Studies On The Effects And Molecular Mechanisms Of Exogenous Carbon Monoxide On Intestinal Barrier Dysfunction In Septic Rats

Background and objective:Sepsis is a systemic inflammatory syndrome with oxidative damage,coagulation disorders and tissue hypoperfusion and may be companied with immune suppression,and severe intestinal mucosal barrier dysfunction.It is a common and serious clinical syndrome in trauma surgery and intensive care unit(ICU),and further develop severe sepsis,septic shock and multiple organ dysfunction syndrome(MODS).It is considered the leading cause of death in ICU.Recently,with further study on sepsis,the mortality of sepsis began to decline,but the mortality of severe sepsis and septic shock remains very high.Therefore,it is an urgent need to look for new treatment of severe sepsis and its complicated MODS.Intestine is one of the earliest organ involved in sepsis,and it is also a stress center.The damage of intestinal lead to intestinal mucosal barrier dysfunction,leading to continuing the intestinal bacteria and toxins into the tissue and blood,inducing excessive uncontrolled systemic inflammatory response,to participate in the occurrence and development of remote organ injury and MODS.Previous studies have confirmed that maintaining tight junction integrity and the normal function of intestinal epithelial cells has become the key to the treatment of sepsis and MODS.CO has been showed to affect several intracellular signaling pathways,and such pathways mediate,in part,the known anti-inflammatory,anti-apoptotic,vasoregulatory and inhibition of platelet aggregation of this gas.In experimental animals,approaches that increase CO levels in the target tissue improve a variety of gastrointestinal disorders,for example,diabetic gastroparesis,inflammatory bowel disease(IBD),sepsis and post-operative ileus and outcomes following intestinal transplant.This research focus on that whether Carbon Monoxide-Releasing Molecule-2(CORM-2)can provide protection against intestinal barrier dysfunction in CLP-induced septic rats and what its mechanism is.It consists of the following two parts:first,the protective effect of exogenous CO on intestinal barrier dysfunction in septic rats;second,the molecular mechanisms of the protection of exogenous CO on intestinal barrier dysfunction in septic rats.Methods:(1).SD rats were randomly divided into four groups:sham operation group(Sham),CLP model group,CLP rats + CORM-2 group(CORM),CLP rats + inactive CORM-2 group(iCORM).Rats were killed at 24h after CLP to collect blood and intestinal tissue.Serum levels of TNF-? and IL-1? were measured by ELISA;pathological changes of intestinal mucosa under light microscopy and ultrastructural changes of intestinal mucosa under transmission electron microscopy(TEM)were observed in each groups;the levels of tight junction proteins(ZO-1,Claudin-1 and Occludin)in the intestinal mucosa were measured by Western blot;intestinal permeability of FITC-Dextran was measured by fluorescence spectrophotometer.The survival rate of 72 hours in each group is also observed.(2).Serum diamine oxidase(DAO)activity and D-lactate level,intestinal mucosa myeloperoxidase(MPO)activity were assessed by available assay kits.Rho kinase(ROCK)-1 and ROCK-2 mRNA of intestinal mucosa were measured by RT-PCR.The levels of proteins of phosphorylation-myosin phosphatase target subunits-1(p-MYPT-1)/MYPT-1 and phosphorylation-myosin light chain(p-MLC)/MLC in the intestinal mucosa were measured by Western blot.Results:(1).Compared with the Sham group,the indicators changed significantly in CLP group,CORM group and iCORM group:the serum levels of TNF-and IL-1? were significantly higher(P<0.05);Degree of injury to intestinal mucosa and tight junction disruption were significantly higher,Chiu's scores were significantly increased(P<0.05);Contents of tight junction proteins(ZO-1,Claudin-1 and Occludin)were significantly lower(P<0.05);the immunofluorescence result showed redistribution of TJ proteins in intestinal mucosa was decreased;the intestinal permeability was increased and the survival rate was decreased(P<0.05).However,compared with the CLP group and iCORM group,the indicators of the CORM group showed:the serum levels of TNF-? and IL-1? were significantly lower(P<0.05);Degree of injury to intestinal mucosa and tight junction disruption were significantly lower,Chiu's scores were significantly decreased(P<0.05);Contents of tight junction proteins(ZO-1,Claudin-1 and Occludin)were significantly higher(P<0.05);the immunofluorescence result showed redistribution of TJ proteins in intestinal mucosa was increased;the intestinal permeability was decreased and the survival rate was increased(P<0.05).(2).Compared with the Sham group,the indicators changed significantly in CLP group,CORM group and iCORM group:the activity of DAO in serum,the serum levels of D-lactate and activity of MPO in intestinal mucosa were significantly higher(P<0.05);intestinal mucosa ROCK-1 and ROCK-2 mRNA expression significantly increased(P<0.05);the ratio of p-MYPT-1/MYPT-1 and p-MLC/MLC significantly increased(P<0.05).However,compared with the CLP group and iCORM group,the indicators of the CORM group showed:the activity of DAO in serum,the serum levels of D-lactate and activity of MPO in intestinal mucosa were significantly lower(P<0.05);intestinal mucosa ROCK-1 mRNA expression significantly decreased(P<0.05),but ROCK-2 mRNA expression was no significant difference;the ratio of p-MYPT-1/MYPT-1 and p-MLC/MLC significantly decreased(P<0.05).Conclusions:(1).CORM-2 can alleviate intestinal pathological damages,inhibit the inflammatory mediators release,protect intercellular tight junction and intestinal mucosal barrier in CLP-induced septic rats.(2).Effect of CORM-2 on CLP-induced septic rats may be related to its inhibition of Rho/ROCK signaling pathway to reduce the expression of inflammation-related gene and protein.