人参总皂苷对D-半乳糖致PC12细胞衰老的改善作用及其机制研究

Study on Improvement Effects of Total Ginsenosides on D-galactose Induced PC12 Cell Senescence and Its Mechanism

目的:研究人参总皂苷对D-半乳糖致PC12细胞衰老的改善作用及其机制。方法:将大鼠嗜铬细胞瘤(PC12)细胞用D-半乳糖处理以建立细胞衰老模型。采用CCK-8法筛选D-半乳糖的建模浓度和人参总皂苷的给药浓度。试验设正常对照组、模型组和人参总皂苷低、高浓度组,检测各组细胞衰老情况、细胞凋亡率、细胞周期和细胞中线粒体膜电位(MMP)、三磷酸腺苷(ATP)、活性氧(ROS)水平以及凋亡相关蛋白[B淋巴细胞瘤2(Bcl-2)及其相关蛋X蛋白(Bax)、细胞色素C(Cyt-C)]和氧化损伤相关蛋白[核因子2相关因子2(Nrf2)、血红素氧合酶1(HO-1)]表达水平,另设阳性药物组[5 mmol/L N-乙酰-L-半胱氨酸(NAC)]和阳性对照组[D-半乳糖+5 mmol/L NAC]比较氧化损伤相关蛋白水平。结果:D-半乳糖能显著抑制PC12细胞的存活率,临界浓度为20 mg/mL;人参总皂苷可显著增加D-半乳糖诱导衰老细胞的存活率,半数效应浓度为65μg/mL,故将后续试验人参总皂苷的低、高浓度设置为55、65μg/mL。与正常对照组比较,模型组衰老细胞数明显增加,细胞凋亡率和G1期细胞比例均显著增加,S期细胞比例、细胞中MMP、ATP含量和Bcl-2蛋白以及线粒体中Cyt-C蛋白表达水平均显著降低,ROS含量和Bax、Nrf2蛋白及胞浆内Cyt-C蛋白表达均显著升高(P<0.05或P<0.01)。与模型组比较,人参总皂苷低、高浓度组衰老细胞数明显减少,细胞凋亡率和G1期细胞比例均显著降低,S期细胞比例、细胞中MMP、ATP(除低浓度组外)含量和Bcl-2、Nrf2、HO-1蛋白以及线粒体中Cyt-C蛋白表达水平均显著升高,ROS(除低浓度组外)含量和Bax蛋白及胞浆内Cyt-C蛋白表达水平均显著降低;阳性对照组细胞内Nrf2、HO-1蛋白表达水平亦显著升高(P<0.05或P<0.01),但均低于人参总皂苷组。结论:人参总皂苷可改善D-半乳糖诱导的P12细胞衰老,其机制可能与激活Nrf2抗氧化信号途径拮抗D-半乳糖诱导的氧化应激、缓解其所致的线粒体功能障碍有关。

OBJECTIVE:To study the improvement effects of total ginsenosides on the senescence of PC12 cells induced by Dgalactose and its mechanism.METHODS:Rat pheochromocytoma(PC12)cells were treated with D-galactose to establish cell senescence model.CCK-8 method was used to screen the D-galactose modeling concentration and total ginsenosides concentration.Normal control group,model group,total ginsenosides low and high concentration groups were set up.Cell senescence,cell apoptosis rate,apoptotic cycle and mitochondrial membrane potential(MMP),cell adenosine triphosphate(ATP)and reactive oxygen species(ROS)levels in each group were detected.The expression of apoptosis related proteins[B lymphoma 2(Bcl-2)and its related egg X protein(Bax),cytochrome C(Cyt-C)]and oxidative damage related proteins[nuclear factor 2 related factor 2(Nrf2),heme oxygenase 1(HO-1)]were detected.In addition,positive drug group[5 mmol/L N-acetyl-L-cysteine(NAC)]and positive control group[D-galactose+5 mmol/L NAC]were set up to compare the levels of oxidative damage related proteins.RESULTS:D-galactose could significantly inhibit the survival rate of PC12 cells,with a critical concentration of 20 mg/mL.The total ginsenosides could significantly increase the survival rate of D-galactose induced senescent cells with a median effective concentration(EC50)of 65μg/mL,and then the low and high concentrations of total ginsenosides were set at 55 and 65μg/mL.Compared with normal control group,the number of aging cells increased,the apoptotic rate and percentage of G1 phase were significantly increased in model group.the percentage of S phase,MMP and ATP contents,the protein expression of Bcl-2 and CytC in mitochondria were decreased significantly,while ROS content,the protein expression of Bax,Nrf2 and Cyt-C protein in endochylema were increased significantly(P<0.05 or P<0.01).Compared with model group,the number of aging cells reduced,the apoptosis rates and percentage of G1 phase were significantly decreased in total ginsenosides low and high concentration groups,the percentage of S phase,the contents of MMP and ATP(except for low concentration group),protein expression of Bcl-2,Nrf2 and HO-1 as well as protein expression of Cyt-C in mitochondria were increased significantly;ROS level(except for low concentration group)and Bax protein as well as protein expression of Cyt-C were decreased significantly.The protein expression of Nrf2 and HO-1 were increased significantly in positive control group(P<0.05 or P<0.01),but it was lower than that of total ginsenosides groups.CONCLUSIONS:Total ginsenosides can improve D-galactose induced senescence of P12 cells,the mechanism of which may be related to activating Nrf2 antioxidant signal pathway to antagonize D-galactose induced oxidative stress and alleviating mitochondrial dysfunction.