Mesenchymal Stem Cells Derived from Different Adipose Tissue Depots Ameliorate Severe Acute Pancreatitis by Inhibiting NF-κB/NLRP3/Caspase-1 Pathways

Abstract

Background

Severe Acute Pancreatitis (SAP) is a critical gastrointestinal inflammatory disease. Mesenchymal stem cells (MSCs), multipotent cells exhibiting diverse biological properties including directional migration, paracrine signaling, immunosuppression, and anti-inflammatory effects. Adipose tissue-derived mesenchymal stem cells (ADSCs) are particularly valuable in regenerative medicine and tissue engineering. Previous studies have demonstrated that ADSCs can mitigate pancreatic damage during acute pancreatitis (AP). However, given the complexity of SAP pathophysiology, which involves a dysregulated systemic inflammatory response and multiorgan failure, the therapeutic differences and underlying mechanisms of ADSCs derived from distinct harvesting sites.

Methods

The SAP rat model was created by retrograde injection of a 4% sodium taurocholate (NaT) solution into the pancreatic duct. Rats were divided into six groups: Sham, SAP, 6 h subcutaneous ADSCs, 12 h subcutaneous ADSCs, 6 h peripancreatic ADSCs, and 12 h peripancreatic ADSCs. A total of 1 × 10⁷/kg body weight of ADSCs was administered via the tail vein at 6 h or 12 h post-model establishment. AR42J cells were stimulated with 200 μM NaT as a cell model of SAP. Serum and supernatants amylase, lipase activity were measured, and inflammatory cytokines were measured using ELISA, while tissue damage was assessed by HE staining and immunohistochemistry. Western blotting (WB) detected NOD-like receptor protein 3 (NLRP3) inflammasome-related proteins, and ADSC homing efficiency was monitored using an in vivo imaging system.

Results

ADSCs from distinct harvesting site significantly attenuated inflammation in SAP rats and cell models. Compared to the SAP group, ADSCs treatment significantly lowered amylase, lipase, IL-1β, and IL-6 levels in serum and supernatants, accompanied by decrease in pancreatic histopathological scores. In vivo imaging demonstrated that peripancreatic ADSCs exhibited a 2.3-fold increase in pancreatic homing efficiency compared with subcutaneous ADSCs. Notably, 6 h peripancreatic ADSCs group showed superior therapeutic efficacy compared to other ADSCs treated rats and cell models. The therapeutic effect of ADSCs in SAP was mediated through the inhibition of NLRP3 inflammasome signaling pathways.

Conclusion

ADSCs reduced SAP-induced pancreatic injury and inflammation by targeting NF-κB/NLRP3/Caspase-1 pathways. Early intervention with peripancreatic ADSCs demonstrated superior therapeutic efficacy, emphasizing the importance of source selection and timing of intervention.

Graphical Abstract

Peripancreatic and subcutaneous ADSCs ameliorate SAP by inhibiting NF-κB/NLRP3/Caspase-1 pathways

Supplementary Information

The online version contains supplementary material available at 10.1007/s12015-025-10922-8.

Introduction

AP is a widespread gastrointestinal disorder globally, with approximately 20% of AP patients progressing to SAP [1]. SAP is characterized by persistent organ failure and the release of inflammatory mediators, which often triggers systemic inflammatory response syndrome (SIRS). This condition can lead to multiple organ dysfunction syndrome (MODS) and even result in fatal outcomes, with mortality rates ranging from 20 to 50% [2, 3]. Currently, conventional clinical treatments for SAP include analgesia, fluid resuscitation, nutritional support, and anti-spasmodic treatment [4]. However, these interventions have not significantly impeded the progression of SAP, rendering its prevention and treatment substantial challenges in clinical practice.

MSCs are multipotent cells that present promising therapeutic options for patients suffering from inflammatory diseases, attributed to their capabilities for self-renewal, multi-lineage differentiation, low immunogenicity, and immunomodulatory properties [5, 6]. MSCs are ubiquitous across nearly all tissues and are frequently isolated from sources such as adipose tissue, umbilical cord, menstrual blood, placenta, and bone marrow [7]. MSCs derived from diverse tissues and organs exert multifaceted protective effects against SAP via coordinated mechanisms, including suppression of hyperactivated immune responses, mitigation of oxidative stress-mediated damage, inhibition of apoptosis, and promotion of tissue regeneration through enhanced paracrine activity [8–12]. However, studies comparing the efficacy of MSCs from different tissue origins in the treatment of pancreatitis have not revealed significant differences, and the precise molecular mechanisms underlying these effects remain to be elucidated. Despite recent advancements in the application of MSCs for SAP treatment, positive therapeutic outcomes have thus far been reported primarily in animal models, with limited evidence from clinical trials. Consequently, the fundamental mechanisms driving these therapeutic effects are still not fully understood.

Pancreatic acinar cells are the primary functional units of the exocrine pancreas. Pathological events occurring within the intra-acinar can lead to acinar cell death, resulting in pancreatic autodigestion and systemic inflammation [13]. The NLRP3 inflammasome is structurally composed of three functional domains: a C-terminal leucine-rich repeat (LRR) domain, a central NACHT (NAIP, CIITA, HET-E, and TP1) domain responsible for nucleotide binding and oligomerization, and an N-terminal pyrin domain (PYD). The PYD mediates homotypic interactions with the ASC adaptor protein via PYD-PYD binding, facilitating subsequent assembly with pro-caspase-1 through CARD-CARD interactions. This molecular architecture enables caspase-1 activation, which proteolytically processes pro-inflammatory cytokines IL-1β and IL-18 into their bioactive forms. Furthermore, the NLRP3 inflammasome functions as a pattern recognition receptor capable of responding to both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) [14]. This process ultimately results in inflammatory cell death, characterized by the loss of membrane integrity and the subsequent release of inflammatory mediators [15]. Numerous studies have demonstrated that the activation of the NLRP3 inflammasome and caspase-1 promote the secretion of inflammatory cytokines, thereby exacerbating AP [16, 17]. Therefore, the NLRP3 inflammasome emerges as a critical nexus between cell death and systemic inflammation, presenting a potential therapeutic target for SAP.

