Effect Of Intestinal Epithelial Ferroptosis On Intestinal Mucosal Barrier Injury And Bacterial Translocation In Severe Acute Pancreatitis

Objective:Severe acute pancreatitis(SAP)is a kind of acute abdomen with acute onset,which is often accompanied by intestinal mucosal barrier injury.Ferroptosis is a non-apoptotic cell death characterized by iron dependence and lipid peroxidation.However,whether ferroptosis is involved in SAP induced intestinal barrier injury is unknown.The aim of this study was to investigate whether ferroptosis occurs in intestinal epithelial cells(IECs)during SAP-induced intestinal barrier injury,and determine whether the inhibition of ferroptosis would ameliorate intestinal barrier injury and prevent bacterial translocation(BT).Methods:Ninety healthy adult male SPF Sprague-Dawley rats were randomly divided into 3groups: sham operation group(SO group),severe acute pancreatitis group(SAP group)and ferroptosis inhibitor Liproxstatin-1 treatment group(SAP+Lip group).The rats in the three groups were subdivided into three subgroups(n = 10 in each group)at 6h,12 h and24h.Sodium taurocholate(5%)was retrogradely perfused into the biliopancreatic duct to establish a rat model of SAP.SAP+Lip group was given Liproxstatin-1(10 mg/kg)one hour before the stablishment of SAP model.After inducing SAP,the rats were anaesthetized again at the specified time point.Serum amylase(AMY),lipase(LIPA),tumor necrosis factor(TNF)-α,interleukin(IL)-6,creatinine(Cr),blood urea nitrogen(BUN),diamine oxidase(DAO)and endotoxin were measured in the rats.In addition,the biochemical and morphological changes associated with ferroptosis were observed,including iron accumulation in intestinal tissue,lipid peroxidation levels,and mitochondrial shrinkage.Hematoxylin and eosin staining was used to assess histological tissue changes.Western blot,RT-PCR,and immunofluorescent staining were performed to analyze the expression of ferroptosis-related proteins and genes as well as tight junction.BT was detected by 16 S r DNA sequencing analysis.Results:The serum indexes of SAP group were significantly increased.The levels of serum AMY,LIPA,TNF-α,IL-6,Cr,BUN,DAO and endotoxin in SAP group were significantly higher than those in SO group at each time point(p<0.01).Compared with SO group,SAP group had obvious intestinal pathological damage.Compared with SO group,the content of iron in ileum of SAP group was significantly increased(p<0.05),the content of MDA in ileum was significantly increased,and the activities of GSH and GPX4 were significantly decreased(p<0.01).The expression of ACSL4 in ileum of SAP group was significantly higher than that of so group(p<0.01),while GPX4 and FTH1 were significantly lower(p<0.01).The mRNA levels of ferroptosisrelated genes ACSL4 and IREB2 in ileum of SAP group were significantly increased(p<0.01).The results indicated that ferroptosis was significantly induced in the IECs from rats with SAP.The levels of serum AMY,LIPA,TNF-α,IL-6,Cr,BUN,DAO and endotoxin in SAP+Lip group were significantly lower than those in SAP group(p<0.01).HE staining showed that there were mild histological changes and lower pathological scores in pancreas and intestine of rats in SAP+Lip group(p<0.05).Treatment with liproxstatin-1lowered levels of serum damage markers,decreased lipid peroxidation,and alleviated intestinal and acute remote organ injury in SAP rats.In addition,inhibition of ferroptosis reduced BT.Conclusion:These findings are the first to demonstrate that ferroptosis of IECs is involved in intestinal mucosal barrier injury in SAP.Inhibition of ferroptosis can reduce intestinal mucosal injury,reduce bacterial translocation,and improve acute remote organ injury.These results suggest that ferroptosis is a potential therapeutic target for SAP-induced intestinal barrier injury.