太白楤木活性成分竹节参皂苷Ⅳa抗氧化及抗衰老作用研究

Antioxidant and anti-aging effect of chikusetsu saponin Ⅳa,an active ingredient in Aralia taibaiensis

目的探讨太白楤木皂苷中的重要成分竹节参皂苷Ⅳa(CHS)抗氧化及抗衰老作用。方法取5~8代的小鼠胚胎成纤维细胞(MEFs),使用过氧化氢(H2O2)诱导细胞氧化应激以及细胞衰老模型。MTT法分别检测不同浓度CHS及H2O2对细胞活力的影响;DCFH-DA染色流式细胞仪检测细胞总活性氧(ROS)水平;Mito SOX染色流式细胞仪检测细胞线粒体ROS水平;衰老相关β-半乳糖苷酶(SA-β-gal)染色检测细胞衰老状态;Western blot法检测衰老标志蛋白p53、p21蛋白表达水平以及关键抗氧化应激Nrf2通路(Nrf2、HO-1、GCLC、GCLM)蛋白表达。结果浓度≤160μg·m L-1的CHS对MEFs细胞无明显毒性作用;H2O2能够显著提高细胞总ROS和线粒体ROS水平,诱导细胞衰老指标SA-β-gal活性增加,提高p53、p21表达水平,提高抗氧化信号通路Nrf2通路表达水平。CHS能够剂量依赖地降低H2O2诱导的细胞总ROS和线粒体ROS水平,减轻H2O2诱导的SA-β-gal活性升高,抑制p53、p21以及Nrf2、HO-1、GCLC、GCLM蛋白表达水平的升高。结论 CHS具有显著的抗氧化应激和抗衰老作用。

Objective To explore the protective effects of chikusetsu saponin IVa （CHS） from Aralia taibaiensis against hydrogen peroxide （H2O2）-induced oxidative stress and aging in mouse embryos fibroblasts （MEFs）. Methods The fifth to eighth generation MEF cells were treated with H2O2 to set up the cellular oxidative stress and aging model. The cell viability in different concentrations of CHS or H2O2 was analyzed by MTT assay. Intracellular reactive oxygen species （ROS） were detected by flow cytometry with 2, 7-dichlorofluorescein-diacetate （DCFH- DA） staining, and mitochondrial ROS with MitoSOX Red Mitochondrial Superoxide Indicator. Cell senescence was detected by senescence associated β-galactosidase staining. Protein levels were detected by Western blot. Results CHS had no obvious toxic effects on MEF cells at concentration than 160μg · mL-l. H2O2 treatment significantly increased the levels of both intracellular ROS and mitochondrial ROS. At the same time, H2O2 obviously increased the percentage of senescence associated fl-galactosidase staining and the expression of protein p53 and p21. The expression of Nrf2 and its targeted downstream proteins （HO-1, GCLC, and GCLM） were increased by H2O2 stimulation. CHS dose-dependently reduced the intracellular ROS and mitochondrial level ROS, decreased the level of senescence associatedfl-galactosidase staining, as well as the expressions of protein p53 and p21. Meanwhile, CHS restrained protein expression levels ofNrf2, HO-1, GCLC and GCLM induced by H2O2. Conclusion CHS shows obvious protection against oxidative stress and oxidative stress-induced aging.