Effect Of Sulfasalazine On Ferroptosis During Intestinal Injury In Rats After Liver Transplantation

The purpose of this study was to establish a rat model of intestinal injury induced by cold ischemia-reperfusion（I/R）after orthotopic liver transplantation（AOLT）,To determine whether there is ferroptosis in the intestinal injury induced by cold I/R in rat AOLT and to observe the protective effect of ferroptosis inhibitor Ferrostain-1 on the intestine of autologous orthotopic liver transplantation in rats.At the same time,to explore the effect of sulfasalazine（SAS）,an activator of ferroptosis,on perioperative intestinal injury in AOLT rats and its possible mechanism.Part one:The role of ferroptosis in perioperative intestinal injury after autologous liver transplantation in rats.Objectives:Using the rat AOLT model,the effect of lipid reactive oxygen species（L-ROS）inhibitor Ferrostain-1 on ferroptosis signal pathway was observed to determine whether ferroptosis occurred in perioperative intestinal injury in rats with AOLT.Methods:Eighteen healthy male adult SD rats of SPF grade,aged 10 to 12weeks,weighing 300±20 g,were randomly divided into three groups:sham operation group（Sham Group）,model group（I/R Group）and ferroptosis inhibitor Ferrostain-1 group（I/R+Fer-1 Group）.The rats in Sham group only underwent laparotomy after anesthesia and closed the abdomen after dissociating the blood vessels and ligaments around the liver.The rats in I/R+Fer-1 group were perfused continuously with 4℃Ringer’s lactate solution into the portal vein after 30±1 min,and the abdominal cavity was closed after washing.The rats in I/R+Fer-1 group received intraperitoneal injection of Fer-15mg/kg one hour before modeling,and the operation was the same as that in I/R group.The inferior hepatic vena cava blood was taken 24 h after reperfusion,and some ileal tissue samples were taken for index detection.Intestinal histopathological changes were observed under high power microscope;serum IL-6 and intestinal injury marker diamine oxidase（DAO）were measured by ELISA;intestinal malondialdehyde（MDA）content and serum MDA concentration were measured by thiobarbituric acid method.The levels of superoxide dismutase（SOD）,reduced glutathione（GSH）,glutathione peroxidase4（GPX4）,ferritin heavy chain（FTH1）and Solute carrier family 7 member 11（SLC7A11）in intestinal tissue were detected by western blot.Results:Compared with sham group,the Chiu’s score and W/D ratio at 24 h after intestinal injury in I/R group and I/R+Fer-1 group increased significantly,the levels of serum IL-6 and MDA increased significantly,the levels of DAO,MDA and Fe²⁺in intestinal tissue increased significantly,the levels of SOD,GSH and GPX4 in intestinal tissue decreased,and the expression of FTH1 and SLC7A11 protein decreased in intestinal tissue.Compared with I/R group,the Chiu’s score and W/D ratio at 24 h after intestinal injury in I/R+Fer-1 group decreased,the levels of serum IL-6 and MDA decreased,the levels of DAO,MDA and Fe²⁺in intestinal tissue decreased significantly,the levels of SOD,GSH and GPX4 in intestinal tissue increased,and the expression of FTH1 and SLC7A11 protein in intestinal tissue increased.Conclusions:Ferroptosis is involved in the pathophysiological process of intestinal injury induced by cold hepatic ischemia-reperfusion in rats.Part two:The effect of sulfasalazine on ferroptosis after intestinal injury in rats with liver transplantation and its mechanism.Objectives:To investigate the effect and possible mechanism of SAS,an activator of ferroptosis,on ferroptosis in rats with intestinal injury induced by cold I/R in the liver of AOLT.Methods:Sixty SPF adult male SD rats were randomly divided into five groups（n=12）:sham operation group（Sham group）,model group（I/R group）,Ferrostain-1 group（I/R+Fer-1 group）,SAS group（I/R+SAS group）and SAS+Ferrostain-1 group（I/R+SAS+Fer-1 group）.six rats were randomly selected at 6 h and 24 h after I/R.One hour before operation,the same volume of DMSO was injected intraperitoneally in I/R group,and each rat was given 1 ml;One hour before operation,Ferrostain-1 was injected intraperitoneally in I/R+Fer-1group with 5 mg/kg per rat.SAS（twice a day,500 mg/kg/time）was injected intraperitoneally for seven days before operation in I/R+SAS group;SAS（twice a day,500 mg/kg/time）was injected intraperitoneally for seven days in I/R+SAS+Fer-1 group one hour before operation,and Ferrostain-1 was injected intraperitoneally one hour before operation.The blood samples of inferior vena cava were taken at 6 h and 24 h after modeling,and the concentrations of MDA and IL-6 in serum were determined by ELISA method,and the ileal tissue sections were observed under light microscope at 6 h and 24 h after modeling.The contents of SOD,MDA,GSH,GPX4 and Fe²⁺were determined by kit,and the protein expression of GPX4 and SLC7A11 was determined by western blot.Results:Compared with the Sham group,the Chiu’s score and W/D ratio increased 6 h and 24 h after modeling in other groups,the levels of serum IL-6and MDA increased significantly,the levels of DAO,MDA and Fe²⁺in intestinal tissue increased significantly,the levels of SOD,GSH and GPX4 in intestinal tissue decreased,and the expression of FTH1 and SLC7A11 protein in intestinal tissue decreased.Compared with I/R group,the Chiu’s score and W/D ratio at 6h and 24 h after modeling in I/R+SAS group increased,the levels of serum IL-6and MDA increased,the levels of DAO,MDA and Fe²⁺in intestinal tissue increased significantly,the levels of SOD,GSH and GPX4 in intestinal tissue decreased,and the expression of FTH1 and SLC7A11 protein decreased in intestinal tissue.Compared with the SAS group,the Chiu’s score and the ratio of W/D at 6 h and 24 h after modeling in the I/R+SAS+Fer-1 group decreased,the levels of serum IL-6 and MDA decreased,the levels of DAO,MDA and Fe²⁺in the intestinal tissue decreased significantly,the levels of SOD,GSH and GPX4in the intestinal tissue increased,and the expression of GPX4 and SLC7A11protein in the intestinal tissue increased.In addition,during the process of AOLT liver I/R,the change trend of all indexes at 6 h and 24 h after reperfusion was basically the same,and the difference was not significant.Conclusions:SAS may induce intestinal injury during perioperative period of liver transplantation by inhibiting SLC7A11/GSH/GPX4 signal axis or iron overload after reperfusion,resulting in a large amount of L-ROS accumulation,activating cellular ferroptosis,and further aggravating intestinal injury.