Role Of Autophagy In Intestinal Ischemia-reperfusion Injury In Mouse

Objective: In this study,mice with intestinal ischemia-reperfusion were used to investigate the effects of autophagy on the intestinal ischemia-reperfusion injury by intraperitoneal injection of autophagy inhibitors(3-methyladenine,3-MA)or Rapamycin(RAP).Methods: Forty-eight healthy male C57BL/6J mice(6-8 weeks)were randomly assigned to 6 groups: S group,IIR group,3-MA group,IIR+3-MA group,RAP group and IIR + RAP group.The model of intestinal ischemia-reperfusion was established.Part of groups were injected with 3-MA or RAP before laparotomy.Open the abdomen along the midline of the abdomen,separate the superior mesenteric artery and clamp it with a micro-arterial clamp for 45 minutes.Blood was collected after 2 hours reperfusion.After blood collection,the mice were sacrificed and the duodenum portion of the mouse were taken.The levels of IL-6 and TNF-α were detected by ELISA.MDA,GSH-Px were measured by kits.The expression of autophagy marker protein(LC3BII/I)、PINK1、Parkin in intestinal were detected by western blot.Furthermore,after paraffin embedding,the changes of intestinal morphological were observed by optical microscope with hematoxylin and eosin staining and the apoptosis index was ealuated by TUNEL at the same time.Results: 1.Compared with S group,the degree of small intestine injury and Chiu’s score in IIR group,IIR+3-MA group and IIR+RAP group were increased(P<0.05),but the difference in 3-MA group and RAP group was not statistically significant.Compared with the IIR group,the degree of injury and Chiu’s score in the IIR+3-MA group were more seriously(P<0.05),while the degree of intestinal tissue damage and Chiu’s score were significantly decreased in the IIR+RAP group(P < 0.05).2.Compared with the S group,there was no significant change in serum biochemical parameters in the 3-MA group and the RAP group,and there was not significantly difference.After intestinal ischemia-reperfusion,the levels of IL-6,TNF-α and MDA in serum increased significantly(P<0.05),while the levels of GSH-Px decreased(P<0.05),along with the same increase trend of apoptotic cells(P<0.05).Among them,these changs were more significant when IIR+3-MA group compared to IIR group(P<0.05),while in IIR+RAP group,the magnitude of changes were smaller(P<0.05).3.Compared with the S group,LC3BII/I,PINK1,and Parkin protein expression levels in the RAP group increased(P<0.05),however,the changes in the 3-MA group were reversed(P<0.05).After intestinal ischemia-reperfusion,compared with the corresponding sham operation group,the expression of LC3BII/I,PINK1,Parkin protein increased(P<0.05).The expression of LC3BII/I,PINK1,Parkin protein decreased in the IIR+3-MA group compared with the IIR group(P< 0.05),while the expression of LC3BII/I,PINK1,Parkin protein in IIR+RAP group were further up-regulated when compared with IIR group(P<0.05).Conclusions: Intestinal ischemia-reperfusion can cause serious damage on intestinal tissue.The autophagy agonist RAP could reduce damage by increasing autophagy,reducing inflammation and oxidative stress levels.While intestinal tissue damage was aggravated after application of autophagy inhibitor 3-MA.Therefore,autophagy plays an important role in intestinal ischemia-reperfusion injury,and its mechanism may be related to the PINK1/Parkin signaling pathway.