1,25(OH)_(2)D_(3)调控维生素D受体表达对H_(2)O_(2)诱导的髓核细胞氧化应激损伤的影响  

Effects of 1,25(OH)_(2)D_(3) regulation of vitamin D receptor expression on H_(2)O_(2)-induced oxidative stress injury in nucleus pulposus cells

作　　者：沈哲[1] 蓝涛[1] 郭伟壮[1] SHEN Zhe;LAN Tao;GUO Wei-zhuang(Department of Spinal Surgery,the Second People’s Hospital of Shenzhen,Shenzhen 518035,Guangdong Province,China)

机构地区：[1]深圳市第二人民医院脊柱外科,广东深圳518035

出　　处：《中国临床药理学杂志》2024年第3期373-377,共5页The Chinese Journal of Clinical Pharmacology

基　　金：广东省卫生健康委医学科研基金资助项目(B2022061)。

摘　　要：目的 探讨1,25(OH)_(2)D_(3)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、H_(2)O_(2)组(400μmol·L^(-1 )H_(2)O_(2))、1,25(OH)_(2)D_(3)组[100.00 nmol·L^(-1) 1,25(OH)_(2)D_(3)+400μmol·L^(-1 )H_(2)O_(2)],1,25(OH)_(2)D_(3)+si-NC组[100.00 nmol·L^(-1) 1,25(OH)_(2)D_(3)+400μmol·L^(-1 )H_(2)O_(2)+si-NC],1,25(OH)_(2)D_(3)+si-VDR组[100.00 nmol·L^(-1) 1,25(OH)_(2)D_(3)+400μmol·L^(-1 )H_(2)O_(2)+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、H_(2)O_(2)组、1,25(OH)_(2)D_(3)组、1,25(OH)_(2)D_(3)+si-NC组和1,25(OH)_(2)D_(3)+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/LC3Ⅰ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。H_(2)O_(2)组的上述指标与对照组比较,1,25(OH)_(2)D_(3)组的上述指标与H_(2)O_(2)组比较,1,25(OH)_(2)D_(3)+si-VDR组的上述指标与1,25(OH)_(2)D_(3)+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)_(2)D_(3)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。Objective To investigate the effect of 1,25(OH)_(2)D_(3)-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,H_2O_(2) group(400 μmol·L^(-1) H_2O_2),1,25(OH)_2D_(3) group [100.00 nmol·L^(-1) 1,25(OH)_(2)D_(3)+400 μmol·L^(-1 )H_(2)O_(2)],1,25(OH)_2D_(3)+si-NC group [100.00 nmol·L^(-1) 1,25(OH)_(2)D_(3)+400 μmol·L^(-1) H_(2)O_(2)+si-NC],1,25(OH)_(2)D_(3)+ si-VDR group [100.00 nmol ·L^(-1)1,25(OH)_(2)D_(3)+ 400 μmol ·L^(-1)H_(2)O_(2) + 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,H_(2)O_(2) group,1,25(OH)_(2)D_(3)group,1,25(OH)_(2)D_(3)+ si-NC group and 1,25(OH)_(2)D_(3)+ 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 and0.42 ± 0.05,respectively.Compared H_(2)O_(2) group with control group,compared 1,25(OH)_(2)D_(3)group with H_(2)O_(2) group,compared 1,25(OH)_(2)D_(3)+ si-VDR group with 1,25(OH)_(

关 键 词：1 25(OH)_(2)D_(3) 髓核细胞 维生素D受体 氧化应激 自噬 

分 类 号：R977.24[医药卫生—药品]