Anti-inflammatory effects of Hericium erinaceus hot water extract via the JAK1/STAT3 pathway in LPS-stimulated RAW 264.7 macrophages

Abstract

Several individual bioactive compounds isolated from Hericium erinaceus exhibit various physiological and biochemical activities, including neuroprotective, neurotrophic, gastrointestinal protective, immunoregulatory, and anti-inflammatory effects. However, the specific anti-inflammatory effects of H. erinaceus extracts, which can vary significantly depending on the extraction solvent, require further investigation. In this study, we aimed to investigate the anti-inflammatory effects of H. erinaceus hot water extract (HWE). Therefore, we evaluated nitric oxide (NO) production, the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), the expression and release of pro- and anti-inflammatory cytokines, and changes in inflammation-related signaling pathways in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. HWE effectively suppressed NO production and iNOS expression; however, COX-2 expression exhibited a biphasic response, increasing at 1.25 mg/ml and decreasing at 2.5 mg/ml. Additionally, HWE reduced the expression and release of the pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and increased the expression and release of the anti-inflammatory cytokine IL-10. Furthermore, HWE attenuated the phosphorylation of IKK-α/β and NF-κB p65 and ameliorated LPS-induced inflammation by inhibiting the phosphorylation of JAK1 and STAT3. These findings suggest that HWE may serve as a valuable source for anti-inflammatory agents or health functional food ingredients.

INTRODUCTION

Hericium erinaceus, an edible mushroom belonging to the Hericiaceae family, is widely found in East Asian countries and possesses pharmacological activities (1). It is commonly known as Hou Tou Gu in China, Yamabushitake in Japan, and the Lion’s mane mushroom in Western countries. In recent years, the bioactive compounds in mushrooms of the Hericium genus have been extensively investigated for their physiological and biochemical activities (2). In particular, H. erinaceus has been reported to exhibit antioxidant (3, 4), neurotrophic (5-7), organo-protective (8, 9), gut microbiota-enhancing (10-12), anticancer (13-16), and immunoregulatory effects (17).

Among immunoregulatory mechanisms, inflammation is an essential biological response that defends tissues and organs against internal signals from damaged or senescent cells and external pathogenic invasion (18). However, excessive inflammatory response results in acute or chronic inflammation, potentially leading to the onset of various diseases. Extensive research has focused on the development of novel anti-inflammatory agents derived from natural products and the elucidation of their underlying regulatory mechanisms (19, 20). Several studies have reported distinct immunoregulatory and anti-inflammatory effects of H. erinaceus; however, these studies have been limited to measuring the expression of transcription factors. The reported effects appear to be dependent on the solvent utilized for extraction. For example, an extract of H. erinaceus prepared with organic solvents, such as ethanol, stimulated innate immune cells to activate the immune response, thereby preventing liver damage and delaying mortality in mice infected with Salmonella Typhimurium (21).

In contrast, H. erinaceus polysaccharides obtained from hot water extraction suppressed colitis in an in vivo mouse model by modulating oxidative damage, inflammation-related signaling pathways, and the gut microbiota (22). Furthermore, H. erinaceus extracts suppressed lipopolysaccharide (LPS)-induced inflammation and H₂O₂-induced oxidative damage in HT22 and BV2 cells (23), and a hot water extract of H. erinaceus exhibited antioxidant and anti-inflammatory effects in RAW 264.7 macrophages (24). In particular, regarding the anti-inflammatory effects of the hot water extract of H. erinaceus, only its nitrite scavenging activity has been demonstrated to date, while mechanistic studies—such as those involving inflammation-related signaling pathways—remain largely unexplored.

Thus, considering that the anti-inflammatory effects of H. erinaceus can differ depending on the extraction method and its resulting chemical profile, a more detailed investigation into its specific mechanisms of action is necessary. In this study, we aimed to investigate the anti-inflammatory effects of H. erinaceus hot water extract (HWE) in LPS-stimulated RAW 264.7 macrophage cells, with a particular focus on the JAK1/STAT3 signaling pathway as a potential mechanism of action.

