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

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(100μmol/L TBHP)、ANDRO低剂量组(100μmol/L TBHP+9μmol/L ANDRO)、ANDRO中剂量组(100μmol/L TBHP+18μmol/L ANDRO)和ANDRO高剂量组(100μmol/L TBHP+36μmol/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。

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(100μmol/L TBHP),low-dose ANDRO group(100μmol/L TBHP+9μmol/L ANDRO),middle-dose ANDRO group(100μmol/L TBHP+18μmol/L ANDRO),and high-dose ANDRO group(100μmol/L TBHP+36μmol/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.