In summary, inflammation represents a critical pathophysiological response at both local and systemic levels in the context of SAP. Systemic damage arises from dysregulated and disproportionate systemic inflammation in reaction to local injury. In patients with SAP, systemic inflammation initially manifests as SIRS. Those with persistent SIRS are at an increased risk of developing systemic organ dysfunction, ultimately leading to multiple organ failure (MOF). Consequently, we hypothesize that the infusion of ADSCs could inhibit the NLRP3 inflammasome, thereby mitigating systemic inflammation and slowing the progression of SAP. Moreover, existing literature indicates that MSCs derived from various sources exhibit distinct biological properties [18]. The therapeutic effects of MSCs sourced from different tissues on the same disease can vary significantly [19, 20]. In the case of ADSCs, the site of adipose tissue harvesting is critical, as it may influence the intrinsic characteristics of the cells and enhance their differentiation potential, thereby affecting their functional capabilities and therapeutic efficacy in treating different diseases. This variability is attributed to regional differences in cell types and the inherent characteristics of adipocyte progenitors [21–23]. Therefore, we further hypothesize that peripancreatic ADSCs may offer a greater therapeutic advantage over subcutaneous ADSCs in the treatment of SAP.

Methods

Materials

Osteogenesis-(Cat. No. RAXMD-90021), adipogenesis-(Cat. No. RAXMD-90031), and chondrogenesis-(Cat. No. RAXMD-90041) induced differentiation kits and surface marker assay kits specialized for ADSCs were purchased from Cyagen Biotechnology Co., Ltd. (Guangdong, China). SPF male rats (weighing 220 g ± 20 g) were purchased from HFK Biotechnology Co., Ltd. (Beijing, China). The rat pancreatic acinar cell line AR42J was obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China). Sodium taurocholate (NaT) was purchased from Sigma-Aldrich (Cat. No. 86339-1G, Taufkirchen, GER). 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO, Cat. No. C1038) was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Transwell 6‐well plates (Cat. No. 14111) were purchased from Labgic Biotechnology Co., Ltd. (Beijing, China). An interleukin-6 (IL-6, Cat. No. ZC-36404) rat ELISA kit and interleukin- IL-1β (IL-1β, Cat. No. ZC-36391) rat ELISA kit were purchased from from ZCIBIO Technology Co., Ltd (Shanghai, China). A hematoxylin and eosin (H&E) staining kit was purchased from Beyotime Biotechnology Co., Ltd. (Cat. No. C0105S, Shanghai, China). Anti-NLRP3 (Cat. No. 30109–1-AP), anti-Caspase-1 (Cat. No. 22915–1-AP), anti-NF-κB (Cat. No. bs-0465R), anti-MPO (Cat. No. 22225–1-AP), anti-GSDMD (Cat. No. 20770–1-AP), anti-IL-1β (Cat. No. 16806–1-AP), anti-GAPDH (Cat. No. 60004–1-Ig), goat anti-rabbit (HRP, Cat. No. RGAR001), goat anti-mouse (HRP, Cat. No. RGAM001) antibodies were purchased from Proteintech Biotechnology Co., Ltd. (Wuhan, China). Anti-ASC antibodies (Cat. No. 67824 T) were purchased from Cell Signaling Technology, Inc. (Massachusetts, USA). A 0.1% type I collagenase (Cat. No. 17100017), 0.05% trypsin (Cat. No. 00–4222-57), low-glucose Dulbecco’s modified Eagle’s medium (DMEM, Cat. No. 11995065), Ham's F-12 K medium (Cat. No. 21127022), fetal bovine serum (FBS, Cat. No. A5670701) and 1% penicillin–streptomycin (Cat. No. 15140122) were purchased from Gibco (Grand Island, NY, USA).

SAP rat Model and Grouping

Throughout the surgical procedures, researchers strictly adhered to aseptic techniques. To avoid perioperative gastrointestinal adverse reactions, all rats were fasted for 12 h before surgery. Surgical instruments were sterilized using high-pressure steam (0.14 MPa, 126.0 °C, 60 min) before use. Isoflurane vapor was inhaled for induction (3.0%, v/v) and maintenance (2.0%, v/v) of anesthesia in all rats during surgery. This study was approved by the Ethics Committee of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences (IRM/2-IACUC-2411–015).

Forty rats were randomly assigned to the Sham group (n = 8), 12 h SAP group (n = 8), 24 h SAP group (n = 8), 48 h SAP group (n = 8), and 72 h SAP group (n = 8). Rats in the Sham group were sacrificed at 72 h, while rats in the SAP groups were euthanized at 12 h, 24 h, 48 h, and 72 h after SAP model establishment, and blood, pancreas samples were properly collected and preserved for subsequent experiments. Another forty-eight rats were randomly divided into the Sham group (n = 8), SAP group (n = 8), 6 h subcutaneous ADSCs group (n = 8), 12 h subcutaneous ADSCs group (n = 8), 6 h peripancreatic ADSCs group (n = 8), and 12 h peripancreatic ADSCs group (n = 8). In the Sham group, anesthetized rats were fixed in the supine position on the operating table, the abdominal skin was prepared and disinfected with povidone-iodine (1.0%, w/v). After making an incision along the midline of the abdominal wall, the pancreas was flipped several times with a sterile cotton swab, and then the abdominal cavity was closed. After closure of the abdominal cavity, 1 mL PBS solution was injected into the rats via the tail vein at 6 h and 12 h post-surgery.

