The Role And Mechanism Of Heme Oxygenase-1 In Intestinal Mucosal Injury Associated With Severe Acute Pancreatitis

Objective: Severe acute pancreatitis(SAP)is a critically ill disease with a high clinical mortality rate.The main causes of death are severe pancreatic infection and systemic multiple organ failure.SAP-induced intestinal mucosal damage may cause intestinal bacterial translocation that induces or aggravates systemic infections.The pathological mechanism of intestinal mucosal injury due to SAP is complex and has not been fully elucidated.Heme oxygenase-1(HO-1)is a potent antioxidant and cytoprotective agent.HO-1 has a protective effect on intestinal mucosa,but its effect on intestinal barrier damage in SAP and its mechanism are not clear.The aim of this study is to investigate the role and mechanism of HO-1 on intestinal mucosal barrier injury in SAP rats,and to provide new insights for the diagnosis and treatment of intestinal mucosal injury.Methods: We selected 40 healthy male SD rats.The rats were randomly divided into 4groups of 10 rats each: sham-operated group(SO group),severe acute pancreatitis group(SAP),HO-1 activation group(SAP+Hemin)and HO-1 inhibition group(SAP+Znpp).SAP rats were developed by retrograde pancreaticobiliary duct injection of 5% sodium taurocholate.SO group rats were injected with equal amount of saline into the pancreaticobiliary duct.rats in the HO-1 activation and HO-1 inhibition groups were injected with Hemin and Znpp intraperitoneally 24 h before modeling,and the rest groups were injected with equal amounts of saline intraperitoneally.All groups of rats were reanesthetized at 24 h postoperatively,and blood,intestinal and pancreatic tissues were collected from the inferior vena cava for subsequent analysis.Measurement of serum amylase(AMY)and lipase(LPS)levels in rats using a fully automated biochemical analyzer.We measured tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-10(IL-10),diamine oxidase(DAO)activity,D-lactate(D-LA),fatty acid-binding protein(FABP2)and endotoxin levels simultaneously with standard diagnostic kits.The levels of malondialdehyde(MDA)and diamine oxidase(SOD)and glutathione peroxidase 4(GPX4)were measured in ileal tissue using standard diagnostic kits.The pathological changes of pancreas and ileum were observed by hematoxylin-eosin(HE)staining,and the pathological score was also done.The levels of HO-1,Occludin,ZO-1,MLCK,MLC,Caspase-3,Bax and Bcl-2 were detected by Western blot;the distribution of HO-1 and Occludin,ZO-1 were detected by immunofluorescence staining;The level of apoptosis of intestinal epithelial cells was detected using TUNEL and the apoptosis rate was counted.Results: The rat model of intestinal mucosal injury in severe acute pancreatitis was successfully established.AMY,LPS,TNF-α,IL-6,DAO activity as well as D-LA,FABP2 and endotoxin levels were significantly higher in the SAP group than in the SO group,and IL-10 was significantly lower than in the SO group(all P < 0.05),and the expression level of intestinal HO-1 was significantly higher in the SAP group than in the SO group(P < 0.05).Meanwhile,compared with the SO group,the SAP group exhibited severe histopathological damage to the pancreas and ileum.The expression of HO-1 was significantly increased in the SAP+Hemin group compared with the SAP group(P < 0.05)and the levels of IL-10,SOD and GPX were significantly increased(P < 0.05).However,the levels of TNF-α,IL-6,DAO,D-LA,FABP2 and endotoxin,MDA and intestinal epithelial cell apoptosis were significantly decreased(all P < 0.05).The pancreatic and ileal tissues of rats in the SAP+Hemin group exhibited milder pathological damage and lower pathological scores.In addition,the expression levels of Occludin and ZO-1 were markedly increased in ileal tissues compared with the SAP group(P < 0.05),apoptosis-related proteins Bax and Caspase-3 were significantly lower(P < 0.05),Bcl-2expression was significantly higher(P < 0.05),and MLCK/p-MLC signaling pathway-related proteins MLCK and P-MLC expression were significantly reduced(P <0.05).In contrast,the expression of HO-1 was significantly decreased in the SAP+Znpp group compared with the SAP group(P < 0.05)and the levels of IL-10,SOD and GPX were significantly decreased(P < 0.05).However,the levels of TNF-α,IL-6,DAO,D-LA,FABP2 and endotoxin,MDA and intestinal epithelial cell apoptosis were significantly increased(all P < 0.05).Compared with the SAP group,the pancreatic and ileal tissues of rats in the SAP+Znpp group exhibited severe pathological damage and higher pathological scores.In addition,the expression levels of Occludin and ZO-1 were markedly decreased in ileal tissues compared with the SAP group(P < 0.05),apoptosis-related proteins Bax and Caspase-3 were significantly higher(P < 0.05),Bcl-2expression was significantly lower(P < 0.05),and MLCK/p-MLC signaling pathway-related proteins MLCK and P-MLC expression were significantly increased(P <0.05).Conclusion: In this study,we showed that upregulation of HO-1 expression attenuated intestinal mucosal injury in SAP through anti-inflammatory,antioxidant and anti-apoptotic pathways,and explored that HO-1 protected intestinal mucosa by inhibiting the MLCK/p-MLC signaling pathway.Our research provides new insights into potential targets for combating intestinal mucosal damage.