麝香对Aβ_(1-42)诱导PC12细胞氧化应激损伤的保护作用研究

Protective Effect of Shexiang(麝香)on PC12 Cells Oxidative Stress Injury Induced by Aβ_(1-42)

目的:探讨麝香对阿尔兹海默症(AD)细胞模型氧化应激损伤及凋亡的保护作用。方法:使用LC-MS/MS法分析麝香化学成分。通过β-淀粉样蛋白1-42(Aβ_(1-42))诱导PC12细胞建立体外AD细胞模型,采用CCK-8法测定PC12细胞存活率,考察Aβ_(1-42)对PC12的细胞毒性及麝香对Aβ_(1-42)诱导PC12细胞损伤的保护作用;采用Annexin V-FITC/PI双染色法检测麝香对Aβ_(1-42)致PC12细胞凋亡的影响,JC-1法检测麝香对Aβ_(1-42)致PC12细胞线粒体膜电位的影响;采用探针DCFH-DA检测麝香对Aβ_(1-42)致PC12细胞内活性氧(ROS)的影响;采用超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)试剂盒检测麝香对Aβ_(1-42)致PC12细胞内抗氧化酶活性水平的影响;采用Western blot法检测PC12细胞内相关凋亡蛋白BAX、BCL-2和Cleaved caspase-3的表达以及核因子红细胞2相关因子2(Nrf-2)、血红素加氧酶-1(HO-1)和Kelch样ECH相关蛋白1(Keap 1)的表达。结果:通过LC-MS/MS法鉴定出麝香的7种化学成分分别为:次黄嘌呤、雄烯二酮、表睾酮、十六碳酰胺、麝香酮、油酸酰胺、硬脂酰胺。与空白对照组比较,模型对照组PC12细胞存活率显著降低(P<0.01),细胞内ROS水平、细胞凋亡率显著升高(P<0.01),线粒体膜电位明显降低(P<0.05),SOD和GSH-Px活力明显降低(P<0.05或P<0.01),BAX、Cleaved caspase-3和Keap 1蛋白表达明显上调,BCL-2、Nrf-2和HO-1蛋白表达明显下调(P<0.05或P<0.01);与模型对照组相比,麝香0.025、0.05、0.1 mg/mL组细胞存活率明显升高(P<0.05或P<0.01),细胞内ROS水平、细胞凋亡显著减少(P<0.01),SOD活力明显升高(P<0.05或P<0.01);其中0.05和0.1 mg/mL组中线粒体膜电位显著升高(P<0.01),BAX、Cleaved caspase-3和Keap 1蛋白表达明显下调(P<0.05或P<0.01);麝香0.1 mg/mL组GSH-Px活力显著升高(P<0.01),BCL-2、Nrf-2和HO-1蛋白表达明显上调(P<0.05或P<0.01)。结论:麝香通过Nrf-2/HO-1通路显著改善Aβ_(1-42)诱导的AD细胞模型的氧化应激和凋亡。

Objective:To explore the protective effect of Shexiang(麝香)on oxidative stress injury and apoptosis in Alzheimer's disease(AD)cell models.Methods:The chemical components of Shexiang were analyzed by LC-MS/MS assay.An in vitro AD cell model was built by Aβ_(1-42)induced PC12 cells,and the survival rate of PC12 cells was determined by the CCK-8 assay to explore the cytotoxicity of Aβ_(1-42)on PC12 and the protective effect of Shexiang on Aβ_(1-42)induced PC12 cell injury.The effect of Shexiang on Aβ_(1-42)induced apoptosis in PC12 cells was detected by Annexin V-FITC/PI double staining,and that of Shexiang on Aβ_(1-42)induced mitochondrial membrane potential in PC12 cells was detected by the JC-1 method.The effect of Shexiang on reactive oxygen species(ROS)in Aβ_(1-42)induced PC12 cells was detected by the DCFH-DA probe.Superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)kits were utilized to determine the effect of Shexiang on the activity levels of antioxidant enzyme in Aβ_(1-42)induced PC12 cells.Western blot was applied to detect the expressions of relevant apoptotic proteins Bax,Bcl-2,Cleaved caspase-3,Nuclear factor erythroid 2-related factor 2(Nrf-2),Heme oxygenase 1(HO-1),and Kelch-like ECH-associated protein 1(Keap 1)in PC12 cells.Results:The seven chemical components of Shexiang were identified by LC-MS/MS:Hypoxanthine,Androstenedione,Epitestosterone,Hexadecanamide,Muscone,Oleamide,and Stearamide.Compared with the blank control group,the model control group achieved a significantly reduced survival rate of PC12 cells(P<0.01),significantly increased intracellular ROS level and apoptosis rate(P<0.01),significantly decreased mitochondrial membrane potential(P<0.05),and significantly reduced activities of SOD and GSH-Px(P<0.05 or P<0.01).The expressions of Bax,Cleaved caspase-3,and Keap 1 protein were significantly upregulated,while the expressions of Bcl-2,Nrf-2,and HO-1 protein were significantly downregulated(P<0.05 or P<0.01).Compared with the model control group,the survival rate of cells was significantly increased in the 0.025,0.05,and 0.1 mg/mL Shexiang groups(P<0.05 or P<0.01),the intracellular ROS level and apoptosis was significantly reduced(P<0.01),and the activity of SOD was significantly increased(P<0.05 or P<0.01).Specifically,mitochondrial membrane potential was significantly increased(P<0.01)and the protein expressions of Bax,Cleaved caspase-3,and Keap 1 were significantly downregulated(P<0.05 or P<0.01)in the 0.05 and 0.1 mg/mL Shexiang group.In addition,the activity of GSH-Px was significantly increased(P<0.01)in the 0.1 mg/mL Shexiang group,and protein expressions of Bcl-2,Nrf-2,and HO-1 were significantly upregulated(P<0.05 or P<0.01).Conclusion:Shexiang significantly improves the oxidative stress and apoptosis of the Aβ_(1-42)induced AD cell model through the Nrf-2/HO-1 pathway.