In the SAP group, after opening the abdominal cavity as described above, a 4.0% (w/v) NaT solution (1.0 mL/kg bw, 0.1 mL/min) was retrogradely injected into the pancreatic duct using a microinjection pump, then the abdominal cavity was closed. After closure of the abdominal cavity, the same volume of PBS solution was injected via the tail vein at 6 h and 12 h post-surgery as described above. In the 6 h subcutaneous ADSCs and peripancreatic ADSCs groups, a suspension of subcutaneous ADSCs or peripancreatic ADSCs (1.0 × 10⁷ cells/kg bw) was injected into each rat via the tail vein at 6 h post-model establishment as described, and 1 mL PBS solution was injected into the rats via the tail vein at 12 h post-surgery. In the 12 h subcutaneous ADSCs and peripancreatic ADSCs groups, 1 mL PBS solution was injected into the rats via the tail vein at 6 h post-surgery, and a suspension of subcutaneous ADSCs or peripancreatic ADSCs (1.0 × 10⁷ cells/kg bw) was injected into each rat via the tail vein at 12 h post-model establishment.72 h after model establishment, rats from all groups were euthanized, and blood and pancreas samples were collected and preserved for subsequent experiments. The experimental process is illustrated in Fig. 2A.

Fig. 2.

NLRP3 inflammasome is activated in the pancreas during SAP. A Schematic illustration of the animal experimental procedure. B WB images of NLRP3, GSDMD, ASC, pro-Caspase-1 and cleaved Caspase-1 in the pancreas from rats in Sham group and SAP group at different time points. C Statistical analysis of pro-Caspase-1 in the pancreas from rats in Sham group and SAP group at different time points. D Statistical analysis of cleaved Caspase-1 in the pancreas from rats in Sham group and SAP group at different time points. E Statistical analysis of NLRP3 in the pancreas from rats in Sham group and SAP group at different time points. F Statistical analysis of GSDMD in the pancreas from rats in Sham group and SAP group at different time points. G Statistical analysis of ASC in the pancreas from rats in Sham group and SAP group at different time points. (One-way ANOVA with Tukey test was performed to compare data among multiple groups. n = 3; mean ± se; *P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001)

Cells Culture and Characterization

ADSCs were isolated, purified, and identified as described previously [24]. Male Sprague–Dawley (SD) rats were selected and anaesthetized by isoflurane vapor. After disinfection with 75% alcohol, a longitudinal incision was made along the abdominal midline of the rats, the subcutaneous and peripancreatic adipose tissue were removed. Adipose tissue was washed three times with sterile phosphate buffer solution (PBS) and minced. The extracellular matrix (ECM) was digested with 0.1% type I collagenase and 0.05% trypsin and centrifuged at 1500 rpm for 10 min. The sediment was suspended with DMEM containing 10% FBS and 1% penicillin–streptomycin, then transferred into a dish and cultured at 37 °C in 5% CO₂. After 48 h, non-adherent cells were removed, the medium was replaced, and the culture medium was replaced twice a week. Passage 3 ADSCs were used and phenotyped. For characterization, ADSCs were first observed morphologically using a microscope. They were then assessed for adipogenesis, osteogenesis, and chondrogenesis under appropriate culture conditions. Finally, flow cytometry was used to detect the surface-specific antigens of ADSCs, including CD11b, CD29, CD45, CD90.

Coculture of isolated ADSCs with AR42J cells

All coculture experiments were conducted in six-well cell culture plates containing 0.4-μm pore-sized transwell inserts. AR42J cells were seeded in the bottom well of the transwell 6‐well plates at a density of 3 × 10⁵ cells/cm² with Ham's F-12 K medium containing 20% FBS and 1% penicillin–streptomycin. After the attachment of cells to the plates, the cells were divided into the following six groups: (1) control group: normal AR42J cells were incubated for additional 24 h, and transwell inserts were supplemented with ADSC-free medium. (2) SAP group: AR42J cells were stimulated with NaT (200 μM) for 20 min [25]. Then the cell medium was changed to fresh medium, the cells were cultured for additional 24 h and transwell inserts were supplemented with ADSC-free medium. (3) 6 h peripancreatic ADSCs treated SAP group: AR42J cells were stimulated with NaT (200 μM) for 20 min, then the cell medium was changed to fresh medium, 4 × 10⁵ peripancreatic ADSCs were seeded onto the transwell inserts after 6 h. (4) 12 h peripancreatic ADSCs treated SAP group: AR42J cells were stimulated with NaT (200 μM) for 20 min, then the cell medium was changed to fresh medium, 4 × 10⁵ peripancreatic ADSCs were seeded onto the transwell inserts after 12 h. (5) 6 h subcutaneous ADSCs treated SAP group: AR42J cells were stimulated with NaT (200 μM) for 20 min, then the cell medium was changed to fresh medium, 4 × 10⁵ subcutaneous ADSCs were seeded onto the transwell inserts after 6 h. (4) 12 h subcutaneous ADSCs treated SAP group: AR42J cells were stimulated with NaT (200 μM) for 20 min, then the cell medium was changed to fresh medium, 4 × 10⁵ subcutaneous ADSCs were seeded onto the transwell inserts after 12 h. The co-culture system was maintained at 37 °C in a humidified atmosphere with 5% CO₂. After 24 h of coculture, AR42J cells and supernatants were collected for further analysis.

In Vivo Tracing of ADSCs

According to the manufacturer's protocol, subcutaneous ADSCs and peripancreatic ADSCs were labeled with the fluorescent probe DiO. For in vivo tracing, 6 h after SAP model rats establishment, DiO-labeled subcutaneous ADSCs or peripancreatic ADSCs suspensions (1 × 10⁷ cells/kg body weight) were injected into SAP model rats via the tail vein. The distribution of subcutaneous and peripancreatic ADSCs in SAP model rats was tracked 24 h after the first injection using a small animal imaging system.

Survival Assessment

All rats were housed in appropriate living environments post-surgery, with free access to potable water and standard chow. Additionally, the survival of the rats in each group was recorded before the rats were euthanized.

