[The investigation of the mechanism underlying the impairment of the intestinal mucous barrier in severe hypertriglyceridemia acute pancreatitis].

OBJECTIVE: To investigate the structural and functional alterations of intestinal goblet cells and the mucus barrier in rats with hypertriglyceridemia acute pancreatitis (HTG-AP), and to evaluate the potential mechanism involved in the progression to severe HTG-AP.

METHODS: Twenty-four specific pathogen-free male Sprague-Dawley rats were divided into control group, biliary acute pancreatitis (BAP) group, hypertriglyceridemia (HTG) group, and HTG-AP group using a random number table method, with six rats in each group. In the BAP group, the BAP model was established by retrograde injection of 3.5% sodium taurocholate (0.3 mL/min) at 2 mL/kg through the pancreaticobiliary duct. In the HTG group, a polyethylene glycol-polypropylene glycol block copolymer was administered intraperitoneally at 0.50 g/kg once daily for 7 days to establish the HTG model. In the HTG-AP group, after the HTG model had been established, caerulein was administered intraperitoneally at 50 μg/kg once every one hour on day eight, and injections were continued for 7 days to induce HTG-AP. The control group received an equal volume of normal saline (2 mL/kg). All rats were sacrificed at 24 hours after modeling. Hematoxylin-eosin (HE) staining was performed to assess histopathological changes in the pancreas, ileum, and colon, and pathological scores were calculated. Western blotting was performed to determine the expressions of CD4, CD8, ZO-1, and Occludin. Enzyme linked immunosorbent assay (ELISA) was performed to measure secretory immunoglobulin A (sIgA) level in intestinal mucosa. Alcian blue/periodic acid-Schiff (AB/PAS) staining was performed to evaluate mucus layer structure and to measure mucus layer thickness and goblet cell number. Double immunofluorescence staining was performed to detect NOD-like receptor protein 6 (NLRP6) and mucin 2 (MUC2) expression and their colocalization.

RESULTS: 1) Compared with the control group, the BAP, HTG, and HTG-AP groups showed intestinal inflammatory changes of varying degrees. The HTG-AP group exhibited the most severe intestinal inflammation, accompanied by extensive acinar necrosis, hemorrhage, and marked edema; the pancreatic histopathological score was higher in the HTG-AP group than that in the BAP group (12.95±1.31 vs. 7.30±1.59, P<0.05). In addition, inflammatory cell infiltration in the ileum and colon extended through the full thickness of the intestinal wall, with massive loss of crypt goblet cells. 2) Intestinal immune function and barrier function were impaired in the BAP, HTG, and HTG-AP groups, and the impairment was most pronounced in the HTG-AP group. Compared with the BAP group, the HTG-AP group showed higher CD4 and CD8 expressions in the ileum (CD4/β-actin: 6.664±0.034 vs. 3.524±0.042, CD8/β-actin: 3.135±0.034 vs. 1.118±0.028, both P<0.05), a lower sIgA level in ileal mucosal supernatant (ng/L: 150.00±44.72 vs. 410.00±54.89, P<0.05), and decreased ZO-1 and Occludin expressions in the colon (ZO-1/β-actin: 0.46±0.13 vs. 0.59±0.20, Occludin/β-actin: 0.33±0.12 vs. 0.60±0.07, both P<0.05). 3) AB/PAS staining showed that the mucus layer of the colonic mucosal epithelium was altered in the BAP, HTG, and HTG-AP groups; the HTG-AP group exhibited severe mucus layer injury with more evident mucus depletion in goblet cells. Compared with the BAP group, the HTG-AP group had a thinner colonic mucus layer (μm: 10.20±4.69 vs. 16.80±4.38, P<0.05) and fewer mucus-containing goblet cells (cells/crypt: 4.30±1.34 vs. 7.52±1.93, P<0.05). 4) Double immunofluorescence staining showed that, compared with the control group, colonic NLRP6 and MUC2 expression and their colocalization signals were decreased in the BAP, HTG, and HTG-AP groups, and the decrease was most evident in the HTG-AP group.

CONCLUSIONS: Disruption of the intestinal mucus barrier may be an important mechanism contributing to the progression to severe HTG-AP, and the NLRP6/MUC2 axis may serve as a potential therapeutic target.