RESULTS

HWE suppressed LPS-induced inflammation by inhibiting NO production and regulating inflammation mediators in RAW 264.7 macrophages

First, we evaluated the cytotoxicity of HWE on RAW 264.7 cells to determine the maximal concentration for subsequent experiments. As measured via a cell counting kit-8 (CCK-8) assay after 24 h of treatment with various concentrations of HWE, the half of maximal growth inhibition (GI₅₀) value was 3.885 mg/ml. In terms of RAW 264.7 cell viability, HWE showed no significant toxicity at 2.5 mg/ml (73.13 ± 5.94%) compared to the control group. However, we observed decrease in cell viability at 5 mg/ml of HWE (38.85 ± 2.40%), indicating cytotoxicity (Fig. 1A). Therefore, the maximum concentration of HWE used in subsequent experiments was set to 2.5 mg/ml.

Fig. 1.

Effects of Hericium erinaceus hot water extract (HWE) on cell viability and NO production in LPS-stimulated RAW 264.7 macrophages. (A) RAW 264.7 macrophages were treated with various concentrations of HWE for 24 h. Cell viability was measured using CCK-8 assay. The error bars represent the mean ± SD (n = 3). ***P < 0.001 and ****P < 0.0001 vs. control group. (B) RAW 264.7 macrophages were pre-treated with various concentrations of HWE or 5 μM dexamethasone (DEX) for 1 h and stimulated by 500 ng/ml of LPS for 24 h. The nitrite concentration was measured using Griess reagent. The error bars represent the mean ± SD (n = 3). ****P < 0.0001 vs. control group; ^####P < 0.0001 vs. LPS-stimulated group.

Next, we investigated the inhibitory effect of HWE on LPS-induced nitric oxide (NO) production. Compared to the LPS-treated group (18.939 ± 0.278 μM), HWE at a concentration of 2.5 mg/ml significantly reduced LPS-induced NO production to 1.879 ± 0.278 μM, which was almost equivalent to that of the control group (1.667 ± 0.105 μM). Furthermore, HWE demonstrated a more superior inhibitory effect on NO production compared to the positive control, dexamethasone (DEX) (8.030 ± 0.105 μM) (Fig. 1B).

Next, we examined the protein expression of the inflammatory mediators inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) via western blot analysis. Specifically, HWE at 2.5 mg/ml significantly reduced iNOS protein levels by approximately 40% compared to the LPS-stimulated group. This inhibitory effect was similar to that observed with 5 μM DEX. Conversely, COX-2 expression showed a more complex biphasic response to HWE. Lower concentrations of HWE (0.3125-1.25 mg/ml) significantly increased COX-2 protein levels, with the highest expression observed at 1.25 mg/ml. However, this effect was diminished at the highest concentration (2.5 mg/ml), where COX-2 expression declined from its peak level, though it still remained approximately 1.9-fold higher than that of the LPS-stimulated group. In contrast, 5 μM DEX effectively suppressed COX-2 expression (Fig. 2A, B).

Fig. 2.

HWE suppressed LPS-induced iNOS expression and attenuated the upregulation of COX-2. RAW 264.7 macrophages were pre-treated with various concentrations of HWE or 5 μM DEX for 1 h and stimulated by 500 ng/ml of LPS for 24 h. (A) Representative western blot images for iNOS, COX-2, and α-tubulin. (B) The relative protein levels of iNOS and COX-2 were quantified via densitometry and normalized to α-tubulin. The normalized values are presented relative to the LPS-stimulated group. The error bars represented as the mean ± SD (n = 3). ****P < 0.0001 vs. control group; ^#P < 0.05 and ^####P < 0.0001 vs. LPS-stimulated group.

HWE inhibited LPS-induced inflammation by decreasing pro-inflammatory cytokines and increasing anti-inflammatory cytokine at mRNA and protein expression levels

Based on the finding that HWE reduced NO production and iNOS expression, we examined the gene and protein expression of key cytokines using reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. HWE treatment suppressed the gene expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in a concentration-dependent manner in LPS-stimulated RAW 264.7 cells. In contrast, the expression of the anti-inflammatory cytokine IL-10 was increased in cells co-treated with HWE and LPS (Fig. 3A).