Measurement of Wet/Dry Weight Ratio

The wet weight of fresh pancreatic tissues were measured and recorded using an electronic balance. The fresh tissues were then dried at 70 °C for 70 h. After drying, the dry weights of the pancreatic tissues were measured again using the electronic balance, and the results were recorded. The wet/dry weight ratio was calculated as the wet weight of the fresh tissue divided by the dry weight of the dried tissue.

Pathological Examination

After euthanizing the rats, they were fixed in the supine position on the operating table, and the abdominal skin was disinfected with a 1% povidone-iodine solution. An incision was made along the midline of the abdominal wall to fully expose the pancreatic tissue, allowing for visual inspection of severe pathological changes such as edema and hemorrhage.

For histopathological examination, fresh pancreatic tissues were fixed with 4.0% paraformaldehyde solution, processed through a gradient of ethanol and xylene, and embedded in paraffin. Sections (4 μm thick) were prepared, deparaffinized, rehydrated, and stained with Hematoxylin and Eosin (H&E). Subsequently, the histopathological scores of the pancreas sections were performed blindly by two pathologists following the criteria reported by Hu et al. [26] respectively (Table S1, Supporting Information).

Enzyme-Linked Immunosorbent Assay

Subsequently, according to the manufacturer's protocol, the concentrations of IL-6, IL-1β in serum and supernatants were measured using appropriate ELISA kits.

Detection of Biochemical Indicators in Serum

The serum and supernatants levels of amylase (Cat. No. C016‐1‐1, Nanjing Jiancheng Bioengineering Institute, China) and lipase (Cat. No. A054‐2‐1, Nanjing Jiancheng Bioengineering Institute, China) were analyzed using commercial kits in accordance with the manufacturer's instructions.

Western Blotting

Total protein was extracted from pancreatic tissue and AR42J cells using a total protein extraction kit, pancreatic tissues and AR42J cells were lysed in ice-cold RIPA lysis buffer (Cat. No. P0013B, Beyotime, Shanghai, China) supplemented with a protease and phosphatase inhibitor cocktail (Cat. No. P1046, Beyotime, Shanghai, China), incubated on ice for 30 min and then centrifuged for 10 min (15, 000 × g, 4 °C). The supernatant was collected, and the protein concentration was calculated using a Pierce BCA protein assay kit (Cat. No. P0399M, Beyotime, Shanghai, China), and protein denaturation with loading buffer in preparation for subsequent protein experiments. Equal amounts of samples were electrophoresed in sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE), and the separated proteins were transferred to methanol-saturated polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were then blocked with blocking buffer at room temperature for 1 h.

For protein immunoblotting, the PVDF membranes were co-incubated overnight at 4.0 °C with the following primary antibodies: anti-NLRP3 antibody (1/2,000 dilution), anti-ASC antibody (1/3,000 dilution), anti-caspase-1 antibody (1/2,000 dilution), anti-GSDMD antibody (1/2,000 dilution), anti-IL-1β antibody (1/1,000 dilution), anti-NF-κB antibody (1/5,000 dilution), and anti-GAPDH antibody (1/10,000 dilution). The next day, the PVDF membranes were washed three times with Tris-buffered saline containing Tween-20 (TBST) and then co-incubated at room temperature with the following secondary antibody: goat anti-rabbit (HRP) antibody (1/10,000 dilution), goat anti-mouse (HRP) antibody (1/10,000 dilution) for 1 h. After incubation, the PVDF membranes were washed three more times with TBST, and the bands were visualized using diaminobenzidine and imaged using a gel imaging system. Finally, the intensity of the target protein bands was semi-quantitatively analyzed using Image J.

Immunohistochemistry

The deparaffinized sections were incubated with 3% hydrogen peroxide for 30 min, followed by citric acid buffer. After antigen retrieval in boiling water for 10 min, sections were treated with anti-MPO (1:100), anti-IL-1β (1:100), overnight at 4 °C. Sections were rinsed with PBS and incubated with peroxidase-labeled secondary antibody at room temperature for 1 h. Finally, sections were stained with 3,3′- diaminobenzidine tetrahydrochloride (DAB) and hematoxylin. After mounting, sections were scored by two pathologists independently under a light microscope. Any discrepancy was resolved by consulting the third party. Finally, the images were analyzed semi-quantitatively using ImageJ.

Statistical Analysis

All data in this study were statistically analyzed using GraphPad Prism 9.5.0. The Measurement data were expressed as mean ± standard error of the mean (SEM) and were tested for normality and homogeneity of variance. The one-way analysis of variance (ANOVA) was used when the data conformed to a normal distribution and exhibited homoscedasticity, and Welch’s ANOVA was used when the data conformed to a normal distribution but did not exhibit homoscedasticity. Differences were considered statistically significant when p < 0.05.

Results

Pancreatic Tissue Damage Tends to Increase During SAP

Rats were euthanized at 12 h, 24 h, 48 h, and 72 h after establishing the SAP model, and blood and pancreatic tissue were collected for analysis. The pancreas of rats in the SAP group displayed varying degrees of edema, hemorrhage and necrosis at different time points, while the pancreas of rats in the Sham group exhibited normal color and gross morphology (Fig. 1A). Compared with the Sham group, the SAP group showed increased disorganization of pancreatic structure, more interlobular edema, inflammatory cell infiltration, acinar cell necrosis, and vacuolar degeneration at different time points (Fig. 1B-E).

Fig. 1.

Pancreatic tissue damage tends to increase during SAP. A Representative gross morphology images of the pancreas from rats in the Sham group and SAP group at different time points. Histopathologic images (B) and pathological scores(C-F) for the pancreas tissues from rats in Sham group and SAP group at different time points. Serum amylase (G) and lipase (H) activities in rats in Sham group and SAP group at different time points. Serum concentrations of IL-1β (I) and IL-6 (J) of rats in Sham group and SAP group at different time points. (n = 4-6; mean ± se; scale bar = 100 μm)

Pancreatic damage was further assessed by measuring serum amylase and lipase activities. Both biochemical parameters significantly increased in the SAP group (Fig. 1G, H). Furthermore, serum concentrations of IL-6 and IL-1β were significantly elevated in the SAP group compared to the Sham group (Fig. 1I, J).

