Effects Of Norcantharidin On The Apoptosis Of Human Mesangial Cells Induced By High Concentration Of Fetal Bovine Serum

Objective:This study was to determine the effect of norcantharidin(NCTD)on the proliferation and apoptosis in human glomerular mesangial cells(HMCs)and its mechanism.Methods:1 MTT assay: By detecting the effect of the gradient concentration fetal bovine serum on the proliferation of HMCs,the best time and concentration of promoting the proliferation of HMCs were determined.2 Hoechest 33258 assay: through the observation of the cell morphology changes under fluorescence microscope to identify the effects of NCTD on apoptosis of HMCs after different time.3 JC-1 fluorescence staining assay: mitochondrial membrane potential changes of HMCs which effected by NCTD in 12 h were determined by fluorescence microscopy and flow cytometry.4 ELISA assay:the expression of Cyt-C in the cytoplasm of HMCs which have been effected by NCTD for 12 h were detected to explore the pathway of apoptosis of HMCs initially.Results:1 The MTT experiment showed that the proliferation of HMCs induced by FBS was time-dependent and dose-dependent.The cell viability of 12 h treated with 25%FBS or 30%FBS was better than the other group and the proliferation rate is(61.13±4.73)% and(68.10±9.15)%,there is no significant deviation between the two concentration.Compared with the blank control group,the difference was significant(P < 0.05).Otherwise,There is a significant time-effect and dose-effect relationship of the inhibition on the growth of HMCs by NCTD.IC50 is 10.03ug/ml of NCTD at the point of 24 h and 7.58ug/ml at the point of 48 h calculated with nonlinear fitting.2 Hoechest 33258 experiments showed that the cells of control group and proliferation group displayed uniform size,and the HMCs of NCTD group with different concentrations was presented by nuclear condensation and apoptotic bodies.The apoptosis rate of 2.5ug/ml NCTD treated for 6h,12 h,24h was 4.63 ± 1.55%,5.22 ± 2.27% and 10.55 ± 3.41%.compared with the control group and the proliferation group there were statistically significant diviation(P<0.05);The apoptosis rate of 5ug/ml NCTD treated for 6h,12 h,24h was 15.61±4.67%,18.13±2.78%,31.51±7.64%.compared with the control group(1.49±0.47%)and the proliferation group(1.39±0.85%)there were statistically significant diviation(P<0.05);The apoptosis rate of 10ug/ml NCTD treated for 6h,12 h,24h was 33.24±13.15%?45.53±6.24%?78.92±11.12%.compared with the control group(5.48±3.24%)and theproliferation group(8.12±10.55%)there were statistically significant diviation(P<0.05).This assay revealed that NCTD can induce apoptosis of HMCs,and the apoptosis rate was time-dependently and dose-dependently increased.3 JC-1 fluorescence staining results showed that compared with control group and proliferation group,mitochondrial membrane potential of HMCs with NCTD treated for 12 h were shown for significantly Decrease.changes of mitochondrial membrane potential of HMCs were detected by flow cytometry.compared with the control group(1.71 + 0.25),JC-1 polymer /JC-1 monomer value of HMCs in each experimental group decreased significantly in a concentration dependent manner(0.47±0.14,0.37±0.19,0.22±0.11,0.14±0.04),(P < 0.05),but the difference between each experimental group is not significant(P >0.05).4 Cyt-C ELISA experiment showed that,compared with the control group and the proliferation group,the release of cytochrome C can significantly increase in the cytoplasm of HMCs in experiment groups after 12 h NCTD treated.The cytochrome C were 15.95±0.29,17.96±0.28,43.98±2.10,56.07±2.35 of experiment groups respectively,compared with the proliferation group(0.99±1.40)were statistically significant(P < 0.05).However,compared with the control group(11.35±6.22),2.5?g/ml and 5?g/ml doses of NCTD groups were not statistically significant,while 10 ?g/ml and 20 ?g/ml dose of NCTD groups were statistically significant(P < 0.05).This suggests thatmitochondrial membrane permeability may be destroyed by NCTD,causing the release of cytochrome C,which induce the apoptosis of HMCs.Conclusion:1 HMCs were in time and dose-dependent significantly proliferation was found after induced by gradient high concentration of fetal bovine serum,With the intervention of 25%FBS 12 h is preferred.2 within a certain range of time and concentration NCTD can inhibit proliferation and induce apoptosis of HMCs which induced by high concentration FBS in time and dose-dependence significantly.3 NCTD can reduce the mitochondrial membrane potential of HMCs.the apoptosis program of HMCs were started by the release of Cyt-C from mitochondrial to cytoplasm.