穿山龙总皂苷通过Nrf2信号通路对阿霉素诱导MPC-5细胞氧化应激损伤保护作用研究

Protective Effect of Total Saponins of Pangolin on Doxorubicin-induced Oxidative Stress Injury in MPC-5 Cells Based on Nrf2 Signal Pathway

目的探讨穿山龙总皂苷对阿霉素诱导MPC-5细胞损伤的保护作用及其机制研究。方法通过阿霉素诱导MPC-5细胞建立细胞损伤模型,采用MTT法测定细胞活力,考察阿霉素对MPC-5细胞的毒性及穿山龙总皂苷对阿霉素诱导MPC-5细胞损伤的保护作用,AnnexinV-FITC/PI双染色法检测穿山龙总皂苷对阿霉素诱导MPC-5细胞凋亡的影响;采用探针DCFH-DA检测穿山龙总皂苷对阿霉素诱导MPC-5细胞内活性氧(ROS)的影响;采用超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)试剂盒检测穿山龙总皂苷对阿霉素诱导MPC-5细胞内氧化应激因子的影响;采用Western Blot法检测MPC-5细胞内相关凋亡蛋白Bax、Bcl-2表达以及细胞质、细胞核和细胞整体内核因子红细胞2相关因子2(Nrf-2)蛋白的表达。结果与空白组相比,阿霉素模型组中细胞活力显著降低(P<0.01);细胞内MDA含量、ROS含量、细胞凋亡率显著升高(P<0.01),SOD活性、GSH-Px含量显著降低(P<0.01);细胞核和细胞总体中Nrf2蛋白表达显著升高(P<0.01);细胞总体中Bax蛋白表达显著升高(P<0.01),Bcl-2蛋白表达显著降低(P<0.01)。与阿霉素模型组相比,穿山龙总皂苷低、中、高剂量组细胞活力显著升高(P<0.05);细胞内MDA含量、ROS含量、细胞凋亡率显著降低(P<0.05,P<0.01),SOD活性、GSHPx含量显著升高(P<0.05,P<0.01);细胞核和细胞总体中Nrf2蛋白表达显著升高(P<0.05,P<0.01);细胞总体中Bax蛋白表达显著降低(P<0.05,P<0.01),Bcl-2蛋白表达显著升高(P<0.05,P<0.01)。结论穿山龙总皂苷通过调节Nrf2信号通路保护MPC-5细胞对抗阿霉素诱导的氧化应激损伤和细胞凋亡。

Objective To investigate the protective effect of total saponins from pangolin on doxorubicininduced injury of MPC-5 cells and its mechanism.Methods MPC-5 cells were induced by doxorubicin to establish a cell injury model,and the cell viability was determined by MTT assay.Protective effect,AnnexinV-FITC/PI double staining method was used to detect the effect of total saponins on doxorubicininduced apoptosis of MPC-5 cells;probe DCFH-DA was used to detect the effect of total saponins on doxorubicin-induced MPC-5 cells.Effects of reactive oxygen species(ROS);superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and malondialdehyde(MDA)kits were used to detect total saponins of pangolin on doxorubicin-induced MPC-5.The influence of intracellular oxidative stress factors;Western Blot was used to detect the expression of related apoptosis proteins Bax and Bcl-2 in MPC-5 cells,as well as the cytoplasmic,nuclear and overall nuclear factor erythrocyte 2-related factor 2(Nrf-2)pathway related proteins Nrf-2 protein expression.Results Compared with the normal group,the cell viability in the adriamycin model group was significantly decreased(P<0.01),the intracellular MDA content,ROS content,and apoptosis rate were significantly increased(P<0.01),SOD activity,GSH-Px content were significantly decreased(P<0.01),Nrf2 protein expressions in nucleus and overall cell were significantly increased(P<0.01),and Bax protein expression was significantly increased in overall cell(P<0.01),Bcl-2 protein expression was significantly decreased(P<0.01).Compared with the model group,the cell viability of the low,medium and high dose groups of total saponins of pangolin significantly increased(P<0.05),the intracellular MDA content,ROS content,and apoptosis rate were significantly decreased(P<0.05,P<0.01),and the SOD activity and GSH-Px content were significantly increased(P<0.05,P<0.01),Nrf2 protein expressions in nucleus and cell population were significantly decreased(P<0.05,P<0.01),and Bax protein expression was significantly decreased in cell population(P<0.05,P<0.01),the expression of Bcl-2 protein was significantly increased(P<0.05,P<0.01).Conclusion Total saponins from pangolin protect MPC-5 cells against doxorubicin-induced oxidative stress injury and apoptosis by regulating Nrf2 signaling pathway.