NLRP3 Inflammasome is Activated in The Pancreas During SAP

These data indicate that the SAP rat model was successfully established by retrograde injection of a 4.0% (w/v) NaT solution into the pancreatic duct, with the most severe damage occurring at 24 h.

Western blot analysis showed that the expression of NLRP3 inflammasome-associated proteins in the pancreas of the SAP group was significantly higher than that of the Sham group at different time points. More importantly, the expression of NLRP3 inflammasome-associated proteins peaked at 24 h and remained elevated at 48 h (Fig. 2B). Considering the activation of intracellular Caspase-1 and the release of its active form, we further examined the levels of cleaved Caspase-1 in the pancreas. The levels of Caspase-1 and cleaved Caspase-1 in the pancreas of the SAP group were significantly higher than those of the Sham group at different time points, peaking at 24 h (Fig. 2C, D).

Notably, the expression of NLRP3, ASC and GSDMD in the pancreas of the SAP group was significantly upregulated at different time points compared to the Sham group, peaking at 24 h and remaining elevated at 48 h (Fig. 2E-G).

Characterization and In Vivo Distribution of ADSCs

We characterized subcutaneous ADSCs and peripancreatic ADSCs previously and investigated their ability for in vivo tracing and homing in SAP model rats. Both subcutaneous ADSCs and peripancreatic ADSCs exhibited a swirling arrangement and typical fibroblast-like morphology during culture, and both demonstrated good proliferation capacity (Fig. 3A). Subsequently, the three-lineage differentiation of subcutaneous ADSCs and peripancreatic ADSCs was further induced to examine their differentiation potential. The results indicated that both subcutaneous ADSCs and peripancreatic ADSCs retained the ability to differentiate into osteogenic, adipogenic, and chondrogenic lineages (Fig. 3B-D). And subcutaneous ADSCs and peripancreatic ADSCs appear to have comparable capabilities in terms of osteogenic, adipogenic, chondrogenic differentiation potential, as well as proliferative capacity (Fig. 3E).

Fig. 3.

Characterization and in vivo distribution of ADSCs. A Representative morphological images of peripancreatic ADSCs and subcutaneous ADSCs at 48 h and 7 days; representative image of colony formation of peripancreatic ADSCs and subcutaneous ADSCs. B Representative light microscopic images of the third-passage cultured induced and uninduced cells for qualitative assessment of the osteogenic differentiation. C Representative light microscopic images of the third-passage cultured induced and uninduced cells for qualitative assessment of the adipogenic differentiation. D Representative light microscopic images of the third-passage cultured induced and uninduced cells for qualitative assessment of the chondrogenic differentiation. E Statistical analysis of the stained areas using crystal violet, red S, oil Red O, and alcian blue for peripancreatic ADSCs and subcutaneous ADSCs. F Representative histograms for the expression of various surface markers: CD 90 and CD 29 with positive expression, and CD 45 and CD11b with negative expression. G Values of the expression % of each marker. H Fluorescence images of DiO-labeled peripancreatic ADSCs and subcutaneous ADSCs in the duodenum, pancreas, lungs, liver, spleen, and kidney of SAP model rats after 24 h. I Statistical analysis of DiO-labeled peripancreatic ADSCs and subcutaneous ADSCs in the duodenum, pancreas, lungs, liver, spleen, and kidney of SAP model rats after 24 h. (n = 4; mean ± se; **** P < 0.0001)

Importantly, surface-specific antigens of subcutaneous ADSCs and peripancreatic ADSCs were analyzed to evaluate their phenotypic characteristics. These cells were positive for CD29 and CD90, but negative for CD11b and CD45 (Fig. 3F, G).

We injected DiO-labeled subcutaneous ADSCs or peripancreatic ADSCs into SAP model rats via the tail vein and observed their biodistribution in vivo after 24 h. Fluorescent signals were primarily distributed in the duodenum, pancreas, lungs, liver, spleen, and kidney of SAP model rats, and peripancreatic ADSCs exhibited a higher degree of enrichment in the damaged pancreatic tissue (Fig. 3H, I). These data suggest that subcutaneous ADSCs and peripancreatic ADSCs home to the injured pancreas during SAP. To investigate the role of subcutaneous ADSCs and peripancreatic ADSCs in SAP rats, we selected 72 h as the time point for more detailed study.

Subcutaneous ADSCs and Peripancreatic ADSCs Attenuate Inflammation and Inhibit NF-κB/NLRP3/Caspase-1 Pathways in AR42J cells

NaT may activate the inflammasome via NRLP3 which inflammatory vesicles are critical inflammatory signaling molecular complexes in SAP. Consequently, the effects of subcutaneous ADSCs and peripancreatic ADSCs on SAP were studied in vitro.

We examined the protein expression levels of the downstream NF-κB/NLRP3/Caspase-1 in AR42J cells by performing WB. WB results showed that compared with control group, the proteins expression levels of NF-κB, NLRP3, GSDMD, Caspase-1, IL-1β, ASC, and cleaved Caspase-1in AR42J cells were increased in SAP group (Fig. 4A). In contrast, both peripancreatic and subcutaneous ADSCs treated SAP groups at 6 h or 12 h exhibited significantly lower protein expression levels of NF-κB, NLRP3, GSDMD, Caspase-1, IL-1β, ASC, and cleaved Caspase-1 compared to NaT model group. Notably, 6 h peripancreatic ADSCs treated SAP group demonstrated the most pronounced inhibitory effect on these inflammatory and NLRP3 inflammasome-associated proteins (Fig. 4B-E). Similar to these results, the expression levels of AMY, IL-6, and IL-1β in the supernatants showed the same trend (Fig. 4F-G).

