Study On The Role Of Autophagy In The Apoptosis Of Human Kidney Tubular Epithelial HK-2 Cells Induced By Triptolide

Objective:Triptolide(TP)is one of the major active components of Tripterygium wilfordii Hood.F(TWHF),which possesses multiple pharmacological activities including anti-inflammatory,anti-oxidative,immunomodulatory and anti-tumor.Meanwhile,TP has been verified that there are serious toxicity in heart,liver and kidney.Studies showed that the toxic effects of TP were closely related to the induction of membrane damage,mitochondrial injury,endoplasmic reticulum stress and so on,leading to metabolic dysfunction and oxidative stress,and eventually resulting in apoptosis.It has been reported that TP could induce apoptosis in Human Kidney Tubular Epithelial HK-2 cells,but the mechanisms underlying nephrotoxicity are still not fully understood.Autophagy exists widely in eukaryotic cells and plays a crucial role in the maintenance of intracellular homeostasis.The imbalance of autophagy regulation is closely related to the occurrence of a variety of diseases.Therefore,the effect and relationship of apoptosis and autophagy were explored by treating HK-2 cells with TP in vitro.A novel insight and theoretical basis were provided for the prevention and treatment of TP nephrotoxicity.Methods:1.The viability,morphology and apoptosis rates of HK-2 cells treated with TP were detected by CCK-8 assay,AO/EB staining and Annexin V-FITC /PI double staining assay,respectively.Meanwhile,the effect of TP on the mitochondrial membrane potential in HK-2 cells was detected by JC-1 fluorescent probe,and the expression of apoptosis-related proteins cleaved caspase 9,Bax and Bcl-2 were examined by Western blot.2.Activation of autophagy in HK-2 cells were observed by immunofluorescence,and the expression of autophagy marker proteins LC3Ⅱ,Beclin 1 and p62(SQSTM1)were measured by Western blot after TP-treated HK-2 cells.3.The effects of autophagy agonist rapamycin(Rap)or autophagy inhibitor3-methyladenine(3-MA)with TP on the expression of LC3Ⅱ and p62(SQSTM1)proteins and the cell viability were detected by Western blot and CCK-8 assay,respectively.The morphology and the rate of apoptosis in HK-2 cells treated with Rap or 3-MA and TP for24 h were detected by AO/EB staining and Annexin V-FITC/PI double staining assay,respectively.The effects of Rap or 3-MA with TP on the ratio of cleaved caspase 9/caspase9 in HK-2 cells were tested by Western blot.Results:1.TP significantly inhibited the viability of HK-2 cells in a concentration-and time-dependent manner.The results of AO/EB staining and Annexin V-FITC/PI double staining showed that the rate of apoptosis gradually elevated after HK-2 cells treated with different concentrations of TP for 24 h.In addition,the results of JC-1 fluorescence probe assay showed that TP could decrease the mitochondrial membrane potential in HK-2 cells.Western blotting analysis showed that TP could increase ratios of cleaved caspase9/caspase 9 and Bax/Bcl-2.2.Immunofluorescence staining showed that the fluorescence intensity and the punctate aggregation of LC3Ⅱ increased in the TP group.Western blot showed that TP could increase the ratio of LC3Ⅱ/LC3Ⅰ and the expression of Beclin 1,and decrease the expression of p62(SQSTM1).3.Western blot results showed that the ratio of LC3Ⅱ/LC3Ⅰ in cells treated with Rap and TP was higher than that treated with TP alone,and the expression of p62(SQSTM1)was further decreased,while the ratio of LC3Ⅱ/LC3Ⅰ in cells treated with 3-MA and TP was lower than that treated with TP alone,and the expression of p62(SQSTM1)was further increased.The CCK-8 assay showed that Rap-induced autophagy could decrease the cell viability of HK-2 cells exposed to TP,while 3-MA-inhibited autophagy could increased the cell viability of HK-2 cells exposed to TP.AO/EB staining and Annexin V-FITC/PI double staining showed that Rap could enhance TP-induced apoptosis,while3-MA could reduce TP-induced apoptosis in HK-2 cells.Furthermore,Western blot results showed that the ratio of cleaved caspase 9/caspase 9 in HK-2 cells treated with Rap and TP was higher than TP group.On the contrary,the ratio of cleaved caspase 9/caspase 9 in HK-2 cells treated with 3-MA and TP was lower than TP group.Conclusion:CCK-8 assay showed that TP could induce toxicity in a dose-and time-dependent manner in HK-2 cells.And the results of AO/EB staining and Annexin V-FITC/PI double staining showed that TP could induce apoptosis in HK-2 cells.JC-1 fluorescent probe detection results showed that TP could reduce the mitochondrial membrane potential in HK-2 cells.Western blot results showed that TP could increase the ratios of cleaved caspase 9/caspase 9 and Bax/Bcl-2,suggested that TP could regulate apoptosis in HK-2cells via mitochondrial pathway.In addition,immunofluorescence staining and Western blot showed that TP could induce autophagy in HK-2 cells.The autophagy agonist Rap increased cytotoxicity and apoptosis in HK-2 cells.Autophagy inhibitor 3-MA decreased cytotoxicity and apoptosis in HK-2 cells.It suggested that we could reduce the nephrotoxicity caused by TP by autophagy inhibition.