Fig. 3.

HWE regulated pro-inflammatory cytokines and anti-inflammatory cytokine at both mRNA and protein expression levels. RAW 264.7 macrophages were pre-treated various concentrations of HWE or 5 μM DEX for 1 h and stimulated by 500 ng/ml of LPS. (A) Cytokine gene expression levels were examined using RT-PCR at 16 h post LPS stimulation. (B-E) Secreted protein levels of IL-1β, IL-6, IL-10, and TNF-α in the culture supernatant were measured using ELISA at 24 h post LPS stimulation. Cytokine expression levels were normalized to that in the LPS-stimulated group. The error bars represent the mean ± SD (n = 3). ***P < 0.001 and ****P < 0.0001 vs. control group; ^##P < 0.01, ^###P < 0.001 and ^####P < 0.0001 vs. LPS-stimulated group.

Next, to confirm whether these changes in gene expression corresponded to protein expression levels, ELISA was performed. The results were consistent with the gene expression data, showing that HWE treatment decreased the secretion of IL-1β, IL-6, and TNF-α, while increasing the secretion of IL-10. Compared to the LPS-stimulated group, HWE at a concentration of 2.5 mg/ml significantly reduced the relative expression ratios of LPS-induced IL-1β, IL-6, and TNF-α to 0.310 ± 0.270, 0.054 ± 0.004, and 0.377 ± 0.011, respectively. In contrast, for IL-10, its relative expression ratio increased to 6.458 ± 1.800 at a concentration of 1.25 mg/ml compared to the LPS-stimulated group (Fig. 3B-E). These results confirmed that the regulation of cytokine protein secretion was consistent with the gene expression data. Thus, our findings indicate that HWE exerts its anti-inflammatory effect by suppressing the expression of pro-inflammatory cytokines and enhancing the expression of an anti-inflammatory cytokine.

HWE attenuated LPS-induced inflammation by inhibiting NF-κB p65 and IKK-α/β phosphorylation in the NF-κB signaling pathway

Consistent with previous studies by Kim et al. reporting that various solvent extracts of H. erinaceus inhibited the LPS-activated NF-κB and the mitogen-activated protein kinase (MAPK) pathways (25) and considering the known time-course of NF-κB activation in macrophages (26), we investigated whether HWE used in our study could reproduce this inhibitory activity. HWE suppressed the phosphorylation of NF-κB p65 and IKK-α/β in LPS-stimulated RAW 264.7 cells (Fig. 4A, B). Specifically, p-NF-κB p65 and p-IKK-α/β levels were reduced by 59.45 ± 17.51% and 69.23 ± 2.93%, respectively, in the HWE-treated group compared to the LPS-stimulated group. Furthermore, HWE exhibited a superior inhibitory effect on the phosphorylation of these proteins compared to the positive control DEX. These results indicate that HWE partly exerts its anti-inflammatory effects by inhibiting the activation of key proteins in the NF-κB signaling pathway.

Fig. 4.

HWE inhibited LPS-induced NF-κB p65 and IKK-α/β phosphorylation. RAW 264.7 macrophages were pre-treated with 2.5 mg/ml of HWE or 5 μM DEX for 1 h, followed by stimulation with 500 ng/ml of LPS for 30 min. (A) Representative western blot images for p-NF-κB p65, NF-κB p65, p-IKK-α/β, IKK-β, and α-tubulin. (B) Relative protein expression levels of p-NF-κB p65 and p-IKK-α/β were quantified using densitometry and normalized to their total protein levels (NF-κB p65 and IKK-β). The normalized values are presented relative to the LPS-stimulated group. The error bars represent the mean ± SD (n = 3). *P < 0.05 and ****P < 0.0001 vs. LPS-stimulated group.