Fig. 4.

Subcutaneous ADSCs and peripancreatic ADSCs attenuate inflammation and inhibit NF-κB/NLRP3/Caspase-1 pathways in AR42J cells. A WB images of IL-1β, NF-κB, NLRP3, GSDMD, ASC, pro-Caspase-1 and cleaved Caspase-1 in AR42J cells of each group. B Statistical analysis of IL-1β in AR42J cells of each group. C Statistical analysis of NLRP3, GSDMD and ASC in AR42J cells of each group. D Statistical analysis of pro-Caspase-1 and cleaved Caspase-1 in AR42J cells of each group. E Statistical analysis of NF-κB in AR42J cells of each group. F The levels of amylase activity in the supernatant of AR42J cells in each group G The levels of IL-6 and IL-1β in the supernatant of AR42J cells in each group. (One-way ANOVA with Tukey test was performed to compare data among multiple groups. n = 3–8; mean ± se; *P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001)

Subcutaneous ADSCs and Peripancreatic ADSCs Attenuate Systemic Inflammation and Local Injury in SAP

Systemic inflammation and local organ injury are two characteristic features of SAP. We sought to determine the effects of subcutaneous ADSCs and peripancreatic ADSCs on these characteristics (Fig. 5A). First, the 72 h survival was observed. In the SAP group, rats started to die at 6 h post-model establishment. The 72 h survival of rats in the subcutaneous ADSCs or peripancreatic ADSCs groups were significantly higher than those in the SAP group (Fig. 5B).

Fig. 5.

Subcutaneous ADSCs and peripancreatic ADSCs attenuate systemic inflammation and local injury in SAP. A Representative gross morphology and histopathologic images of the pancreas from rats in each group 72 h after model establishment. B Cumulative survival of rats in each group from model establishment to 72 h after model establishment. C IL-6 and IL-1β levels in the serum from rats in each group. D Serum amylase and lipase activities in the rats in each group. E Pathological scores for the pancreas tissues from rats in each group. F The wet/dry weight ratios of the pancreas tissues from rats in each group. G Representative immunohistochemistry images for IL-1β and MPO of the pancreas from rats in each group. H IL-1β and MPO positive rates of the pancreas from rats in each group. (One-way ANOVA with Tukey test was performed to compare data among multiple groups. n = 6–8; mean ± se; scale bar = 100 μm; *P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001)

Serum levels of IL-6, IL-1β were assessed to evaluate systemic inflammation. Compared to the Sham group, serum concentrations of IL-6 and IL-1β were significantly elevated in the SAP group. There was no significant difference in the reduction of serum IL-6 and IL-1β levels between the subcutaneous ADSCs and peripancreatic ADSCs groups. However, the effects were more pronounced with the 6 h tail vein infusion compared to the 12 h injection (Fig. 5C).

Pancreatic injury was assessed by measuring serum amylase and lipase activities. The biochemical parameters in the subcutaneous ADSCs and peripancreatic ADSCs groups were significantly lower than those in the SAP group. Furthermore, the biochemical parameters in the peripancreatic ADSCs group were significantly lower than those in the subcutaneous ADSCs group, with better effects observed with early infusion (Fig. 5D). To better understand the effects of subcutaneous ADSCs and peripancreatic ADSCs, we analyzed the pancreatic micropathology. Compared to the SAP group, the subcutaneous ADSCs and peripancreatic ADSCs groups exhibited reduced pancreatic disorganization, less interlobular edema, acinar cell necrosis, and hemorrhage, with better therapeutic effects observed with early infusion (Fig. 5E). Edema was further assessed by measuring the wet/dry weight ratios of the pancreatic tissues. Compared to the SAP group, the subcutaneous ADSCs and peripancreatic ADSCs groups exhibited lower pancreatic tissue edema (Fig. 5F). Immunohistochemistry showed that the levels of MPO and IL-1β in the pancreas were significantly lower in the subcutaneous ADSCs and peripancreatic ADSCs groups compared to the SAP group after transplantation, with better effects observed with early infusion. Furthermore, early peripancreatic ADSCs were more effective in reducing IL-1β expression compared to subcutaneous ADSCs (Fig. 5G, H). These results indicate that subcutaneous ADSCs and peripancreatic ADSCs effectively attenuate systemic inflammation and local pancreatic injury in SAP model rats, with early tail vein infusion potentially providing better therapeutic effects.

Subcutaneous ADSCs and Peripancreatic ADSCs Inhibit NF-κB/NLRP3/Caspase-1 Pathways in SAP

We verified the effects of subcutaneous ADSCs and peripancreatic ADSCs on the expression of NF-κB/NLRP3/Caspase-1 pathways in the pancreas. WB analysis showed that the expression of NF-κB/NLRP3/Caspase-1 pathways in the subcutaneous ADSCs and peripancreatic ADSCs groups were significantly lower than that in the SAP group (Fig. 6A). The levels of IL-1β in the subcutaneous ADSCs and peripancreatic ADSCs groups were significantly lower than those in the SAP group (Fig. 6B). The expression of NLRP3 inflammasome-associated proteins, Caspase-1 and cleaved Caspase-1 in the subcutaneous and peripancreatic ADSCs groups were significantly lower than in the SAP group (Fig. 6C, D).

Fig. 6.

