摘要
目的 探讨1,25(OH)2D3介导维生素D受体(VDR)表达对髓核细胞氧化应激损伤的影响.方法 将人椎间盘髓核细胞随机分为对照组、H2O2组(400 µmol·L-1 H2O2)、1,25(OH)2D3组[100.00 nmol·L-1 1,25(OH)2D3+400μmol·L-1H2O2],1,25(OH)2D3+si-NC 组[100.00 nmol·L-1 1,25(OH)2D3+400 μmol·L-1 H2 O2+si-NC],1,25(OH)2 D3+si-VDR 组[100.00 nmol·L-1 1,25(OH)2D3+400 μmol·L-1H2O2+si-VDR].以噻唑蓝(MTT)法检测细胞存活率,以蛋白质印迹法检测各组细胞蛋白表达水平,以流式细胞术、JC-1荧光探针法、DCFH-DA探针法分别检测细胞凋亡、线粒体膜电位、活性氧(ROS)水平,以试剂盒检测丙二醛(MDA)和超氧化物歧化酶(SOD)水平.结果 对照组、H2O2组、1,25(OH)2D3组、1,25(OH)2D3+si-NC 组和 1,25(OH)2 D3+si-VDR组的细胞存活率分别为(100.00±2.43)％、(63.79±5.21)％、(90.11±7.24)％、(88.79±6.48)％和(55.31±4.65)％,VDR 蛋白相对表达水平分别为 0.79±0.06、0.28±0.03、0.68±0.05、0.69±0.06和0.34±0.04,细胞 凋亡率 分别为(4.12±0.26)％、(19.37±1.21)％、(8.49±0.57)％、(8.23±0.60)％和(14.68±1.37)％,线粒体膜电位水平分别为(100.00±4.31)％、(49.46±5.02)％、(82.14±7.02)％、(83.14±5.12)％和(67.16±5.48)％,ROS 水平分别为 1.79±0.12、7.98±0.51、3.87±0.34、3.92±0.22 和5.79±0.28,Beclin-1 蛋白相对表达水平分别为 0.86±0.09、0.35±0.04、0.76±0.07、0.75±0.08和0.46±0.05,LC3-Ⅱ/LC3 Ⅰ 蛋白相对表达水平分别为 1.00±0.07、0.25±0.04、0.78±0.07、0.85±0.08 和 0.42±0.05.H2O2组的上述指标与对照组比较,1,25(OH)2D3组的上述指标与H2O2组比较,1,25(OH)2D3+si-VDR组的上述指标与1,25(OH)2D3+si-NC组比较,在统计学上差异均有统计学意义(均P＜0.05).结论 1,25(OH)2D3可能上调VDR表达促进髓核细胞自噬、抑制细胞凋亡,发挥抗氧化应激损伤.
Abstract
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.
基金项目
广东省卫生健康委医学科研基金资助项目(B2022061)
国家科技期刊平台
NSTL
万方数据