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目的:探讨参芎葡萄糖注射液(Shenxiong glucose injection,SGI)对过氧化氢(H2O2)诱导的H9c2细胞氧化损伤的保护作用及其机制。方法:体外培养H9c2心肌细胞,用H2O2(300μmol·L-1)处理0.5 h,建立H9c2心肌细胞H2O2氧化损伤模型,SGI组:H9c2细胞用(100,200,400μmol·L-1)SGI处理6 h后,另设空白组与模型组,再用H2O2处理0.5 h。利用流式细胞术检测细胞内活性氧(ROS)和线粒体膜电位(MMP);用MTS法检测细胞存活率;用酶联免疫吸附测定(ELISA)法检测乳酸脱氢酶(LDH)漏出量、丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)活性;用蛋白质免疫印迹(Western blot)技术检测凋亡相关基因B细胞淋巴瘤/白血病-2(Bcl-2),Bcl-2相关X蛋白(Bax)和半胱氨酸蛋白酶(Caspase-3)的表达情况。结果:在300μmol·L-1H2O2作用细胞0.5 h的情况下,细胞存活率降低至50%左右,降低的程度合适,且实验结果重复性好,因此后续实验用该条件建立氧化损伤模型。与模型组比较,SGI预处理6 h能显著升高细胞存活率(P<0.05,P<0.01),减少LDH的外漏和MDA的生成(P<0.05,P<0.01),显著增加SOD,GSH-Px和CAT的活性(P<0.01),明显减少H9c2细胞内ROS含量(P<0.01),并使MMP丢失减少(P<0.01),Western blot表明,SGI能显著上调Bcl-2的表达,下调Caspase-3的表达(P<0.05,P<0.01)。结论:参芎葡萄糖注射液能保护H9c2心肌细胞对抗H2O2诱导的氧化损伤,其作用机制可能与其提高细胞清除ROS能力和抑制细胞凋亡有关。
Objective: To investigate the protective effect of Shenxiong glucose injection (SGI) on oxidative damage of H9c2 cells induced by hydrogen peroxide (H2O2) and its mechanism. Methods: H9c2 cardiomyocytes were cultured in vitro and treated with H2O2 (300μmol·L-1) for 0.5 h. H2O2 oxidative injury model was established in H9c2 cardiomyocytes. SGI group: H9c2 cells were treated with SGI at 100, 200, 400μmol·L-1 for 6 h, Another blank group and model group, and then treated with H2O2 0.5 h. Cell viability (ROS) and mitochondrial membrane potential (MMP) were assayed by flow cytometry. Cell viability was measured by MTS assay. Lactate dehydrogenase (LDH) leakage was measured by enzyme linked immunosorbent assay (ELISA) (MDA), superoxide dismutase (SOD) activity, catalase (CAT) and glutathione peroxidase (GSH-Px) activity were determined by Western blotting The expressions of Bcl-2, Bcl-2 and Caspase-3 were detected by flow cytometry. Results: After treated with 300μmol·L-1H2O2 for 0.5 h, the cell viability decreased to about 50%, and the degree of reduction was appropriate. The results of the experiment showed good reproducibility. Therefore, the oxidative damage model was established in the following experiment. Compared with model group, SGI pretreatment for 6 h significantly increased cell viability (P <0.05, P <0.01), decreased leakage of LDH and MDA (P <0.05, P <0.01) GSH-Px and CAT (P <0.01), and significantly decreased the ROS content in H9c2 cells (P <0.01) and decreased MMP loss (P <0.01). Western blot showed that SGI could up-regulate the expression of Bcl- , Down-regulated Caspase-3 expression (P <0.05, P <0.01). Conclusion: Shenxiong glucose injection can protect H9c2 cardiomyocytes against oxidative damage induced by H2O2. Its mechanism may be related to its ability of increasing ROS level and inhibiting apoptosis of cells.