Intervention Of Schistosoma Japonicum Cysteine Protease Inhibitor In Treatment Of Sepsis Induced By LPS In Mice And The Preliminary Study On Immunological Mechanism

Sepsis is a systemic inflammatory response syndrome formed by the organism infected with pathogenic microbes that even lead to shock and a high mortality rate of patient.However,there is no effective drug for sepsis.Therefore,it is particularly important to find a new effective drug for sepsis.In recent years,the relationship between helminth and sepsis has attracted much attention.It has been proved that helminth infection can effectively relieve systemic inflammatory response in sepsis.However,using the infection of helminth to treat sepsis is not accepted by the patient.Therefore,using worm-derived protein instead of helminth infection to treat sepsis not only avoid the shortcomings of the helminth infection but can play a therapeutic role.RSjCystatin is a highly effective inhibitor of cysteine protease of Schistosoma japonicum.As a worm-derived protein,it has been proved that it has some anti-inflammatory effects in vitro,but the mechanism of rSjCystatin in vivo has not been reported.Therefore,this subject is intended to use rSjCystatin to observe the therapeutic effect of sepsis and to explore its immunological mechanism,so as to provide experimental basis for the study of rSjCystatin as a new drug for the treatment of sepsis.Objective:The main purpose of this study was to observe the effect of rSjCystatin on sepsis and the difference between different time periods after treatment.Through the general condition of mice,survival rate,pathological evaluation of tissues and organs,combined with mice liver and kidney function,inflammatory factor and the change of immune regulatory factor,explore rSjCystatin for potential applications for the treatment of sepsis.Methods:(1)Express and purify rSjCystatins protein and determine the protein concentration.(2)Construct mouse sepsis model.24 mices were randomly divided into the normal control group(PBS group),sepsis group(LPS group)and treatment group(LPS+rSjCystatin group),observing the general state changes state and 72h-survival rate of mice after modeling.(3)Construct mouse sepsis model.48 mice were randomly divided into 6 groups:the normal control group(PBS group),rSjCystatin group(rSjCystatin group),12 h sepsis(LPS group)and 12 h treatment group(LPS + rSjCystatin group),24 h sepsis group(LPS group)and 24 h treatment group(LPS+rSjCystatin group).Kill the different group mices under the condition of anaesthesia at different time period after modeling,collect specimens: mice serum,liver,lung,kidney.HE staining the mices,liver,lung,kidney.ELISA detected levels of TNF-α,IL-6 and IL-10 in serum of mice,and the levels of ALT,AST,BUN and Cr in serum of mice were detected by automatic biochemical analyzer.Results:(1)The normal control group mice had good mental activities,normal activities,normal eating and water intake,smooth hair and bright color.In the sepsis group,the mice were depressed,the activity was reduced,the mice were cluttered,the body temperature was reduced,the body hair was less or no food,the body hair was disorderly and luster,the yellow and white secretions in the eyes of the mice increased,and some mice had bloody stools around the anus,and the 72h-survival rate was 0.The mices of rSjCystatin protein treatment group was better than that of the LPS group,although the symptoms of poor spirit,disheveled body hair,less activity and less food were also found,but the mice had no bloody stool in the perianal and less eye secretions,and the 72h-survival rate was 36%,which was significantly higher than that in the LPS group.(2)Obvious organ damage occurred in the sepsis group(12h and 24h),the hepatic cell cord have lose the normal structure,hepatocyte edema,and the inflammatory cells infiltrate hepatic lobule under the microscope.Part of glomus contracted kidney tubular cells,edema,lung markings thickening.The levels of ALT,AST,BUN,Cr,IL-6 and TNF-α in the serum are significantly higher than those in the control group.Although the IL-10 increased,there is no significant difference(p<0.05),and there is no significant difference between the groups at different time periods.(3)Compared with the control group,the levels of ALT,AST,BUN,Cr,IL-6,TNF-α in the serum of the treatment group(12h and 24h)are higher,and the level of IL-10 decreased(p<0.05),except for IL-6,there is no significant difference between the groups of different time groups.(4)Compared with 12 h and 24 h sepsis group,the damage of liver,kidney and lung tissue in 12 h and 24 h treatment group is significantly reduced,and the serum levels of ALT,AST,BUN,TNF-α,IL-6 decreased obviously,and IL-10 level increased significantly(p<0.05).(5)Compared with the control group,the rSjCystatin protein group had no significant difference in liver,kidney,lung tissue and liver and kidney function,and the level of IL-10 factor increased significantly(p<0.05).Conclusion : RSjCystatin protein can improve the general condition of sepsis,improve the survival rate of mice,ameliorate the pathological damage of organ tissue,reduce the level of TNF-α and IL-6 in serum,increase the level of immunomodulatory factor IL-10,and have a significant therapeutic effect on sepsis.