穿心莲内酯对TBHP诱导的椎间盘髓核细胞凋亡的作用及机制研究

Effect of andrographolide on TBHP-induced apoptosis of intervertebral disc nucleus pulposus cells and its mechanism

摘要:

目的 探讨穿心莲内酯（andrographolide，ANDRO）对叔丁基过氧化氢（tert-butyl hydroperoxide，TBHP）诱导的椎间盘髓核细胞（nucleus pulposus cells，NPC）凋亡的作用及其机制。方法 用TBHP诱导NPC建立细胞模型。将NPC分为5组：对照组、模型组（100 μmol/L TBHP）、ANDRO低剂量组（100 μmol/L TBHP+9 μmol/L ANDRO）、ANDRO中剂量组（100 μmol/L TBHP+18 μmol/L ANDRO）和ANDRO高剂量组（100 μmol/L TBHP+36 μmol/L ANDRO）。流式细胞术检测细胞凋亡和活性氧（reactive oxygen species，ROS）水平，ELISA试剂盒检测氧化应激相关指标超氧化物歧化酶（superoxide dismutase，SOD）、谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-PX）和过氧化氢酶（catalase，CAT）水平，JC-1探针法检测线粒体膜电位，Western blot检测动力相关蛋白1（dynamin-related protein 1，Drp1）、p-Drp1和线粒体融合蛋白2（mitofusin 2，Mfn2）的表达水平。结果 与对照组相比，模型组NPC凋亡率、ROS水平和线粒体膜电位下降细胞比例明显升高（均P=0.000），SOD、GSH-PX和CAT水平明显降低（均P=0.000），p-Drp1/Drp1水平明显上调（P=0.000），Mfn2蛋白表达明显下调（P=0.000）。与模型组相比，经ANDRO高剂量干预后，NPC凋亡率［（41.37±0.84）% vs.（26.36±1.33）%］、ROS水平［（42.62±0.71）% vs.（22.50±0.37）%］和线粒体膜电位下降细胞比例［（43.85±0.71）% vs.（24.96±1.04）%］明显降低（均P=0.000），SOD［（288.16±14.59） ng/mL vs.（658.87±13.22） ng/mL］、GSH-PX［（6.66±0.37） ng/mL vs.（15.66±0.56） ng/mL］和CAT［（40.89±1.04） pg/mL vs.（87.91±1.56） pg/mL］水平明显升高（均P=0.000），p-Drp1/Drp1水平（0.96±0.05 vs. 0.63±0.01）明显下调（P=0.001、0.000），Mfn2蛋白表达（0.39±0.01 vs. 0.64±0.02）明显上调（均P=0.000）。结论 ANDRO具有抑制TBHP诱导的NPC细胞凋亡的作用，其机制可能与调节线粒体功能，缓解氧化应激有关。

Abstract:

Objective To investigate the effect and mechanism of andrographolide（ANDRO） on intervertebral disc nucleus pulposus cell（NPC） apoptosis induced by tert-butyl hydroperoxide（TBHP）.Methods The cell model of NPCs induced by TBHP was established. NPCs were divided into five groups： control group，model group（100 μmol/L TBHP），low-dose ANDRO group（100 μmol/L TBHP+9 μmol/L ANDRO），middle-dose ANDRO group（100 μmol/L TBHP+18 μmol/L ANDRO），and high-dose ANDRO group（100 μmol/L TBHP+36 μmol/L ANDRO）. Cell apoptosis and reactive oxygen species（ROS） levels were determined by flow cytometry. The levels of oxidative stress related indicators，such as superoxide dismutase（SOD），glutathione peroxidase（GSH-PX），and catalase（CAT），were determined by an enzyme-linked immunosorbent assay kit. Mitochondrial membrane potential was measured by a JC-1 probe. Western blot was used to determine the expression levels of dynamin-related protein 1（Drp1），phosphorylated Drp1（p-Drp1），and mitofusin 2（Mfn2）.Results Compared with the control group，the model group had significantly increased apoptosis rate of NPCs，ROS level，and proportion of cells with decreased mitochondrial membrane potential（all P=0.000），significantly decreased SOD，GSH-PX，and CAT levels（all P=0.000），a significantly up-regulated p-Drp1/Drp1 level（P=0.000），and significantly down-regulated protein expression of Mfn2（P=0.000）. Compared with the model group，the high-dose ANDRO group had significantly decreased apoptosis rate of NPCs［（41.37±0.84）% vs.（26.36±1.33）%］，ROS level［（42.62±0.71）% vs.（22.50±0.37）%］，and proportion of cells with decreased mitochondrial membrane potential［（43.85±0.71）% vs.（24.96±1.04）%］（all P=0.000），significantly increased SOD［（288.16±14.59） ng/mL vs.（658.87±13.22） ng/mL］，GSH-PX［（6.66±0.37） ng/mL vs. （15.66±0.56） ng/mL］，and CAT［（40.89±1.04） pg/mL vs. （87.91±1.56） pg/mL］ levels（all P=0.000），a significantly down-regulated p-Drp1/Drp1 level（0.96±0.05 vs. 0.63±0.01）（P=0.001，0.000），and significantly up-regulated protein expression of Mfn2（0.39±0.01 vs. 0.64±0.02）（all P=0.000）.Conclusion ANDRO can inhibit TBHP-induced NPC apoptosis，and the mechanism may be related to the regulation of mitochondrial function and the relief of oxidative stress.