Subcutaneous ADSCs and peripancreatic ADSCs Inhibit NLRP3 inflammasome in SAP. A WB images of IL-1β, NF-κB, NLRP3, GSDMD, ASC, pro-Caspase-1 and cleaved Caspase-1 in the pancreas from rats in each group. B Statistical analysis of IL-1β in the pancreas from rats in each group. C Statistical analysis of NLRP3, GSDMD and ASC in the pancreas from rats in each group. D Statistical analysis of pro-Caspase-1 and cleaved Caspase-1 in the pancreas from rats in each group. E Statistical analysis of NF-κB in the pancreas from rats in each group. (One-way ANOVA with Tukey test was performed to compare data among multiple groups. n = 3; mean ± se; *P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001)

Although there were no statistically significant differences in the expression of GSDMD and cleaved Caspase-1 between the subcutaneous and peripancreatic ADSCs groups at the same time point, the expression of NNLRP3 in pancreatic tissue was significantly reduced after 6 h tail vein infusion compared to 12 h infusion, with early infusion of peripancreatic ADSCs showing better effects. Interestingly, we found that the expression of NF-κB appeared to follow a similar trend to that of the NLRP3 inflammasome-associated proteins (Fig. 6E). Overall, these results indicate that subcutaneous and peripancreatic ADSCs ameliorate severe acute pancreatitis by inhibiting NF-κB/NLRP3/Caspase-1 pathways.

Discussion

Inflammatory cytokines play a crucial role in the onset and progression of AP [27]. In treating SAP, the key is to reduce the release of inflammatory cytokines. Zhao et al. [28] demonstrated that transplantation of MSCs significantly reduced the expression of tumor necrosis factor α (TNF-α) and IL-1β in the pancreas and lungs. Additionally, research showed the therapeutic efficacy of MSCs in AP/SAP rat models, where transplantation of MSCs significantly reduced levels of inflammatory cytokines, such as transforming growth factor β (TGF-β), TNF-α, and interferon γ (IFN-γ), and significantly improved pancreatic tissue histopathology scores [20]. However, the specific anti-inflammatory mechanisms by which MSC transplantation exerts protective effects against SAP remain to be further investigated. With ongoing research, the role of the NLRP3 inflammasome in the pathological process of AP has become increasingly recognized. Research indicates that the NLRP3 inflammasome is pivotal in the inflammation induced by SAP, and its activation is closely related to the outcomes of SAP [29, 30]. Sheng et al. [31] found that the administration of proanthocyanidins alleviated pancreatic inflammation in SAP mice by suppressing the NLRP3 inflammasome in pancreatic tissue. This study aims to investigate whether transplantation of ADSCs from different harvesting sites can exert protective effects on SAP by inhibiting NF-κB/NLRP3/Caspase-1 pathways to explore the differences in these effects.

The NLRP3 inflammasome has garnered the most significant research attention among innate immune complexes, distinguished by its exceptional capacity to sense diverse stimuli spanning host-derived metabolic signals, environmental stressors, and pathogenic microorganisms [32]. NLRP3, Caspase-1, ASC, and GSDMD play crucial roles in AP [33]. Activation of the NLRP3 inflammasome pathway promotes the secretion of IL-18 and IL-1β, enhancing the inflammatory response [34]. Studies have shown that inhibiting GSDMD significantly reduces the release of IL-1β in pancreatic cells, while activation of GSDMD promotes acinar cell pyroptosis, thereby exacerbating AP [35, 36]. Interestingly, activation of the Caspase-1 pathway has also been observed in pancreatic acinar cells, and inhibition of Caspase-1 reduces the secretion of pro-inflammatory cytokines and the occurrence of acinar cell pyroptosis in AP, thus alleviating the severity of AP [37]. In this study, we found that the expression of NLRP3 inflammasome-associated proteins in the SAP group tended to increase over time, peaking at around 24 h and then decreasing, consistent with the findings of Lyu et al. [38]. We also observed that the trends of NF-κB and IL-1β levels were similar to those of NLRP3 inflammasome-associated proteins. Therefore, inhibiting NF-κB/NLRP3/Caspase-1 pathways may be a potential therapeutic strategy for treating SAP.

The mechanisms by which MSCs exert therapeutic effects in disease are multifaceted. Previous extensive studies have shown that MSCs can home to immunoregulatory organs and damaged tissues, reducing inflammatory cascades and exerting therapeutic effects [39, 40]. SAP leads to inflammatory injury, and the injured tissue may chemoattract MSCs for homing [41]. Previous studies have shown that after the infusion of MSCs, the number of MSCs enriched in pancreatic tissue following infusion without pancreatic injury is significantly lower than that in the infusion group with pancreatic damage. Additionally, the number of labeled MSCs in the lungs, liver, spleen, and kidneys is much lower compared to that in the pancreas [42]. Our research indicated that systemic transplantation of both subcutaneous ADSCs and peripancreatic ADSCs at different times inhibited NLRP3 inflammasome in SAP rats, reduced systemic inflammatory parameters, and pancreatic injury, with early systemic transplantation of peripancreatic ADSCs showing the most significant effect. This may be an important basis for the superior therapeutic effect of peripancreatic ADSCs compared to subcutaneous ADSCs. In our study, peripancreatic ADSCs demonstrated significantly enhanced homing capacity to damaged pancreatic tissues in SAP rats compared to subcutaneous ADSCs. We propose that this phenomenon may be attributed to the injured pancreas releases substantial inflammatory cytokines (e.g., IL-6 and IL-1β), local inflammatory responses trigger the release of cytokines, which naturally recruit ADSCs to sites of inflammation, injury, and necrosis [43]. ADSCs derived from different harvesting sites preferentially migrate to the injured pancreas without the need for systemic immunosuppression. They are retained in the organ, colonize it, and may contribute to the healing of SAP. ADSCs from different harvesting sites may exhibit variations in surface markers and cytokine expression, peripancreatic ADSCs may overexpress homing factors such as ICAM-1, CXCR4, and CXCR7 compared to their subcutaneous ADSCs [44–46]. These characteristics could influence their immune regulatory functions and responses to inflammatory signals, thereby affecting their homing efficiency. The intrinsic homing capability of ADSCs, coupled with their ability to migrate to injured tissues following allogeneic transplantation, underpins their therapeutic potential. However, efficiently recruiting ADSCs to specific injury sites remains a critical challenge, the specific mechanisms underlying these differences require further investigation.

