitrobenzenesulfonic acid

TNF-α

tumor necrosis factor alpha

TNFR1

tumor necrosis factor receptor 1

TRAF6

TNF-receptor-associated factor 6

UD

ultrasonic degradation-derived molecular-weight fraction

Wnt

Wingless-related integration site

XELOX

oxaliplatin/capecitabine

ZO-1

zonula occludens-1

Funding Statement

Funding: No external funding received.