Insulin Alleviates Intestinal Barrier Dysfunction During Sepsis Involving Downregulation Of Uncoupling Protein 2

Research backgroundSepsis is a severe disease with high incidence and high mortality.The cases of intraperitoneal infection,such as appendicitis perforation,intestinal fistula or necrotizing enterocolitis,take up 20%of that of sepsis.However the mortality of this kind of diseases nearly reaches 60%.Gastrointestine(GI)has two-side effect on onset and development of sepsis.On one hand,GI may be one of the most organ dysfunction involved in sepsis and always be damaged by sepsis due to its pro-inflammatory and ischemia-reperfusion effect;on the other hand,gastrointestinal dysfunction caused by kinds of diseases,including trauma,bleeding,burn,et al,itself can cause or aggravate sepsis leading by intestinal epithelial barrier damage which can lead intestinal contents,such as intestinal bacteria,toxins and Antigen,to transfer across the mucosal barrier into the body circulation,and then induced multiple organ dysfunction(MODF).Glucose-insulin-potassium(GIK)therapy has been reported to decrease mortality among critically ill patients due to its suppression of inflammatory and apoptotic effects,and has been used clinically for decades and Insulin,which has now been confirmed to be the major bioactive component of GIK,can decrease pyruvate production and glycolysis and increase glycogen synthesis,which may result in reduced production of lactate during sepsis.Uncoupling protein 2(UCP2),a mitochondrial inner membrane protein,is widely expressed in various tissues,including intestinal tissue,and has been reported to have an important role in the maintenance of the intestinal mucosal barrier during inflammatory or ischemia/reperfusion conditions.Recently,it is reported that the mortality rate of wild-type rats with cecal ligation and puncture(CLP)-induced sepsis was higher than that of UCP2-knockout rats,and that UCP2 may increase the expression of fatty acid synthase(FASN)and promote protein kinase B,p38 mitogen-activated protein kinase and NLR family-pyrin domain-containing 3 inflammasome activation during sepsis.Research purposesIt has not been confirmed whether UCP2 plays a role in intestinal mucosal injury during septic conditions.However,there appears to be some relationship between insulin and UCP2.Therefore,the purpose of the present study was to confirm the potency of the effects of GIK using in vitro and in vivo models of intestinal mucosal barrier injury,and to elucidate the association between GIK therapy and the expression of UCP2 in these models.Research methodsTwo models of sepsis were established in vivo and in vitro.SD rats conducting cecal puncture and ligation(CLP)were adopted as in vivo sepsis model and were randomly divided into five groups:control group,sham CLP group,CLP group,CLP+ GIK group and CLP + GK group.the following contents were studied:72-hour mortality,serum IL-1β/TNF-α level,levels of IL-1β/TNF-α/IL-6/IL-10 in the ileum tissue,Chiu’s score of HE staining in the ileum tissue,serum FD-4 method essessing ileum tissue permeability function in the ileum tissue,tight junction protein expression(occludin,claudin-1 and zonula occludens-1)in the ileum tissue,UCP2 and NLRP3 mRNA levels in the ileum tissue,UCP2,NLRP3 and Caspasel p10 protein expression in the ileum tissue.Human colon cancer cell lines(Caco2)treated by lipopolysaccharide(LPS)was adopted as in vitro sepsis model.Monolayer cell permeability test was conducted to evaluate the integrity function of cells.Caco2 cells were treated with Oug/ml,0.1ug/ml,1.0ug/ml,lOug/ml and 100ug/ml LPS to study monolayer cell permeability,UCP2 and p-p38 MAPK protein expression.The cells were pretreated with 100 ug/ml LPS solution and then treated with 1.0nmol/ml,10nmol/ml and 100nmol/ml insulin.Monolayer cell permeability test,expression of UCP2 and p-p3 8 MAPK protein were studied 24 hours later.Research result1.Insulin can lower the 72-hour mortality and serum pro-inflammatory cytokines IL-1β and TNF-a production in rats subjected to of CLP-induced polymicrobial sepsis.2.Insulin can alleviate intestinal mucosal injuries,lower pro-inflammatory cytokine production and increase anti-inflammatory cytokine production in rats following CLP-induced polymicrobial sepsis.3.Insulin can decrease intestine permeability and increase ZO-1,Occludin and Claudin-1 protein expression in rats following CLP-induced polymicrobial sepsis.4.Insulin can decrease relative expression of UCP2 and NLRP3 mRNA and UCP2,NLRP3 and Caspasel p10 protein expressions in rats following CLP-induced polymicrobial sepsis.5.Monolayer cell permeability and expression of UCP2 and p-p38 MAPK protein increased in Caco2 cells following LPS stimulation in a concentration-dependent manner.6.Monolayer cell permeability and expression of UCP2 and p-p38 MAPK protein decreased in LPS-pretreated Caco2 cell subjected to insulin treatment in a concentration-dependent manner.ConclusionInsulin can decrease serum pro-inflammation and mortality in CLP-induced sepsis model,alleviate pro-inflammatory level and damages of ileal epithelial tissue,increase the expression of tight junction protein and enhance the barrier function of ileal epithelial tissue.The main mechanism is to down-regulate UCP2 protein and inhibit the activation of downstream p38 MAPK and NLRP3/ASC/Caspasel signaling pathways.