Protective Effect Of Schisandrinb On Cisplatin Induced Human Kidney Proxiamal Tubular Epithelial Cells Apoptosis And Its Possible Mechanism

Objective: This experiment is to observe the protective effect of low,medium and high concentration of schisandrin B(Sch B)on cisplatin induced human proximal tubular epithelial cells(HK-2)apoptosis in vitro,and to explore its potential mechanism of action.Methods: Human renal proximal tubule epithelial cells were cultured in vitro and divided into five groups:control group:HK-2 does not do any dosing;CDDP group: in which cells were incubated with 50μmol/L CDDP for 24 hours;Sch B pre-incubation group(CDDP +Sch B): 5μmol/L,10μmol/L and 20μmol/L Sch B were added to HK-2 medium for 2 hours pre-incubation and then 50μmol/L CDDP continued to be incubated for 24 hours.CCK-8 kit were used to detect the cell viability of five groups.Flow cytometry was used to detect the apoptotic rate of five groups.The morphology of cells was observed by inverted microscope.The protein expressions of P21、Apaf-1 and Caspase-3 were assessed by Western blot assay.The expression of Bcl-2 m RNA was determined by RT-PCR.The expression of P21 was detected by immunofluorescence.Results: The results of CCK-8 showed that HK-2 activity was significantly decreased in cisplatin group compared with ctrl group.The activity of HK-2 in Sch B group was significantly higher than that in cisplatin group,and the activity of HK-2 was dependent on Sch B concentration.The apoptotic rate of HK-2 in cisplatin group was significantly higher than that of ctrl group,and the apoptotic rate of Sch B pre-incubation group was significantly higher than that of cisplatin group.The apoptotic rate of 10μmol/L and 20μmol/L Sch B pre-incubation group was significantly different from that of cisplatin group.The number of HK-2 in cisplatin group was significantly lower than that in ctrl group which was observed by inverted microscope.The number of HK-2 in the Sch B group was significantly higher than that in the cisplatin group.The number of HK-2 increases stepwise in the form of Sch B concentration.Compared with the control group,the expression of Apaf-1 and cleaved caspase-3 in cisplatin group increased significantly and the expression of P21 protein was increased.The expression of Apaf-1 and cleaved caspase-3 in SchB pre-incubation group was significantly lower than that in cisplatin group,while the expression of P21 protein increased significantly,There was significant difference between medium and high concentrations of Sch B pre-incubation group and the cisplatin group.The expression of Bcl-2 m RNA in cisplatin group was significantly lower than that in control group,and the expression of Bcl-2 m RNA in Sch B pre-incubation group was significantly higher than that in cisplatin group.10μmol/L and 20μmol/L Sch B pre-incubation group were significantly different from the cisplatin group.Immunofluorescence assay showed that P21 in cisplatin group was expressed in the nucleus and showed a slight distribution in the nucleus,and no expression or deletion in some regions.The expression of P21 in Sch B pre-incubation group was significantly enhanced than that in cisplatin group in granular distribution in the nucleus.Conclusion: 5μmol/L、10μmol/L and 20μmol/L Sch B have protective effects on cisplatin-induced apoptosis of HK-2 cells.The mechanism may be related t o the up-regulation of Bcl-2 and down-regulation of Apaf-1 and Caspase-3,an d inhibition of mitochondrial pathway apoptosis,and it was also related to the up-regulation of P21 and the inhibition of cyclin-dependent kinase.The protecti ve effect of 10μmol/L and 20μmol/L Sch B on HK-2 cell apoptosis was more obvious.