The Effect Of Hypoxia Induced By Cobalt Chloride On Apoptosis Of Renal Tubular Epithelial Cells Induced By Vancomycin

Objective:This study used Vancomycin（VAN）to intervene renal tubular epithelial cells（HK-2）to explore the mechanism of VAN nephrotoxicity and the effect of hypoxia induced by cobalt chloride on HK-2 cells proliferation and apoptosis,and to explore the role of hypoxia inducible factor 1α（HIF-1α）,and then further to find a theoretical basis for the clinical efficacy of VAN nephrotoxicity treatment.Methods:Human proximal renal tubular epithelial cells（HK-2）were selected as the study object,the concentration and time of VAN and and CoCl₂were selected for CCK8cytotoxicity assay,and were divided into Control group,VAN group（40μg/m L VAN for48 h）and VAN+CoCl₂group（50μmol/L CoCl₂pretreatment for 6 h+40μg/m L VAN for48 h）.By intervening HK-2 cells with VAN and CoCl₂observe the effects on the growth and apoptosis of HK-2 cells.（1）CCK8 cytotoxicity assay was performed to observe the effect of VAN and CoCl₂on the proliferation of HK-2 cells;（2）flow cytometry was used to observate the apoptosis rate of HK-2 cells intervened by VAN and CoCl₂;（3）RT-PCR and Western Blot detection of CoCl₂on the expression of HIF-1αand apoptosis-related genes（Caspase-3,Bcl-2）in HK-2 cells.Results:（1）The results of CCK-8 assay for cell suppression rate showed that the effect of VAN and CoCl₂on cell proliferation is concentration-dependent,the higher the concentration,the stronger the inhibitory effect.40μg/m L VAN and 50μmol/L CoCl₂were selected as the drugs dose of this experiment.（2）The analysis of apoptosis rate by flow cytometry showed that the apoptosis rate of VAN group was higher than Ctrl group（35.5%vs 3.13%,P<0.05）.The apoptosis rate of VAN+CoCl₂group was lower than Ctrl group（18.9%vs 3.13%,P<0.05）.The apoptosis rate of VAN group was higher than VAN+CoCl₂group（18.9%vs 35.5%,P<0.05）.（3）PCR and Western blot results showed that there was no significant difference in the expression of HIF-1αm RNA in VAN group compared with Ctrl group（P>0.05）.Compared with Ctrl group and VAN group,the expression of HIF-1αin VAN+CoCl₂group was up-regulated（P<0.05）.Compared with Ctrl group,the expression of Caspase-3 in VAN group and VAN+CoCl₂group was up-regulated（P<0.05）.Compared with VAN group,the expression of Caspase-3 in VAN+CoCl₂group was down-regulated（P<0.05）.Compared with Ctrl group,the expression of Bcl-2 in VAN group was down-regulated（P<0.05）.Compared with Ctrl group and VAN group,the expression of Bcl-2 in VAN+CoCl₂group was up-regulated（P<0.05）.Conclusion:（1）VAN can inhibit the growth and proliferation of HK-2 cells,and it has direct relationship with the apoptosis of cells and the expression of apoptotic proteins.（2）High concentration cobalt chloride can inhibit the growth and proliferation of HK-2cells.At appropriate range,the effects of hypoxia induced by cobalt chloride can regulate the expression of apoptosis related proteins and mitigate apoptosis induced by VAN.This process may be related to the up regulation of HIF-1αmediated by cobalt chloride.