目的 探讨氧化苦参碱(oxymatrine,OMT)对H9c2心肌细胞氧化应激条件下的保护作用。方法 建立H9c2心肌细胞氧化应激模型,加入OMT进行预处理。取对数生长期细胞,分为:对照组,不给于任何处理;模型组,在细胞培养液中加入终浓度为100 μmol/L的H2O2培养4 h;2个OMT预处理组,分别用含有10 μmol/L和50 μmol/L OMT且不含有血清的DMEM完全培养液预处理12 h,再向其加入终浓度为100 μmol/L的H2O2培养4 h。通过MTT法检测细胞代谢活力;HE染色观察细胞形态;收集细胞上清液检测乳酸脱氢酶(LDH)含量,检测细胞内丙二醛(MDA)、过氧化氢酶(CAT)与超氧化物歧化酶(SOD)含量;Hoechst染色检测细胞凋亡率;通过RT-PCR检测细胞中Bcl-2 mRNA、Bax mRNA、Casepase3 mRNA、Nrf2 mRNA、HO-1 mRNA表达水平;Western blot法检测Nrf2、HO-1蛋白表达水平。结果 与对照组比较,模型组细胞存活率(39.53%±0.25%)下降,LDH漏出量[(472.22±15.54)U/L]增加,SOD[(64.51±5.37)U/mgprot]等含量降低,MDA[(6.70±0.05)nmol/mgprot]含量升高;RT-PCR与Western blot 结果均显示Nrf2、HO-1表达升高,差异有统计学意义(P<0.05);与模型组比较,OMT低、高浓度预处理组细胞存活率(57.40%±0.12%、77.51%±0.23%)升高,LDH漏出量[(367.72±12.53)U/L、(275.35±12.48)U/L]降低,细胞内SOD[(90.55±2.27)U/mgprot、(130.52±5.94)U/mgprot]等含量增加,MDA[(4.75±0.09)nmol/mgprot、(3.22±0.03)nmol/mgprot]含量下降;RT-PCR与Western blot 结果均显示Nrf2、HO-1表达升高,差异有统计学意义(P<0.05)。结论 氧化苦参碱通过抑制线粒体凋亡途径和激活Nrf2/HO-1信号通路,保护氧化应激损伤的H9c2心肌细胞。
Abstract
Objective To investigate the protective effects and mechanisms of oxymatrine (OMT) on H9c2 cardiomyocytes induced by oxidative stress. Methods An oxidative stress model of H9c2 cardiomyocytes was established and pretreated with oxymatrine. Logarithmic growth phase cells were taken and divided into four groups: the control group that was left untreated,model group in which 100 μmol/L H2O2 was added to the cell culture medium for 4 h,and two oxymatrine pretreatment groups that were pretreated for 12 h with complete DMEM medium containing 10 μmol/L and 50 μmol/L oxymatrine respectively before 100 μmol/L H2O2 was added for 4 h. Cell viability was detected by MTT assay while cell morphology was observed by HE staining. The cell supernatant was collected to determine the content of lactate dehydrogenase (LDH) and the levels of malondialdehyde (MDA), catalase (CAT) and superoxide dismutase (SOD) in the cells. The rate of apoptosis was measured by Hoechst. The expression levels of Bcl-2 mRNA, Bax mRNA, Casepase3 mRNA, Nrf2 mRNA and HO-1 mRNA were analyzed by RT-PCR. Protein expressions of Nrf2 and HO-1 were analyzed by Western blot. Results Compared with the control group, the rate of cell viability (39.53%±0.25%) was obviously decreased, the amount of LDH leakage[ (472.22±15.54) U/L]was increased, the content of SOD[ (64.51±5.37) U/mgprot]was decreased and the content of MDA[ (6.70±0.05) nmol/mgprot] was elevated in the model group. The results by RT-PCR and Western blot showed that the expressions of Nrf2 and HO-1 were increased (P<0.05). Compared with the model group, different concentrations in OMT pretreatment groups ameliorated cell viability (57.40%±0.12%,77.51%±0.23%). The amount of LDH leakage [ (367.72±12.53) U/L, (275.35±12.48) U/L] was decreased, the content of SOD[ (90.55±2.27) U/mgprot, (130.52±5.94) U/mgprot] was increased and the content of MDA[ (4.75±0.09) nmol/mgprot, (3.22±0.03) nmol/mgprot] was reduced. The results by RT-PCR and Western blot showed that the expressions of Nrf2 and HO-1 were increased (P<0.05). Conclusions Oxymatrine can protect H9c2 cardiomyocytes induced by oxidative stress by inhibiting mitochondrial apoptosis pathway and activating Nrf2/HO-1 signaling pathway.
关键词
氧化苦参碱 /
氧化应激 /
心肌细胞 /
保护
Key words
oxymatrine /
oxidative stress /
cardiomyocyte /
protection
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基金
天津市自然科学基金重点项目(16JCZDJC31900);天津市科技计划项目(15ZXLCSY00040)
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