HWE suppressed LPS-induced inflammatory responses by suppressing the phosphorylation of JAK1 and STAT3

The janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway is involved in various cellular functions, including immune regulation, and its hyperactivation enhances inflammatory responses (27). Specifically, STAT3 phosphorylation is recognized for its crucial role in inflammation, with phosphorylation at Ser727 known to maximize its transcriptional activity and exacerbate inflammatory responses (28). In the present study, LPS stimulation significantly increased the phosphorylation levels of both JAK1 and STAT3 in LPS-stimulated RAW 264.7 cells. However, treatment with 2.5 mg/ml of HWE ameliorated LPS-induced inflammatory responses by significantly suppressing the phosphorylation of JAK1 and STAT3 compared to the LPS-stimulated group (Fig. 5A, B). Specifically, p-JAK1 and p-STAT3 levels were reduced by 42.02 ± 19.41% and 72.91 ± 8.15%, respectively, in the HWE-treated group compared to the LPS-stimulated group. Furthermore, HWE exhibited a superior inhibitory effect on the phosphorylation of these proteins compared to the positive control DEX. Importantly, the total protein levels of JAK1 and STAT3 remained unchanged across all treatment groups, indicating that HWE specifically targeted the phosphorylation process rather than protein degradation.

Fig. 5.

HWE suppressed LPS-induced inflammatory responses via the JAK1/STAT3 pathway. RAW 264.7 macrophages were pre-treated with 2.5 mg/ml of HWE or 5 μM DEX for 1 h, followed by stimulation with 500 ng/ml of LPS. (A) Representative western blot images for p-JAK1, JAK1, p-STAT3, STAT3, and α-tubulin. (B) Relative protein expression levels of p-JAK1 and p-STAT3 were quantified using densitometry and normalized to their total protein levels (JAK1 and STAT3). The normalized values are presented relative to the LPS-stimulated group. The error bars represent the mean ± SD (n = 3). **P < 0.01 vs. LPS-stimulated group.

Consequently, these results indicate that HWE inhibits the activation of the JAK1/STAT3 signaling pathway in LPS-induced inflammatory responses. In our study, HWE effectively suppressed LPS-induced JAK1 and STAT3 phosphorylation, whereas the positive control DEX showed only a marginal effect. This result aligns with the understanding that anti-inflammatory mechanisms of DEX are largely independent of the direct inhibition of the JAK/STAT pathway. Instead, DEX primarily targets the NF-κB, AP-1, and MAPK pathways for its anti-inflammatory actions (29-31). This suggests a distinct mechanism of action for HWE involving the JAK/STAT pathway.

DISCUSSION

Many phytochemicals, including compounds derived from H. erinaceus, exhibit anti-inflammatory and immunomodulatory activities depending on the concentration used, sometimes resulting in a biphasic dose-response where opposing effects occur at different concentrations (32). Furthermore, the properties of the extract vary, depending on the extraction solvent (distilled water, ethyl alcohol, methyl alcohol, chloroform, or hexane). In particular, COX-2 is known to be involved in physiological responses such as inflammation, pain, and fever. Biphasic expression of COX-2 suggests a multi-target effect, which is a phenomenon commonly observed with natural product extracts. For HWE, its different constituents may exert distinct or oven opposing effects on various signaling pathways at different concentrations (33). The distinct IL-10 peak reflects a sensitive regulatory feedback loop activated within an optimal range. At the highest dose, the strong suppression of pro-inflammatory signals may have negated the trigger for this compensatory IL-10 production (34). In our study, expression levels of both COX-2 and IL-10 increased up to 1.25 mg/ml of HWE, but then decreased at 2.5 mg/ml, suggesting that the same substance can elicit opposing biological effects at low and high concentrations.

