Role And Mechanism Of CysRS In Pyroptosis Death Of Renal Tubular Epithelial Cells Induced By Ischemia-reperfusion Injure

Objective: Ischemia reperfusion injury(IRI)is a kind of is characterized by rapid drop in kidney function of potential fatal syndrome,as acute kidney injury(AKI)one of the main causes of closely associated with high morbidity and mortality,and lack of specific treatment methods,based on this we think the IRI caused by the specific pathogenesis of AKI and seeking early intervention treatment targets is very important.We previously screened significantly down-regulated molecular cysteinyl-tRNA synthetase(CysRS)in renal tubular epithelial cell hypoxia/reoxygenation model by label-free proteomics.CysRS are involved in various biological reactions such as mitochondrial functional metabolism,synthesis and decomposition of Adenosine triphosphate(ATP),and regulation of RNA translation.The role of CysRS in ischemia/reperfusion injury remains unclear.Therefore,this study will explore the possible mechanism of CysRS affecting ischemia-reperfusion injury,and lay a foundation for the study of IRI-induced AKI and the search for early intervention targets.Methods: 1.In vitro experiment: The hypoxia/reoxygenation(H/R)model of human renal tubular epithelial cells(HK-2)was established by using the MIC-101 hypoxia modular-incubation chamber system of Billups Rosenberg Company,USA.The expression of CysRS in HK-2 cells at different hypoxia time points was verified by Western blot.To investigate the mechanism of CysRS influencing H/R by affecting pyrotosis,Western blot was used to detect the expressions of pyrotosis related proteins Pro-Caspase 1,Caspase 1,Pro-IL-1β and NLRP3 in HK-2 cells at different hypoxia time points.The concentration of ATP and AMP at different time points of hypoxia was detected by colorimetric method.H/R cells were treated with pyroptosis agonist and pyroptosis inhibitor,and the pyroptosis activity and CysRS expression of HK-2 cells were observed under different treatment conditions.Sh RNA knockdown and overexpression of CysRS were used to detect the degree of pyroptosis by Western blot,and then agonists and inhibitors were used to observe the degree of pyroptosis.2.In vivo experiment: a mouse IRI model was successfully constructed by clamping bilateral renal arteries and veins of mice with non-invasive microvascular clip.CysRS were immunohistochemical staining was used to observe the changes of CysRS after ischemia at different time points.In vivo transduction of super purified adenovirus was used to conduct IRI modeling after CysRS knockdown in mice,and then the pyroptosis activity and the change of IRI degree were observed.Results: Both in vitro and in vivo experiments showed that with the extension of hypoxic-ischemia time,CysRS protein increased first and then decreased,and with the trend of pyroptosis activity and metabolism of ATP and AMP increased first and then decreased,the occurrence of pyroptosis could be affected by treatment with pyroptosis agonists and inhibitors.After CysRS were knocked down,the protein volume expression of some related indexes of pyroptosis could be reduced to a certain extent,and overexpression of CysRS could also affect the protein volume expression of some related indexes of pyroptosis to a certain extent.Conclusions: Renal tubular epithelial cells in ischemia-reperfusion injury when use disorders associated with the occurrence of pyroptosis and ATP,CysRS is a kind of direct participation in ATP metabolism of protein,its expression is closely related to the occurrence of pyroptosis.In the future,it may be possible to inhibit the pyrotosis of renal tubular epithelial cells,reduce renal tissue damage and delay renal function loss induced by IRI by regulating the expression of CysRS,so provide a new idea and target for the future management of IRI induced AKI.