珠子参总皂苷通过促进Nrf2转位拮抗新生大鼠心肌细胞氧化应激损伤

目的观察珠子参总皂苷H2O2所致氧化应激损伤保护效应并探讨其机制。方法 Wistar乳鼠心肌细胞原代培养,用H2O2建立氧化应激损伤模型,然后用珠子参总皂苷(100、200μg/ml)孵育24 h后,观察细胞搏动频率,MTT法检测珠子参总皂苷对细胞活力的影响,流式细胞仪和Hoechst33258染色检测珠子参总皂苷对氧化应激诱导细胞凋亡及胞内ROS含量的影响,比色法测定培养液中乳酸脱氢酶(LDH)、肌酸激酶(CK)含量,同时检测培养液中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)的活性及丙二醛(MDA)含量,实时定量PCR技术检测珠子参总皂苷对SOD/GPX/CAT反应系统SOD1、SOD2、SOD3、CAT和GPX1基因表达的影响,Western blot检测珠子参总皂苷对细胞质和细胞核Nrf2蛋白表达的影响。结果 H2O2明显影响细胞活力和诱导凋亡(IC50为500μmol/L),珠子参总皂苷(100、200μg/ml)处理能显著增加心肌细胞搏动频率和存活率(P<0.01),抑制心肌细胞凋亡和改善细胞形态,降低细胞内ROS含量(P<0.01);减轻H2O2所致乳鼠心肌细胞培养液中LDH、CK释放量,同时能够升高SOD、CAT和GSH-Px活性,降低MDA含量(P<0.05,P<0.01);上调SOD/GPX/CAT反应系统基因表达水平和促进Nrf2核移位,增加心肌细胞核内Nrf2的表达水平(P<0.05,P<0.01)。结论珠子参总皂苷抑制心肌细胞氧化应激损伤,与Nrf2抗氧化途径的激活和清除ROS有关。

Total saponin from Rhizoma Panacis majoris protects neonatal rat cardiomyocytes against oxidative stress-induced injuries by improving Nrf2 translocation

Objective To determine the effect of total saponin from Rhizoma Panacis majoris on the oxidative stress-induced injuries in neonatal rat cardiomyocytes.Methods Neonatal rat cardiomyocytes were primarily isolated from 1-to 3-day-old Wistar rats and then treated with H2O2 at the IC50 of 50% to induce Oxidative stress-induced injury.The injuried cells were then treated with the total saponin(100 and 200 μg/ml) for 24 h.The beating rates of cardiomyocytes were observed by inverted microscopy,viability of cells and oxidative stress-induced apoptosis measured by by MTT assay,cell apoptotic rate and ROS content detected by flow cytomety,and apoptosis morphous of cells were observed by hoechst33258 through fluorescence microscopy.The levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),hydrogen peroxidase(CAT),malondialdehyde(MDA),lactate dehydrogenase(LDH),and creatine kinase(CK) in the culture medium were also measured.Real-time polymerase chain reaction was applied to detect the mRNA expression of SOD10-SOD3,CAT and GPX1 antioxidative genes,and expression of Nrf2 in the cell cytoplasm and nucleus were detected by Western blotting.Results H2O2 at the concentration of 200 to 800 μmol/L significantly inhibited the viability and apoptosis of cells(IC50=500 μmol/L).Total saponin from Rhizoma Panacis majoris(100 and 200 μg/ml) significantly increased the beating rates of cardiomyocytes and cell viability(P<0.01),reduced the apoptosis rate,improved apoptosis morphous,decreased intra-cellular ROS contents(P<0.01),improved the activities of SOD,GSH-Px and CAT,and decreased the levels of LDH,CK and MDA(P<0.05,P<0.01).Moreover,the mRNA expression of SOD1-SOD3,CAT and GPX1 in the cardiomyocytes was decreased remarkably in the model group,while they were up-regulated in total saponin-treatment groups(P<0.05,P<0.01).The protein expression of Nrf2 in the nucleus were significantly increased compared with model group(P<0.05,P<0.01) after treatment with the total saponin.Conclusion Total saponin from Rhizoma Panacis majoris effectively inhibits oxidative stress-induced injuries of cardiomyocytes by activating anti-oxidative pathway of Nrf2 and removing ROS.