In the present study, our findings confirmed that HWE exhibits a distinct anti-inflammatory effect within an LPS-induced inflammatory environment, in contrast to the immunoregulatory effects reported for H. erinaceus extracts obtained using organic solvents such as ethanol or methanol (17, 21). Specifically, our HWE markedly suppressed NO production and downregulated iNOS and COX-2 expression, while also inhibiting the phosphorylation of NF-κB p65 and IKK-α/β in the NF-κB signaling pathway. In contrast, ethanol extracts of H. erinaceus have been shown to increase NO production and upregulate inflammatory cytokines such as IL-6 and TNF-α (17, 21). These opposing outcomes underscore the critical influence of the extraction method on the biochemical composition and functional activity of H. erinaceus extracts. The hot water extraction method employed in our study not only avoids residual solvent toxicity but also selectively yields bioactive components with anti-inflammatory properties, making it a safer and more industrially applicable approach. Furthermore, although LPS-activated NF-κB p65 is not known to directly influence nuclear accumulation of STAT3, it can contribute to STAT3 expression and activation through interconnected signaling cascades, which were also attenuated by HWE in our study (35).

In conclusion, our findings partially suggest that HWE exerts its anti-inflammatory effects by inhibiting the NF-κB signaling pathway and, in turn, modulates the indirectly activated JAK1/STAT3 signaling pathway. However, further research, including nuclear-cytoplasmic fractionation and a detailed analysis of phosphorylation patterns, will be required to fully elucidate the precise mechanism of action. While previous literature has consistently reported that the polysaccharides and their yield are abundant in hot water extracts (36), we did not perform a detailed compositional analysis, such as polysaccharide or phenolic profiling. This represents a limitation of the present study. Further research should aim to identify the specific bioactive compounds within HWE to elucidate the precise mechanisms underlying their effects.

MATERIALS AND METHODS

Reagents and antibodies

Dulbecco’s Modified Eagle’s Medium (DMEM), 100X penicillin-streptomycin, 1 M HEPES, and RIPA cell lysis buffer were purchased from GenDEPOT (Baker, TX, USA). Fetal bovine serum (FBS) and Dulbecco’s phosphate-buffered saline (DPBS) were purchased from Welgene (Gyeongsan, Republic of Korea). LPS from Escherichia coli K12 was purchased from InvivoGen (San Diego, CA, USA), and CCK-8 was purchased from DOJINDO (Kumamoto, Japan). The BCA protein assay kit and Griess reagent were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Phenylmethylsulfonyl fluoride (PMSF) was obtained from Roche (Basel, Switzerland), and DEX was obtained from Merck (Darmstadt, Germany). The antibodies used for western blot analysis and their respective manufacturers are detailed in Supplementary Table 1.

Preparation of HWE

Dried fruiting bodies of Korean H. erinaceus were provided by CNGbio Corp. (Cheongju, Republic of Korea). For the primary extraction, the mushrooms were weighed, mixed with 15 volumes (w/v) of distilled water, and heated for 8 h at 100 ± 3°C. The remaining solids were collected, and a secondary extraction was performed by adding 5 volumes (w/v) of distilled water and heating for 4 h under the same temperature conditions. The combined hot water extract was filtered through a SACAR 1 μm wound filter (SAEWONFILTEC, Gimpo, Republic of Korea) and concentrated at 55-65°C and a reduced pressure of 350-550 mmHg. Prior to experimental use, the obtained extract was sterilized by sequential filtration through 0.8 μm, 0.45 μm, and 0.22 μm polyethersulfone filters from Merck.

Cell culture and cell cytotoxicity assay

The RAW 264.7 murine macrophage cell line was obtained from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea). The cells were cultured in DMEM supplemented with 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 25 mM HEPES. All cells were maintained at 37°C in an incubator containing 5% CO₂. Cell viability was measured using the CCK-8 assay. For this assay, cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well and incubated overnight (O/N). Subsequently, the cells were treated with various concentrations of HWE and incubated for an additional 24 h. After the incubation period, the medium was removed, and 100 μl of fresh medium containing 10% CCK-8 reagent was added to each well. Then, the plates were incubated for 1 h at 37°C in a 5% CO₂. Cell viability was determined by measuring the absorbance at 450 nm using an Epoch microplate spectrophotometer (BioTek, Winooski, VT, USA). All measurements were performed in triplicate and repeated in three independent experiments.

