The M2olecular Mechanism Of Protective Role Of Thioredoxin-1 In Sepsis By Suppressing Endoplasmic Reticulum Stress

Sepsis is a disorder of host response to infection serious and results in severe organs dysfunction.Sepsis is the common complication of severe injury,burn,infection,ischemia reperfusion injury,acute pancreatitis,and major surgery and the leading cause of death in clinical sever patients.The environmental pollution,the aging of the population,the trauma,the amount use of antibiotics,hormone drugs,as well as organ transplantation,induces the increasing incidence of sepsis year by year.With the development of sepsis,it will be complicated with organ dysfunction,and then develop into severe sepsis,septic shock and multiple organ dysfunction syndrome,(MODS),finally organ failure(MOF),which leads to death.The pathogenesis of sepsis is complex and is involved in a series of pathophysiological processes,its molecular mechanism is still unclear.The study shows that the excessive release of inflammatory mediators,immune cell apoptosis,free radicals,intestinal bacteria and endotoxin translocation,microcirculation failure,mitochondrial dysfunction,and coagulation disorders are associated with the pathogenesis of sepsis in different stages and different degree.Inflammation and immune cell apoptosis play key roles in the development of sepsis.The endoplasmic reticulum(ER)stress is a protecting response and reduces the abnormal protein aggregation through activating unfolded protein response(UPR).The long term and sever ER stress results in cell apoptosis.The reasons involved in ER stress include oxidative stress,calcium homeostasis imbalance,pathogen infection,malnutrition and ischemia reperfusion injury.ER stress is related with various diseases,such as Parkinson disease,infection disease and sepsis.Under the normal condition,the endoplasmic reticulum chaperone Glucose regulated protein 78(GRP78)binds to three transmembrane proteins in the endoplasmic reticulum surface.These three proteins are inositol-requiring enzyme 1(IRE1),activating transcription factor 6(ATF6)and protein kinase RNA-like ER kinase(PERK).GRP78 binds internal ends of these three transmembrane proteins to maintain the inactive state of signal transduction factors.When endoplasmic reticulum stress occurs,the GRP78 is separated from transmembrane proteins and activated,then the UPR is induced through IRE1,PERK and ATF6.When ER stress is prolonged,cell apoptosis will be induced through c-Jun N-terminal kinase(JNK),C/EBP homologous protein(CHOP),Caspase-12 apoptosis signal pathway.Endoplasmic reticulum apoptosis and mitochondrial apoptosis have cross talk.The ER stress not only induces apoptosis,but also plays an important role in inflammation.The ER stress induced inflammation usually through NF-κB signal pathway.Thioredoxin-1(Trx-1)is a 12-kDa multifunctional protein with a conserved sequence:-Cys-Gly-Pro-Cys-.Trx-1 is widely expressed in prokaryotes and eukaryotes.Trx-1 plays roles in suppressing oxidation,inflammation and apoptosis.Trx-1 also regulates ER stress.In the present study,we used cecal ligation and puncture(CLP)to make the model of sepsis and used C57BL/6 mice and Trx-1 transgenic mice to investigate the protective effect of Trx-1 in sepsis.We selected 6-8 weeks male C57BL/6 mice and Trx-1 transgenic mice.Mice were divided randomly into four groups:wild-type sham control group(group WC),human Trx-1 transgenic sham control group(group TC),wild-type sepsis group(group WS)and human Trx-1 transgenic sepsis group(group TS).Survival was monitored for 7 days.The levels of TNF-a and IL-1β in the serum and lung tissue were determined at 12 hours after CLP.The number of bacteria in the peritoneal lavage fluid was determined at 12 h after CLP.Plasma PCT level was measured by using the mouse PCT ELISA kit.We evaluated morphologic changes in the spleen and lung by using H&E staining.Apoptosis of lung and spleen was detected by TUNEL assay.We detected endoplasmic reticulum stress inflammatory signaling pathway related protein GRP78,IRE1,tumor necrosis factor receptor-associated factor 2(TRAF2),CHOP and NF-kappa B related proteins IκBα,p-NF-κB and NF-κB in the lung tissue by Western blot.Furthermore,we examined endoplasmic reticulum stress apoptosis signaling pathway related proteins GRP78,IRE 1α,TRAP2,JNK,CHOP,Caspase-12 and mitochondrial apoptosis pathway related proteins Bcl-2,Bax,Caspase-9,Caspase-3.The main results were as follows:1.Trx-1 overexpression played protective role in septic mice.The survival was longer in sepsis induced by cecal ligation and puncture(CLP)in Trx-1 overexpression transgenic(Tg)mice compared to wild-type mice.Trx-1 overexpression reduced injury of lung and attenuated the pulmonary edema in sepsis.Trx-1 overexpression inhibited the growth of abdominal bacteria and reduced the plasma level of PCT in sepsis.2.Overexpression of Trx-1 in mice inhibited the inflammatory response by inhibiting the endoplasmic reticulum stress inflammatory signaling pathway.The levels of TNF-a and IL-1β were increased in group WS,which were suppressed in Trx-1 TG mice.The expressions of GRP78,IRE1α,TRAF2,CHOP,NF-κB and inhibitor of NF-κBα(IκBα)were increased in lung tissue.The overexpression of Trx-1 in transgenic mice suppressed NF-κB inflammatory signal pathway by inhibiting the activation of these molecules.3.Overexpression of Trx-1 in mice inhibited the endoplasmic reticulum stress signaling pathway and mitochondrial apoptosis signaling pathway in sepsis.The TUNEL positive cells of WS group were significantly increased compared with WC group after CLP.Trx-1 overexpression significantly inhibited apoptosis.The expressions of GRP78,IRE1α,TRAF2,JNK,CHOP,Caspase-12,Bax,Caspase-9,and Caspase-3 were significantly increased in wild-type mice in sepsis,while the overexpression of Trx-1 inhibited the increases of the above proteins.Bcl-2 expression was decreased significantly in the WS group compared with the sham group.Conversely,Bcl-2 was stored in Trx-1 overexpression.These results suggest that Trx-1 inhibits cell apoptosis by suppressing endoplasmic reticulum stress pathway and mitochondrial apoptosis pathways.Conclusion:Trx-1 overexpression played a protective role in resisting sepsis and increased survival after CLP by reducing the inflammatory response,decreasing apoptosis,and improving bacterial clearance,and this protective role may be through suppressing ER stress mediated inflammtion and apoptosis.This study increases our knowledge of the pathogenic mechanisms of sepsis and provides new targets and innovative strategies for the treatment of sepsis.