| Objective: To investigate the effects and its mechanism of carbon monoxide on intestinal mucosal mechanical barrier function to indicate the regulation of CO on pyroptosis and autophagy of intestinal mucosa cells as well as inflammatory cytokines of serum and intestinal mucosa in septic rats,and explore its related mechanism.Methods: A total number of 132(180-200 g)male Sprague Dawley rats were randomly divided into 6 groups: Sham group(n=22),sepsis group(CLP group,n=22),endogenous CO agonist group(Hemin groupn=22),endogenous CO endogenous inhibitor group(ZnPPⅨ group,n=22),CO-releasing moleculer 2 group(CORM-2,n=22),depleted CO of CORM-2 group(inactive CORM-2 or iCORM-2,n =22).Cecal ligation and puncture(CLP)were used to duplicate the sepsis models.Recording the survival time and drawing a 7-day survival curve among 6 groups(n=10).At 24 h after intervention,fluorescence tracer method was used to detect the concentration of FD-4 in serum(n=6).Intestinal injury in each group were evaluated by H&E staining and scored by Chiu’s score system;Western blot and the immune fluorescent confocal methods were used to detecte the levels of intestinal pyroptosis related proteins(caspase1 and caspase11)and autophagy related proteins(LC3B and Beclin1).The levels of inflammatory cytokines,like TNF-α,IL-1β,IL-18,HMGB1 and IL-10 in serum and tissue homogenate supernatant of model rats in each group were detected by ELISA.Results:(1)7-days survival rate of Sham group was 100%,while it was was 0 in CLP group and all rats dead in 5th day,that in Hemin and CORM-2 were 30% and 20% respectively,while the survival rate in ZnPPⅨ group was 0,and all rats dead at 3th day(P<0.05).The average survival time of Sham group,CLP group,Hemin group,ZnPPⅨ group,CORM-2 group and iCORM-2 group were 7 day,2 day,3 day,1 day,4 day,2 day respectively.(2)The concentration of FD-4 in CLP group was obviously elevated compared to Sham group,which was significantly decreased in Hemin group and CORM-2 group and increased significantly in ZnPPⅨ group compared to the CLP group(P<0.05).(3)Intestinal damage increased apparently in CLP group compared to Sham group(P<0.05),Hemin and CORM-2 decreased intestinal injury and ZnPPⅨ aggravated the intestinal damage degree significantly compared to CLP group(P<0.05).(4)The expression of pyroptosis related proteins caspase-1,caspase-1p20,caspase-11 and caspase-11p20 were significantly increased in CLP group(P<0.05),and were decreased in Hemin group and CORM-2 group(P<0.05),and increased obiviously in ZnPPⅨ group compared to CLP group(P<0.05).(5)The expression of autophagy related proteins LC3 B and Beclin1 were significantly increased in Hemin group and CORM-2 group compared to CLP group(P<0.05),while that in ZnPPⅨ group,they were obiviously decreased compared to CLP group(P<0.05).(6)The expression levels of TNF-α,IL-1β,IL-18,HMGB1 and IL-10 in CLP group were significantly increased compared to Sham group;the levels of serum and instinal mucosal IL-10 significantly increased in the Hemin group and CORM-2 group compared to CLP group(P<0.05),and that in ZnPPⅨ group were decreased(P<0.05);the levels of serum and instinal mucosal TNF-α,IL-1β,IL-18 and HMGB1 were significantly decreased in the Hemin group and CORM-2 group,that in ZnPPⅨ group were significantly increased compared to CLP group(P<0.05).Conclusions:(1)Both endogenous CO and exogenous CO intervention could improve the 7-day survival rate of septic rats.(2)CO reduced the damage of intestinal mucosal mechanical barrier and intestinal mucosal permeability of septic rats,which may be attributed to its inhibition of the secretion of proinflammatory cytokines,including TNF-α,IL-β,IL-18 and HMGB1,as well as elevation of the secretion and expression of anti-inflammatory cytokine IL-10.(3)CO reduced the damage of intestinal mucosal mechanical barrier and intestinal mucosal permeability of septic rats through inhibiting intestinal mucosal cell pyroptosis and promoting intestinal mucosal autophagy in septic rats. |
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