Measurement of nitrite production

RAW 264.7 cells were seeded in 6-well cell culture plates at a density of 5 × 10⁵ cells/well and incubated O/N. The following day, the cells were pre-treated for 1 h with various concentrations (0.3125, 0.625, 1.25, and 2.5 mg/ml) of HWE or with 5 μM DEX, which served as a positive control. Immediately after pre-treatment, the cells were stimulated with 500 ng/ml of LPS for 24 h. Afterward, the cell culture supernatants were collected and centrifuged at 15,000 rpm for 5 min at 4°C. The nitrite concentration in the supernatant was measured using a Griess reagent assay kit according to the manufacturer’s protocol. Following the reaction, the absorbance was measured at 548 nm. A standard curve was generated using sodium nitrite to quantify the nitrite concentration in the samples. All measurements were performed in triplicate and repeated in three independent experiments.

RT-PCR

RAW 264.7 cells were seeded in 6-well plates at a density of 5 × 10⁵ cells/well and incubated O/N. The following day, the cells were pre-treated for 1 h with various concentrations (0.3125, 0.625, 1.25, and 2.5 mg/ml) of HWE or with 5 μM DEX as a positive control. Immediately after pre-treatment, the cells were stimulated with 500 ng/ml of LPS for 16 h. After treatment, the cells were washed once with DPBS. Thereafter, total RNA was extracted from the cells using the Universal RNA Extraction kit (BIONEER, Daejeon, Republic of Korea) according to the manufacturer’s instructions. Subsequently, complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the AccuPower® RT PreMix and Oligo-dT₍₂₀₎ primers from BIONEER according to the manufacturer’s instructions. Afterward, RT-PCR was performed using cDNA-specific primers. The primer sequences used for RT-PCR are detailed in Supplementary Table 2. The thermal cycling conditions consisted of an initial pre-denaturation step at 95°C for 10 min; followed by 20 to 30 cycles (depending on the target gene) of denaturation at 95°C for 1 min, annealing at 56.5°C for 1 min, and extension at 72°C for 20 sec; a final extension step was performed at 72°C for 5 min.

ELISA

RAW 264.7 cells were seeded in 6-well plates at a density of 5 × 10⁵ cells/well and incubated O/N. The cells were subsequently pre-treated for 1 h with various concentrations (0.3125, 0.625, 1.25, and 2.5 mg/ml) of HWE or 5 μM DEX as a positive control, followed by stimulation with 500 ng/ml of LPS for 24 h. After incubation, the culture supernatants were collected and centrifuged at 15,000 rpm for 5 min at 4°C. The concentrations of mouse IL-1β, IL-6, IL-10, and TNF-α in the resulting supernatants were measured using sandwich ELISA-based Mouse DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol.

Western blot analysis

RAW 264.7 cells were seeded in 6-well plates at a density of 5 × 10⁵ cells/well and incubated O/N. The cells were pre-treated for 1 h with 2.5 mg/ml of HWE or 5 μM DEX (positive control), followed by stimulation with 500 ng/ml of LPS for the indicated time periods. After treatment, the cells were harvested, and total proteins were extracted using RIPA cell lysis buffer supplemented with 1 mM PMSF, a protease inhibitor cocktail (GenDEPOT), and a phosphatase inhibitor cocktail (GenDEPOT). The protein concentration of the total cell lysates was quantified using the Pierce^TM BCA Protein Assay Kit. Equal amounts of protein from each sample were separated by 8% SDS-PAGE gel and transferred onto a polyvinylidene fluoride membrane. The membranes were blocked for 2 h at room temperature (RT) with 3% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20. After blocking, the membranes were incubated with specific primary antibodies overnight at 4°C. Subsequently, the membranes were washed and incubated with the appropriate secondary antibodies for 1 h at RT. Finally, the protein bands were detected using the WesternBright^TM ECL HRP substrate (Advansta, San Jose, CA, USA).

Statistical analysis

All statistical analyses were performed using Prism software (Version 9.3.1; GraphPad, La Jolla, CA, USA). The significance of differences between the control and treatment groups was determined by a one-way analysis of variance, followed by Tukey’s multiple comparison post-hoc test. P-value < 0.05 was considered statistically significant.