The Mechanism Of Hydrogen Sulfide Protecting Septic Intestinal Injury By Up-regulating Autophagy-related Genes Beclin1 And LC3

Hydrogen sulfide（H₂S）is a newly discovered gas signaling molecule and an agonist of autophagy.It activates autophagy through a variety of signal pathways,thereby improving septic myocardial and liver damage.The intestine is one of the effector organs of sepsis.Whether H₂S can protect the intestinal function of sepsis by regulating the expression of autophagy-related genes is still unclear.Objective To study the protective effect and mechanism of H₂S on intestinal dysfunction in septic rats.Method1.Thirty male rats were randomly divided into Control group（n=5）,Sham operation group（n=5）,sepsis group（CLP group n=5）and CLP+NaHS intervention group（n=15）,of which rats in the Control group,Sham group and CLP group were injected intraperitoneally with only 1ml/kg of normal saline（NS）,and the CLP+NaHS group was divided into 3 groups according to the concentration of NaHS,5 in each group,injected intraperitoneally with 12.5μmol/kg NaHS（low dose）,25μmol/kg NaHS（medium dose）,and 50μmol/kg NaHS（high dose）.2.Enzyme-linked immunosorbent assay was used to detect the expression levels of plasma inflammatory factors HMGB-1 and IL-1βat 24 hours after administration,to observe the effect of NaHS on the expression levels of inflammatory factors in each group of rats.3.Taking the intestinal mucosa tissue of each group of rats,to observe the histopathology of intestinal mucosa under HE staining microscope and the ultrastructure of mitochondria under electron microscope,and detect the expression of CD4 and CD8 protein of intestinal mucosa by immunofluorescence technique;To further observe the local immune function and pathological changes of the intestinal mucosa during sepsis and the influence of NaHS intervention on intestinal function.4.PCR technology was used to detect the expression levels of the intestinal mucosal autophagy-related genes Beclin1 and LC3 in each group;Electron microscopy was used to observe the changes in the number of intestinal mucosal autophagy.In order to detect the changes in the level of intestinal mucosal autophagy when sepsis occurs and the influence of NaHS on the level of intestinal mucosal autophagy.Result1.There were no significant differences in the plasma inflammatory factors HMGB-1 and IL-1β,the protein expression levels of CD4 and CD8 in intestinal mucosa,the number of autophagosomes,and the relative expression levels of autophagy-related genes Beclin1 and LC3 in the Control and Sham groups（P>0.05）.The mucosa of small intestine and colon was intact under HE staining,and there was no obvious abnormality in mitochondrial structure under electron microscopy.2.Compared with the control group and the sham operation group,the expression levels of inflammatory factors HMGB-1 and IL-1βin the CLP group were significantly increased（P<0.05）.The relative expression levels of autophagy-related genes Beclin1 and LC3 in the small intestine and colon mucosa decreased（P<0.05）.The small intestine and colon mucosal tissues were significantly damaged under HE staining light microscope,and a large number of inflammatory cells infiltrated in the mucosal gland layer and basal layer,and the duct structure was disordered.The results of immunofluorescence showed that the expression levels of CD4 and CD8 proteins from the intestinal mucosa were decreased（P<0.05）,and the results of electron microscopy showed that the structure of mitochondria was destroyed,mitochondrial cristae edema is enlarged or vacuolated,and the number of autophagosomes is reduced（P<0.05）.3.Compared with the rats in the CLP group,the postoperative plasma inflammatory factors HMGB-1 and IL-1βexpression levels in rats treated with different concentrations of NaHS intervention were reduced,and the medium-dose NaHS intervention had the most significant effect（P<0.05）;The relative expression levels of CD4 and CD8 proteins in the small intestine and colon mucosa decreased（P<0.05）;The results under HE staining microscope showed that the intestinal mucosal damage of rats in the CLP group was not significantly improved when the low-dose NaHS was administered.However,after the intervention of medium and high doses of NaHS,the infiltration of inflammatory cells in the small intestine and colon mucosa of rats was gradually reduced,the uneven arrangement of intestinal villi was significantly improved,the damage of mitochondrial structure was relieved,and the damage of intestinal mucosa was relieved,Among them,the intestinal mucosal injury in rats intervened with high-dose NaHS was the most relieved.Electron microscopy results showed that the number of autophagosomes increased（P<0.05）,and mitochondrial edema was relieved,and intestinal mucosal damage was reduced,and the relative expression levels of autophagy-related genes Beclin1 and LC3 increased.However,low-dose NaHS intervention had no significant effect（P>0.05）,and the middle and high-dose intervention had significant effects（P<0.05）.Conclusion1.In septic rats,the expression levels of plasma inflammatory factors HMGB-1and IL-1βare elevated,the intestinal mucosa are obviously damaged,and its immune function and autophagy reaction are inhibited.2.Low concentration of H₂S can alleviate the intestinal mucosal damage of septic rats,and the mechanism may be that the autophagy of intestinal mucosal cells is up-regulated by promoting the expression of autophagy-related genes.