Protective Effect And Mechanism Of Panax Notoginseng Saponins On Renal Tubular Epithelial Cells Damaged By Cisplatin

Objective: Cisplatin is a first-line chemotherapeutic agent for clinical tumor treatment,but has severe side-effects.Over 30% of tumor patients have been found to occur acute kidney injury(AKI)after cisplatin treatment.Although several studies have elucidated the pathogenesis for cisplatin-induced AKI,there is still short of drug to effectively treat cisplatin-induced AKI.Panax notoginseng saponins(PNS),a major active ingredient isolated from the natural medicinal plant Sanqi,has effects of anti-oxidation,anti-inflammation and inhibition of cell apoptosis.Our previous study established the rat model of acute renal injury,and found that PNS could protect the rats from cisplatin-induced renal injury,which may be involved with the protective effect against cisplatin-induced AKI by the HIF-1α pathway-mediated mitochondrial autophagy activation.However,the specific cellular and molecular mechanisms have not been elucidated.In the current study,we established an in vitro cisplatin-induced AKI model on renal tubular epithelial cells.HIF-1α-low-expressed renal tubular epithelial(HK-2)cells were constructed by RNA interference(RNAi)technology,followed by medicine intervention of PNS,in the aim to clarify the protective mechanism of PNS on renal tubular epithelial cells.Methods:1.Determination of the proliferation and apoptosis of cisplatin-damaged HK-2 cells after PNS treatment:Normal or HIF-1α si RNA transfected HK-2 cells under the logarithmic growth phase were grouped into the normal cells group,cisplatin(CDDP)group,CDDP+PNS group,Control si RNA group,HIF-1α si RNA transfection+cisplatin(HIF-1α si RNA+CDDP)group,HIF-1α si RNA transfection+cisplatin+PNS(HIF-1α si RNA+CDDP+PNS)group,then treated with 6.25 g/m L of cisplatin and/or 6.25 mg/m L of cisplatin.After incubation for 12 h or 24 h in vitro,proliferation activity of the cells was detected by CCK-8 assay,whereas the apoptosis rate was detected by flow cytometry.Expression levels of apoptosis-related proteins including Cytochrome C,caspase-3,Bax and Bcl2 were determined by Western Blot assay.2.Determination of mitochondrial function in cisplatin-damaged HK-2cells:Normal or HIF-1α si RNA transfected HK-2 cells were treated with the indicated drugs as above grouping and procedures.The mitochondrial membrane potential(MMP)of each group was detected by flow cytometry.Mitochondrial ATP content in each group was detected by using the Mitochondrial ATP kit.3.Investigation on the molecular mechanism about the protective effect of PNS on cisplatin-damaged HK-2 cells:Normal or HIF-1α si RNA transfected HK-2 cells were treated with the indicated drugs as above grouping and procedures.The expression levels of HIF-1α and BNIP3 in each group were detected by Western Blot assay and the cellular immunofluorescence assay.Results:1.PNS has pro-proliferation and anti-apoptotic effects on cisplatin-damaged HK-2 cells in vitro:Compared with the normal cell control group,the cell activity in CDDP group was inhibited,while the apoptotic rate was increased.And the expression of pro-apoptotic proteins Cytchrome C,Cleaved caspase-3 and Bax were increased,while expression of anti-apoptotic protein Bcl2 was decreased,and the ratio of Bax/Bcl2 was increased.Compared with CDDP group,CDDP+PNS group showed increased cell activity,decreased cell apoptosis rate,and down-regulated expression of Cytchrome C,Cleaved caspase-3 and Bax,but upregulated expression of Bcl2.The ratio of Bax/Bcl2 was decreased.After knockdown of HIF-1α expression in HK-2,the cell activity of HIF-1αsi RNA+CDDP group was further decreased,the expression of Cytchrome C,Cleaved caspase-3 and Bax protein were further increased,the expression of Bcl2 protein was further decreased,and the ratio of Bax/Bcl2 was increased,when compared with CDDP group.Cytchrome C,Cleaved caspase-3,and Bax protein expressions were decreased in the HIF-1αsi RNA+CDDP+PNS group compared with the HIF-1α si RNA+CDDP+PNS group,while Bcl2 protein expression was increased,and the Bax/Bcl2 ratio was decreased.2.PNS promotes mitochondrial ATP production and membrane potential recovery in cisplatin-damaged HK-2 cells in vitro:Compared with normal cell control group,ATP and MMP levels in CDDP group were significantly decreased.ATP and MMP levels were both higher in CDDP+PNS group than in CDDP group.After knockdown of HIF-1αexpression,the levels of ATP and MMP were further significantly decreased in HIF-1α si RNA+CDDP group compared with CDDP group.Compared with the HIF-1α si RNA+CDDP group,the levels of ATP and MMP were significantly increased in the HIF-1α si RNA+CDDP+PNS group.3.Molecular mechanisms of PNS protection against cisplatin-induced damage in HK-2 cells:Compared with normal cell control group,the protein expressions of HIF-1α and BNIP3 in CDDP group were significantly increased at 12 h and 24 h,whereas the protein levels of HIF-1α and BNIP3 at 24 h were higher than those at 12 h.Compared with CDDP group,HIF-1α and BNIP3 protein expressions in CDDP+PNS group were further increased.HIF-1α expression was down-regulated in both HIF-1α si RNA+CDDP and HIF-1α si RNA+CDDP+PNS groups after knockdown of HIF-1α gene.Compared with CDDP group,the protein levels of HIF-1α and BNIP3 in HIF-1α si RNA+CDDP group were significantly decreased.Compared with HIF-1α si RNA+CDDP group,the levels of HIF-1α and BNIP3 protein in HIF-1α si RNA+CDDP+PNS group were increased at 24 h.Conclusion:1.PNS could effectively inhibit the cisplatin-induced apoptosis and promote the cell viability recovery and proliferation of the renal tubular epithelial cells.2.PNS could effectively reduce the cellular mitochondrial damaged by cisplatin.3.PNS might play a protective role in human proximal renal tubular epithelial cells damaged by cisplatin through regulating the HIF-1α/BNIP3 pathway.