During SAP, acinar cells undergo damage. Injured acinar cells secrete pro-inflammatory and anti-inflammatory cytokines, and the NLRP3 inflammasome activation of acinar cell is a significant component in promoting pancreatic inflammation, playing a crucial role in SAP [47, 48]. To elucidate the mechanistic basis of peripancreatic ADSCs and subcutaneous ADSCs in pancreatitis, we systematically evaluated their modulatory effects on NF-κB/NLRP3/Caspase-1 pathways. The study showed that both types of ADSCs significantly inhibited the expression of NF-κB/NLRP3/Caspase-1 pathways and also reduced IL-1β and IL-6 levels in SAP rats. We found that SAP administered by peripancreatic ADSCs had a better protective effect. Additionally, the NF-κB pathway may be one of the regulatory pathways mediating the anti-inflammatory effects of peripancreatic and subcutaneous ADSCs on the inflammasome in SAP rats. Although our study demonstrates the superior therapeutic efficacy of peripancreatic ADSCs in SAP, their clinical translation requires careful evaluation of practical challenges. Direct procurement of peripancreatic adipose tissue may be limited to patients undergoing pancreatic surgery. Considering the limitations of cell-based therapies, cell-free derivatives of peripancreatic ADSCs, such as exosomes, may represent a promising alternative approach.

Limitations

There are several limitations. Due to the complex etiology and variable clinical conditions of SAP, the currently established animal and cell models may not fully recapitulate the pathological and physiological features and therapeutic outcomes of the disease in clinical settings. The underlying reasons for the differences in survival time, functional morphology, and homing capacity of peripancreatic and subcutaneous ADSCs after in vivo transplantation remain to be elucidated. Additionally, the mechanisms by which peripancreatic and subcutaneous ADSCs exert their preventive effects and the specific molecular mechanisms involved remain unclear, and these aspects will require further exploration in future studies. Finally, the sample size for biological replicates in certain experiments was relatively small, future studies should increase the sample size to enhance the reliability and generalizability of the findings.

Conclusion

In summary, this study indicates that NLRP3 inflammasome activation may be positively correlated with the severity of SAP progression. Notably, both peripancreatic ADSCs and subcutaneous ADSCs can reduce inflammation by suppressing NF-κB/NLRP3/Caspase-1 pathways, thereby exerting protective effects against SAP. Furthermore, early intravenous injection of peripancreatic ADSCs appears to have a greater effect in SAP model rats, although further exploration of its underlying mechanisms is needed. Our study provides new insights and strategies for the application of cell therapy in the treatment of SAP.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

A W and Y C were responsible for designing the study and formulating its underlying concept. A W, Y A, X W, C W, Z T, and Y C developed and implemented the research methods. A W, Y A, W G, F X, and Y L conducted the experiments and collected data. A W, Y A, and X W performed the data analysis. A W and Y C wrote the original draft and were also responsible for reviewing and editing the manuscript. Y C secured the funding for the study. Y C and W H oversaw the project, guiding the research and ensuring its proper execution. All authors reviewed and revised the manuscript critically, contributing to the final version.

Funding

This study was supported by grants from the Key project of Scientific Research Program of Tianjin Education Commission, Research on the establishment of AI individualized evaluation system for acute pancreatitis complicated with pancreatic necrosis based on pancreatic m6A methylation and CT radiomics (2024ZD043 to Y.C.), and the Key project of Tianjin Public Health Science and Technology, Clinical study of human umbilical cord mesenchymal stem cells in the treatment of severe acute pancreatitis based on minimally invasive individualized integrated traditional Chinese and western medicine surgical treatment system (24ZXGZSY00140 to Y.C.), and the "Unveiling the Leader" Project of Tianjin Cell Ecology Haihe Laboratory, Clinical Research on Umbilical Cord Mesenchymal Stem Cells in the Treatment of Severe Acute Pancreatitis (HH24KYZX0014 to Y.C.), and the Traditional Chinese Medicine Scientific Research Program of Hebei Provincial Administration of Traditional Chinese Medicine, Study on the mechanism of remodeling the peritoneal inflammatory microenvironment of Qingpancreatic decoction based on the regulation of apoptotic EVs derived from adipose MSCs (T2025036 to Y.C.) China Medical and Health Development Foundation, Young and Middle-aged Doctors Excellent Talent, Pei Ying Program, Clinical study on prevention and treatment of exocrine pancreatic insufficiency associated with severe acute pancreatitis (BJ2023YCPYJH003 to Y.C.), and the Tianjin Nankai Hospital integrated Traditional Chinese and Western medicine prevention and treatment key technology and program optimization 2022 key project, A multi-omics study of the microenvironment of abdominal inflammation in acute pancreatitis based on minimally invasive individualized integrated traditional Chinese and Western Medicine surgical treatment system (NKYY-IIT-2022–009-2 to Y.C.), and the Tianjin key areas of traditional Chinese medicine science and technology project, Clinical study of combined treatment of TCM and Western medicine with pancreato-intestinal therapy based on peritoneal microecology in the treatment of acute pancreatitis (2022005 to Y.C.), and the Tianjin Natural Science Foundation key project, Establishment of individualized surgical treatment system for severe acute pancreatitis and intelligent evaluation of multimodal imaging (21JCZDJC00550 to Y.C.), and the Tianjin 131 innovative talent team, innovation team for Diagnosis and treatment of acute abdomen related to biliary and pancreatic diseases, (201938 to Y.C.).

Data Availability

The data that support the findings of this study are available on request from the corresponding author, upon reasonable request.

Declarations

Ethical Approval

This study was approved by the Ethics Committee of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences (IRM/2-IACUC-2411–015).

Consent to Participate

Not Applicable.

Consent to Publish

The work has not been published before and it is not under consideration for publication elsewhere, its publication has been approved by all co-authors.

Competing interests

All the authors report no relevant conflicts of interest for this article.