To investigate the mechanism of bone marrow-derived mesenchymal stem cells (MSCs) in alleviating severe acute pancreatitis (SAP) by inhibiting the pyroptosis pathway in a rat model.

Bone marrow-derived MSCs were isolated from 4-week-old male SPF SD rats [body weight, (100±20) g] using the bone marrow flush technique, followed by culture and characterization. Twenty-four male SPF SD rats [body weight (200±20) g] were randomly assigned to four groups (n=6 per group) using a random number table: a sham-operated group, an SAP model group, an ulinastatin-treated group, and an MSCs-treated group. SAP was induced in the latter three groups via retrograde injection of 5%sodium taurocholate into the biliopancreatic duct. The SAP model group received an injection of 100 μL PBS at 12 h post-modeling. The ulinastatin-treated group received an intraperitoneal injection of ulinastatin solution (5×10⁴ U/kg) immediately after model induction. The MSCs-treated group received an injection of MSC suspension (5×10⁶ cells/kg) at 12 h post-modeling. Sham-operated rats underwent laparotomy with gentle pancreatic manipulation before abdominal closure. Pancreatic histopathology was assessed by HE staining and pathological scoring. Serum amylase and lipase activities were measured using commercial assay kits. Serum levels of IL-10, IL-1β, and TNF-α were determined by ELISA. The ultrastructural changes of pancreatic acinar cells were observed under transmission electron microscopy (TEM). Protein expression of NLRP3, GSDMD, and caspase-1 in pancreatic tissue was evaluated by immunofluorescence. The mRNA levels of NLRP3, GSDMD, and caspase-1 were quantified by qRT-PCR. The relative protein expression levels of NLRP3, GSDMD, GSDMD-N, caspase-1, cleaved caspase-1, and IL-1β were analyzed by Western blotting.

The isolated cells exhibited a typical adherent, spindle-shaped, fibroblast-like morphology. Flow cytometry confirmed they were highly positive for the mesenchymal marker CD29 (99.22%) and nearly negative for the hematopoietic marker CD45 (0.42%). These cells also demonstrated osteogenic and adipogenic differentiation potential and were maintained in good condition. Compared with the sham-operated group, the SAP model group showed significantly increased pancreatic histopathological scores, elevated serum amylase and lipase activities, higher levels of pro-inflammatory cytokines (IL-1β and TNF-α), and a lower level of IL-10 (all P<0.000 1). Both ulinastatin and MSCs treatment significantly attenuated these changes, reducing histopathological scores, serum enzyme activities, IL-1β, and TNF-α (P<0.001), while increasing IL-10 (P<0.05). Importantly, the MSCs-treated group showed significantly greater improvement in all these metrics compared to the ulinastatin-treated group (P<0.05). TEM analysis revealed that MSCs treatment promoted the recovery of mitochondrial cristae structure, improved overall organelle morphology, and reduced endoplasmic reticulum swelling in acinar cells, with these restorative effects being more pronounced than in the ulinastatin group. The immunofluorescence results showed that compared with the SAP model group, the expression levels of NLRP3, GSDMD, and caspase-1 in pancreatic tissue were decreased in both the MSCs-treated group and the ulinastatin-treated group (P<0.000 1). Western blot results indicated that, compared with the SAP model group, the relative protein expression levels of NLRP3, GSDMD, GSDMD-N, caspase-1, cleaved caspase-1, and IL-1β in pancreatic tissue were reduced to varying degrees in both the MSCs-treated group and the ulinastatin-treated group (P<0.000 1). qPR-PCR results demonstrated that, compared with the SAP model group, the mRNA expression levels of NLRP3, GSDMD, and caspase-1 in pancreatic tissue were significantly decreased in both the MSCs-treated group and the ulinastatin-treated group (P<0.01). Moreover, compared with the ulinastatin-treated group, the MSCs-treated group exhibited a more significant decrease in all the aforementioned indicators (P<0.001).

Mesenchymal stem cells alleviate inflammation and tissue damage in severe acute pancreatitis. Their protective mechanism involves inhibiting the NLRP3/GSDMD/IL-1β signaling axis, thereby suppressing acinar cell pyroptosis.