| Background:Chronic drug nephrotoxicity,as one of the side effects of calcineurin inhibitor(CNI)drugs,is a fatal shortcoming of current immunosuppressive programs.Longterm drug toxicity damage can cause focal glomerular sclerosis,renal tubular atrophy,and kidney transplantation.Changes in interstitial fibrosis,etc.,eventually lead to the failure of the transplanted kidney.Up to now,we have mastered a lot of experience in the application of bone marrow mesenchymal stem cells(BMSCs)in clinical treatment.However,its ability to target homing the lesion organs and to survive in the hypoxicischemic microenvironment are both still poor,which can be significantly improved by pretreatment methods such as erythropoietin(EPO)and Heme Oxygenase-1(HO-1),etc.The SIRT1 gene was found not only to participate in regulating the activity of key cell proteins,but also to control cell proliferation,differentiation,apoptosis,antioxidative stress and other cellular physiological and pathological processes.EPO can promote protection by up-regulating the activity of SIRT1.Purpose:In this experiment,a cyclosporine A(CsA)toxicity injury model was constructed through in vitro experiments to simulate the in vivo CsA chronic nephrotoxic fibrosis injury environment,and to verify and explore the following research purposes(1)It is clear that EPO pretreatment enhances the effect of BMSCs against CsA toxic damage;(2)Explore the role of SIRT1 in EPO pretreatment of BMSCs against CsA toxic damage;(3)Explore the mechanism of SIRT1 in EPO pretreatment of BMSCs against CsA toxic damage;(4)To open up new ideas and new measures for the clinical application of BMSCs in the clinical treatment of CsA chronic nephrotoxic fibrosis,prompting to provide a solid and credible experimental data basis and theoretical basis.Methods:First,by comparing with the BMSCs of the control group and the BMSCs of the CsA toxic injury group,after pretreating the BMSCs with EPO,the cytotoxic activity,apoptosis,fibrosis level,inflammatory response level and other indicators in the environment of CsA toxic injury were detected.It is clear that EPO pretreatment enhances the effect of BMSCs against CsA toxic damage.Then continue to use EPO pretreated BMSCs to specifically silence the expression of SIRT1 gene in BMSCs by siRNA small interference technology,and then detect its cytotoxicity,apoptosis,fibrosis level,and inflammation in the environment of CsA toxic injury The response level and other indicators,to explore the role of SIRT1 gene in EPO pretreatment of BMSCs against CsA toxic damage.Then,after EPO pretreatment of BMSCs and siRNA small interference technology specifically silenced the expression of SIRT1 gene in BMSCs,the changes in the autophagy level of BMSCs were detected,and it was clear that the EPO pretreatment of BMSCs mediated the up-regulation of autophagy level and the SIRT1 gene in EPO The role of pretreatment of BMSCs in mediating the up-regulation of autophagy levels.Finally,through the use of autophagy activators and inhibitors,the autophagy level of BMSCs pretreated by EPO is regulated,and then the level of fibrosis of BMSCs under CsA toxicity environment is used to verify the level of autophagy mediated by EPO pretreatment and BMSCs and EPO pretreatment.The relationship between the effects of BMSCs against CsA toxic damage.Finally,based on the experimental results,it is proposed to prove the scientific hypothesis:EPO pretreatment of BMSCs can mediate the enhancement of autophagy,increase cell survival rate,and resist the toxic damage effect of CsA drugs by upregulating the expression of SIRT1 gene.Results:1.BMSCs pretreated with EPO can up-regulate the expression of SIRT1 gene;2.BMSCs pretreated with EPO through regulation enhances the cell viability of BMSCs,reduces cell apoptosis,improves the degree of fibrosis,inhibits the occurrence of inflammation,and achieves resistance and alleviation of CsA’s drug toxicity and damage.3.BMSCs pretreated with EPO regulates the increase of autophagy level by upregulating the SIRT1 gene;4.BMSCs pretreated with EPO can mediate and improve the fibrosis level of BMSCs by up-regulating the level of autophagy activity,and resist the toxic and damaging effects of CsA.Conclusions:BMSCs pretreated with EPO mediates autophagy by up-regulating the expression of SIRT1 to improve cell survival rate and resist CsA toxicity damage. |
