Protective Efficacy Of Schistosoma Japonicum Cystatin On Sepsis-induced Cardiac Dysfunction In Mice And Immunological Mechanism

Sepsis is a serious life-threatening disease that occurs after severe infections,trauma,burns,and major surgery and is the leading cause of death in the intensive care unit.Myocardial dysfunction in sepsis is one of the most common complications of multiple organ failure in septic shock and significantly increases mortality in patients with sepsis.Studies have confirmed that 50% of patients with sepsis have left and right ventricular systolic and diastolic dysfunction,and there are no clinically effective drugs to treat sepsis-induced cardiac dysfunction.Therefore,it is particularly important to find a new effective drug for sepsis-induced cardiac dysfunction.Schistosoma japonicum cysteine protease inhibitor(Sj-Cys),a worm-derived cysteine protease inhibitor,has been confirmed in several experiments to play a protective role in inflammatory diseases,but there is no information about report of Sj-Cys on cardiac dysfunction caused by sepsis.Therefore,our study intends to use rSj-Cys to observe the therapeutic effect on sepsis-induced cardiac dysfunction and to preliminarily explore its immunological mechanism,so as to provide experimental basis for rSj-Cys to become a new type of sepsis-induced cardiac dysfunction treatment drug.Objective: The main purpose of this study was to observe the effect of rSj-Cys on sepsis-induced cardiac dysfunction.The potential application value of rSj-Cys in the treatment of sepsis-induced cardiac dysfunction was investigated by observing the pathological changes,biochemical indexes and inflammatory factors of cardiac tissue in mice.A cardiomyocyte culture system was established in vitro to further study the effect of rSj-Cys on endotoxin-induced cardiomyocyte injury and its possible molecular mechanism.Methods: 1.Express,purify and identify rSj-Cys and determine the protein concentration.2.Construct mouse sepsis-induced cardiac dysfunction model.24 male BALB/c mice were randomly divided into 4 groups: the normal control group(Sham group),rSj-Cys group(r Sj-Cys group),sepsis(CLP group)and treatment group (CLP + rSj-Cys group).The mice were anesthetized and sacrificed 12 hours after modeling,and sera and heart tissue samples were collected.Cardiac function indicators(EF%,FS%,E/A)were measured by a cardiac echocardiograph to evaluate mice heart function.HE staining determined cardiac pathological changes in mice.ELISA detected the levels of inflammatory factors(TNF-?,IL-6, IL-10,TGF-?)and the concentrations of myocardial biochemical indicators(cTnI, NT-proBNP)in sera of mice.The level of the serum Myoglobin(Mb)was detected by automatic biochemical analyzer and the activity of MPO in cardiac tissue was detected by myeloperoxidase(MPO)kit.3.Culture H9C2 cells and divide them into four groups,the Medium control group,rSj-Cys group,LPS and(LPS + rSj-Cys)group.After treatment in different ways, the supernatants of cells were collected to detect inflammatory factors(TNF-?, IL-6,IL-10,TGF-?)and extract cell proteins to detect the expression of MyD88.Results: 1.Echocardiographic test results showed that compared with the Sham group,the left ventricular EF%,FS% and E/A were decreased in the CLP group(p < 0.05).Compared with the CLP group,the left ventricular EF%,FS% and E/A were increased in the CLP + rSj-Cys group(p < 0.05).However,there was no significant difference in the left ventricular EF%,FS% and E/A,between the medium control group and LPS + rSj-Cys group.These results showed that rSj-Cys could significantly alleviate the cardiac function impairment caused by CLP.2.Compared with the Sham group,myocardial fibers lost normal structure,muscle fibers were arranged disorderly,edema,and infiltrated by inflammatory cells.Compared with the Sham group,the levels of cTnI,NT-proBNP,Mb,IL-6,TNF-? in serum,mRNA levels of IL-6 and TNF-? in heart tissues,and MPO levels were significantly higher in the CLP group(p < 0.05).Compared with the CLP group,the damage of cardiac tissue structure in the CLP + rSj-Cys group was significantly reduced,and the levels of cTnI,NT-proBNP,Mb,IL-6 and TNF-? in serum were significantly reduced,while the concentrations of IL-10 and TGF-? conspicuously increased(p < 0.05).Compared with the Sham group,the mice in the rSj-Cys group had no significant difference in cardiac tissue structure and cardiac function,and the levels of IL-10 and TGF-? factor were significantly increased(p < 0.05).3.Compared with the Medium control group,the levels of pro-inflammatory factors IL-6 and TNF-? in the cell supernatant,the apoptosis rate and the protein expression of MyD88 in the cells increased in the LPS group.The levels of IL-6 and TNF-? in cell supernatant and the protein expression of MyD88 in the cells decreased in LPS + rSj-Cys group in comparison to the LPS group.However,there was no significant difference in the levels of IL-6 and TNF-?,the apoptosis rate and the protein expression of MyD88,between the Medium control group and rSj-Cys group.Conclusions: 1.rSj-Cys can reduce LPS-induced cardiomyocyte secretion of pro-inflammatory factors and apoptosis.2.rSj-Cys can promote the transformation of M1 type macrophages into M2 type macrophages.3.Reduce the secretion of pro-inflammatory factors by down-regulating the activity of MyD88 signaling pathway to improve sepsis-induced cardiac dysfunction.