Protective Effect And Intervention Mechanism Of TsadSPI On Acute Kidney Injury Induced By Sepsis In Mice

Background:Acute Renal Injury(AKI)and sepsis are complex diseases with similar risk factors.Sepsis is the most common trigger for AKI,about 50% of AKI patients are associated with sepsis,and the mortality rate of sepsis-associated acute kidney injury(SA-AKI)reaches 70%.In view of the high mortality rate,the treatment of sepsis should give priority to the targeted treatment of SA-AKI.However,at present,there is still no better treatment for SA-AKI.Therefore,it is particularly important to study the mechanism of renal damage in sepsis and to find new treatment strategies for SA-AKI.A large number of epidemiological datas and experimental studies have confirmed that “Helminth therapy”,that is,helminth and their derivatives can be used in the treatment of metabolic disease and inflammatory diseases.TsadSPI(recombinant adult serine protease inhibitor from Trichinella spiralis)is a serine protease inhibitor screened from T.spiralis.It has been proved that this protein can regulate immune response in vitro and treat inflammatory diseases by regulating Th1/Th2 balance and inducing Treg cells amplification in vivo.However,the mechanism of TsadSPI on renal immune regulation in patients with SA-AKI has not been reported.Therefore,this subject induced the SA-AKI model of BALB/c mice by cecal ligation and puncture(CLP)to observe the effect of sepsis on the kidney,and then intervened with TsadSPI to explore whether this protein has protective effect on kidney and its specific mechanism.The purpose of this study is to provide theoretical basis and experimental support for TsadSPI to become a new immunosuppressive drug for the treatment of SA-AKI.Objective:The main purpose of this study was to investigate the protective effect of TsadSPI on SA-AKI.The mechanism of renal injury during sepsis were studied by observing the general state,liver and kidney function,liver and kidney pathological manifestations of septic mice,combined with the changes of pro-inflammatory cytokines and immunomodulatory cytokines,the expression levels of Myd88(myeloid differentiation factor 88)/ NF-kappa B(nuclear factor-κB p65)in the kidney tissues,improving the molecular mechanism of “Helminth therapy” to play the role of immune regulation.Methods:1.Screening,expression and concentration determination of TsadSPI: According to the sequence number(EU263307.1)of TsadSPI in Gen Bank,the corresponding gene was synthesized by PAS(PCR-based Accurate Synthesis).After being double digested by Nde I and Xba I,it was connected to the expression vector p Czn1.The successfully constructed p Czn1-TsadSPI was transferred into the expression bacteria Arctic Express.IPTG induced the expression of the target protein.The target protein was purified and analyzed by 12% SDS-PAGE.The endotoxin of the purified protein was removed,and the protein concentration was determined and stored at-80 ℃.2.Establish SA-AKI mouse model: In this experiment,BALB/c male mice were selected and SA-AKI model was established by CLP method.Eighteen BALB/c mice were stochastically divided into the sham operation group(Sham+PBS group),the model group(CLP+PBS group)and the TsadSPI treatment group(CLP+TsadSPI group),n=6 in each group of 18.In the Sham group,it was only exploratory laparotomy without ligating or perforating the cecal.At 30 minutes after operation,the sham operation group and the model group were intraperitoneally injected with PBS(100 μl),the TsadSPI treatment group was intraperitoneally injected with PBS(100 μl)containing TsadSPI(2 μg).The mice were killed after12 hours.3.The protective effect of TsadSPI on SA-AKI and its immune mechanism: After 12 hours,the therapeutic effect and immune mechanism of TsadSPI on SA-AKI were evaluated by observing the general state of mice;the damage of tissue structure and function of liver and kidney;the changes of pro-inflammatory cytokines(TNF-α,IL-6)and immunomodulatory cytokines(IL-10,TGF-β)in the serum;and the expression levels of Myd88 / NF-κB in the kidney tissue.Results:1.General state: After 12 hours,the mice in the Sham group moved freely and had no obvious changes in general state.While after CLP,the mice were lazy and less active,slow to respond,their bodies were not comfortable shaking and kept warm together,yellowish secretions could be seen around the corners of the eyes,it was difficult to open their eyes and unable to eat and drink.After TsadSPI treatment,the general state of the mice was better than that of the CLP model group,with a little activity and less yellowish secretions in the corners of the eyes,but they were still unable to eat and drink normally.2.Changes of hepatic and renal function in mice: Compared with the Sham group,the levels of liver function ALT,AST,renal function Cr and BUN increased significantly after CLP(P < 0.001),and decreased significantly after TsadSPI intervention(P < 0.001).3.HE results of liver and kidney sections of mice: After CLP,obvious damage occurred in the tissue structure of liver and kidney.Compared with the Sham group,hepatic cords lost normal arrangement structure,hepatic sinusoid congestion,hepatocyte edema,loose cytoplasm,a large number of inflammatory cell infiltration;glomerular ischemia and shrinkage,loss of normal shape,enlargement of glomerular-capsule space,increased exudation of inflammatory cells and swelling of renal tubular epithelial cells in the renal tissue.After TsadSPI treatment,the degree of liver and kidney injury were significantly better than that of CLP group.The structures of hepatic lobule were slightly damaged,the cell edema was alleviated;the glomerular shrinkage was improved,and the glomerular-capsule space were no significant enlarged.4.Changes of TNF-α,IL-6,IL-10 and TGF-β levels in mice: After CLP,the levels of pro-inflammatory cytokines TNF-α and IL-6 were higher than those in the Sham group(P < 0.001),and TNF-α and IL-6 showed a remarkable reduction after TsadSPI treatment(P < 0.001).The levels of IL-10 and TGF-β in mice treated with TsadSPI were significantly higher than those in the CLP group(P < 0.001).5.The expression level of Myd88 in the kidney tissue: Myd88 is a cytoplasmic inflammatory mediator.The cytoplasmic Myd88 in the model group showed a higher expression than that in the Sham group(P < 0.001),and its expression levels after TsadSPI treatment were decreased significantly(P < 0.001).6.The expression level of NF-κB p65 in the kidney tissue: The nuclear positive rate of NF-κB p65 is the proportion of the number of cells with brown granules in the nucleus of the kidney tissue in the total number of cells.After CLP,NF-κB p65 brown granules appeared in the cytoplasm and nucleus of the renal tissue.After TsadSPI treatment,the brown granules in cytoplasm and nucleus were significantly decreased,and the NF-κB p65 nuclear positive rate was significantly lower than that in the CLP group(P < 0.001).Conclusion:TsadSPI can reduce the expression of Myd88 and the nuclear positive rate of NF-κB p65 in the renal tissue,up-regulate the expression of immunomodulatory cytokines and down-regulate the expression of pro-inflammatory cytokines,thereby protecting the kidney from sepsis.It has the potential to become a new immunosuppressive drug for the treatment of SA-AKI.