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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。
Objective To observe the protective effect of total saponin H2O2 induced oxidative stress injury and to explore its mechanism. Methods Wistar neonatal rat cardiomyocytes were cultured in primary culture and oxidative stress injury model was established by H2O2. The cells were incubated with total ginsenoside (100, 200μg / ml) for 24 hours. The cell beating frequency was observed. The total saponin Cell viability, flow cytometry and Hoechst33258 staining were used to detect the effects of total saponins from Bentley Roots on oxidative stress-induced apoptosis and intracellular ROS content. The contents of lactate dehydrogenase (LDH), creatine (CK) and the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) and the content of malondialdehyde (MDA) , Real-time quantitative polymerase chain reaction (RT-PCR) to detect the expression of SOD1, SOD2, SOD3, CAT and GPX1 in SOD / GPX / CAT reaction system and the effect of total ginsenoside on the expression of Nrf2 protein in the cytoplasm and nucleus. Results H 2 O 2 significantly affected cell viability and induced apoptosis (IC50 was 500 μmol / L). Beads total saponin (100 and 200 μg / ml) significantly increased cardiomyocyte pulsatility and survival rate (P <0.01) and inhibited cardiomyocyte apoptosis (P <0.01); reduce the release of LDH and CK in the culture medium of neonatal rat cardiomyocytes induced by H2O2; at the same time, it can increase the activity of SOD, CAT and GSH-Px and decrease the content of MDA (P <0.05, P <0.01). Upregulation of gene expression and Nrf2 nuclear translocation in the SOD / GPX / CAT response system increased Nrf2 expression in cardiomyocytes (P <0.05, P <0.01). Conclusion SSP can inhibit the oxidative stress injury of cardiomyocytes, which is related to the activation of Nrf2 anti-oxidative pathway and ROS scavenging.