Study On The Mechanism Of Exogenous Hydrogen Sulfide Regulating Autophagy In Intestinal Injury In Sepsis

Background and objective:Sepsis is one of the main causes of death in critically ill patients.The intestinal tract is not only the organ easily involved in sepsis,but also the initial organ in the progression of sepsis.Therefore,the improvement of intestinal function is the key to the treatment of sepsis.In recent years,studies have shown that autophagy is involved in the pathological process of sepsis,maintaining mitochondrial function by clearing damaged organelles,inhibiting inflammation,oxidative stress and apoptosis,regulating immunity and maintaining intestinal functional homeostasis,effectively improving the condition and prognosis of sepsis,is an effective target for the treatment of sepsis.Hydrogen sulfide（H₂S）,as a new gas signal molecule,can protect organ functions such as anti-inflammation and antioxidation by regulating autophagy,which has become a new target in the treatment of sepsis.Therefore,the purpose of this study is to explore the role and mechanism of exogenous H₂S regulating autophagy in intestinal injury in sepsis.Method:1.Sixty male SD rats were randomly divided into three groups:control group（Control group,n=20）,sham operation group（Sham group,n=20）and sepsis model group（CLP group,n=20）.The septic rat model was established by cecal ligation and puncture（CLP）.Each group was subdivided into two subgroups:intraperitoneal injection of NS（n=5）and intraperitoneal injection of NaHS（n=15）,and the subgroups of intraperitoneal injection of NaHS were divided into three groups according to different NaHS concentration gradients:CLP+NaHS（12.5μmol/kg）group,CLP+NaHS（25μmol/kg）group and CLP+NaHS（50μmol/kg）group.2.During intestinal injury in septic rats,the expression levels of inflammatory cytokines TNF-αand IL-6 in plasma and cytokines ROS and SOD reflecting oxidative stress injury were significantly increased.After administration of exogenous H₂S donor NaHS,the expression levels of TNF-α,IL-6,ROS and SOD decreased significantly,and the levels of TNF-α,IL-6 and ROS decreased gradually with the increase of NaHS concentration,while the level of SOD increased gradually,and the effect of NaHS was the most significant at 50μmol/kg concentration.3.The expression levels of inflammatory cytokines TNF-αand IL-6 and cytokines ROS and SOD reflecting oxidative stress injury in plasma of rats in each group were detected by ELISA method to study the effect of NaHS on inflammatory response and oxidative stress injury in septic rats.4.The expression levels of autophagy-related proteins LC3B,LAMP-2,Rab7and p62 in intestinal epithelial cells of rats in each group were detected by Western Blot technique,and the situation of autophagosome and autophagy lysosome in intestinal epithelial cells of rats in each group were observed under electron microscope,and the damage of intestinal epithelial cell autophagy flux in septic rats and the effect of NaHS on intestinal epithelial cell autophagy flux in septic rats were comprehensively evaluated.Results:1.Intestinal injury was obvious in the rats with sepsis,mucosal epithelial edema,disordered glandular arrangement,villi shortening and a large number of inflammatory cell infiltration were observed under light microscope.Injury of intestinal epithelial junctions was observed under transmission electron microscopy.The intestinal injury of septic rats was improved after the administration of exogenous H₂S donor NaHS,and the improvement of intestinal injury in the high-dose NaHS group（50μmol/kg NaHS group）was the most significant（P<0.01）.2.In sepsis rats with intestinal injury,the expression levels of TNF-α,IL-6 and ROS in plasma were significantly increased,while the expression levels of SOD in plasma were significantly decreased（P<0.01）.The expression levels of TNF-α,IL-6and ROS were significantly decreased after exogenous H₂S donor NaHS（P<0.01）,the expression level of SOD was significantly increased（P<0.01）;With the increase of NaHS concentration,TNF-α,IL-6 and ROS levels gradually decreased,while SOD levels gradually increased,and the effect of NaHS was the most significant at50μmol/kg（P<0.01）.3.During sepsis,the expression of autophagy-related proteins LC3B and p62 in intestinal epithelial cells increased（P<0.01）,while the expression of LAMP-2 and Rab7 decreased（P<0.01）.Double-membrane autophagosome accumulation was observed under electron microscope,and single-membrane autophagy lysosome could not be formed.After administration of exogenous H₂S donor NaHS,the expression of LC3B,LAMP-2 and Rab7 protein increased（P<0.05）,while the expression of p62protein decreased（P<0.05）,double membrane autophagosome decreased and monolayer autophagy lysosome formation increased.The above effects were concentration-dependent,and the effect of NaHS was the most significant at the concentration of 50μmol/kg（P<0.01）.Conclusions:1.The intestinal tract of rats with sepsis was damaged.Exogenous H₂S had a protective effect on intestinal injury in septic rats,and this protective effect was concentration-dependent.The higher the concentration was,the more significant the protective effect was.2.The autophagy flux of intestinal epithelial cells in sepsis was impaired.Exogenous H₂S can up-regulate the autophagy flux by regulating the expression of autophagy-related proteins,inhibit inflammation and oxidative stress,and then played a protective role in intestinal injury in septic rats.The protective effect was dependent on NaHS concentration,and the higher the concentration was,the more significant the protective effect was.