Effect And Mechanism Of Angiotensin Ⅱ1 Receptor-mediated Oxidative Stress On Intestinal Barrier Function In Rats With Severe Acute Pancreatitis

Purpose:Acute pancreatitis(AP)is a common clinical emergency with pathogenesis associated with dietary and biliary factors.Some cases progress to severe acute pancreatitis(SAP),which can trigger a series of complications and even be life-threatening.SAP is often accompanied by intestinal mucosal barrier dysfunction,resulting in a range of serious consequences,including sepsis.However,the specific mechanisms underlying this phenomenon remain unclear.The angiotensin Ⅱ type 1 receptor(AT1)is a crucial receptor in the reninangiotensin system,which is known to regulate blood pressure and participate in various physiological and pathological processes,including inflammation and oxidative stress.In this study,we aimed to confirm the expression of AT1 in intestinal mucosal cells of patients with SAP and to investigate whether AT1-mediated oxidative stress is involved in intestinal barrier injury in SAP.Method:24 male Wistar rats of SPF level were randomly divided into 3 groups: severe acute pancreatitis group(SAP group),sham operation group(SO group),and Azilsartan intervention group(SAP+AZL group).SAP model was induced by retrograde injection of5% sodium taurocholate into the pancreatic duct,while the sham operation group received an equal amount of normal saline injection.The rats in the SAP+AZL group were treated with 1mg/kg/d Azilsartan suspension by gavage for 7 consecutive days before modeling.After 24 hours of modeling,blood samples were collected from the inferior vena cava,and terminal ileum tissue and pancreatic tissue were taken for subsequent analysis.All rats were euthanized after sample collection.The levels of amylase,lipase,and other indicators were determined by an automated biochemical analyzer.Serum interleukin-6(IL-6),interleukin-10(IL-10),tumor necrosis factor-alpha(TNF-α)were measured using ELISA kits.Serum endotoxin concentration was determined by the EKT-5M assay.Glutathione(GSH),malondialdehyde(MDA),and superoxide dismutase(SOD)activity in the ileum were measured using assay kits.The level of reactive oxygen species(ROS)in the ileum was measured using an assay kit.The pathological damage of the ileum and pancreas was evaluated by HE staining.The expression levels of tight junction(TJ)protein,AT1,and NOX2 in the intestine were detected by Western blot.The expression and distribution of TJ protein,AT1,and NOX2 were validated by immunofluorescence.Results:(1)Results of amylase and serum inflammatory indexes: compared with SO group,the levels of amylase,lipase,IL-6,IL-10,TNF-α and endotoxin in SAP group were significantly increased(P<0.05),and those levels in SAP+AZL group were decreased compared with SAP group(P<0.05).(2)ileum oxidative stress level: compared with SO group,the indexes of GSH and SOD in SAP group were significantly decreased,while the indexes of ROS and MDA were significantly increased(P<0.05).Meanwhile,compared with SAP group,the indexes of GSH and SOD in SAP+AZL group were increased,while the indexes of ROS and MDA were decreased(P<0.05).(3)HE staining and pathological score: compared with SO group,SAP group showed typical damage manifestations in intestine and pancreas,such as edema,bleeding,inflammatory cell infiltration,and pathological score was significantly increased(P<0.05).In SAP+AZL group,the above injury manifestations were alleviated,and the pathological score was significantly decreased(P<0.05).(4)Western-blot results indicated that compared with SO group,TJ protein expression in SAP group decreased,AT1 and NOX2 protein expression increased(P<0.05),and these expressions were reversed in SAP+AZL group(P<0.05).(5)Protein expression in ileum mucosa was detected by immunofluorescence.Compared with the SO group,the expression and distribution of TJ protein in SAP group were decreased,while the expression and distribution of AT1 and NOX2 were increased.In SAP+AZL group,TJ protein levels were increased,AT1 and NOX2 levels were decreased.These changes were consistent with the trends of pathological injury,Western-blot,intestinal oxidative stress level and serum inflammatory markers.Conclusion:Our experiments demonstrated the expression of AT1 in the ileal mucosa,and we demonstrated that AT1-specific inhibitor Azisartan can reduce the level of intestinal oxidative stress.These findings confirmed that AT1-mediated oxidative stress injury is involved in the injury of intestinal mucosal barrier in SAP,and inhibition of this pathway can effectively reduce the intestinal oxidative stress injury.This may be a potential target for the treatment of intestinal barrier damage in SAP.