Effects of 1,25(OH)2 D3 regulation of vitamin D receptor expression on H2 O2-induced oxidative stress injury in nucleus pulposus cells
Objective To investigate the effect of 1,25(OH)2 D3-mediated vitamin D receptor(VDR)expression on oxidative stress injury in nucleus pulposus cells.Methods Human interdisc nucleus pulpocytes were randomly divided into control group,H2O2 group(400 µmol·L-1H2O2),1,25(OH)2 D3 group[100.00 nmol·L-11,25(OH)2D3+400 μmol·L-1H2O2],1,25(OH)2D3+si-NC group[100.00 nmol·L-1 1,25(OH)2D3+400 μmol·L-1 H2O2+si-NC],1,25(OH)2D3+si-VDR group[100.00 nmol·L-1 1,25(OH)2D3+400 μmol·L-1 H2O2+si-VDR].Cell survival rate was measured by methyl thiazolyl tetrazolium(MTT)assay.Western blot assay was used to detect the protein expression level of each group.Apoptosis,mitochondrial membrane potential and reactive oxygen species(ROS)levels were detected by flow cytometry,JC-1 fluorescence probe and DCFH-DA probe,respectively.Malonclialdehyde(MDA)and superoxide dismutase(SOD)levels were detected with the kit.Results The cell survival rates of control group,H2O2 group,1,25(OH)2D3 group,1,25(OH)2D3+si-NC group and 1,25(OH)2D3+si-VDR group were(100.00±2.43)％,(63.79±5.21)％,(90.11±7.24)％,(88.79±6.48)％and(55.31±4.65)％;VDR protein levels were 0.79±0.06,0.28±0.03,0.68±0.05,0.69±0.06 and 0.34±0.04;the apoptosis rates were(4.12±0.26)％,(19.37±1.21)％,(8.49±0.57)％,(8.23±0.60)％and(14.68±1.37)％;mitochondrial membrane potential levels were(100.00±4.31)％,(49.46±5.02)％,(82.14±7.02)％,(83.14±5.12)％and(67.16±5.48)％;ROS levels were 1.79±0.12,7.98±0.51,3.87±0.34,3.92±0.22 and 5.79±0.28;Beclin-1 levels were 0.86±0.09,0.35±0.04,0.76±0.07,0.75±0.08 and 0.46±0.05;LC3-Ⅱ/LC3Ⅰ levels were 1.00±0.07,0.25±0.04,0.78±0.07,0.85±0.08 and 0.42±0.05,respectively.Compared H2 O2 group with control group,compared 1,25(OH)2 D3 group with H2O2 group,compared 1,25(OH)2D3+si-VDR group with 1,25(OH)2D3+si-NC group,the differences of the above indicators were all statistically significant(all P＜0.05).Conclusion 1,25(OH)2 D3 may up-regulate the expression of VDR,promote autophagy and inhibit apoptosis of nucleus pulposus cells,and play an anti-oxidative stress role.
1,25(OH)2D3nucleus pulposus cellsvitamin D receptoroxidative stressautophagy
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