人参糖蛋白对阿霉素所致心肌毒性的保护作用及其机制研究

Protective effect and mechanism of ginseng glycoproteins on cardiotoxicity caused by adriamycin

目的通过体内外实验,探讨人参糖蛋白对阿霉素心脏毒性的保护作用及机制。方法建立SD大鼠心肌损伤模型,给予人参糖蛋白进行干预后,检测血清中乳酸脱氢酶(lactatedehydrogenase,LDH)、肌酸激酶同工酶MB(creatinekinase isoenzymes-MB,CK-MB)、超氧化物歧化酶(superoxide dismutase,SOD)活性及谷胱甘肽(glutathione,GSH)水平;采用苏木素-伊红(HE)染色法观察大鼠心肌组织病理变化。建立心肌细胞H9c2损伤模型,采用CCK-8法检测H9c2细胞活力;通过流式细胞术检测H9c2细胞周期、细胞凋亡、活性氧(reactive oxygen species,ROS)水平和线粒体膜电位变化;采用Western blotting法检测H9c2细胞凋亡相关蛋白、丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路相关蛋白、沉默信息调节因子2相关酶类3(silent mating type information regulation 2 homolog 3,Sirt3)的表达情况。结果心肌损伤大鼠模型中,模型组大鼠心肌纤维排列紊乱,肌纤维严重变性,LDH和CK-MB活性显著升高(P<0.01),SOD活性和GSH水平显著降低(P<0.05、0.01);与模型组比较,人参糖蛋白高剂量组心肌肌束排列较为整齐,LDH和CK-MB活性显著下降(P<0.05),GSH水平显著升高(P<0.05),人参糖蛋白对阿霉素所致的心肌组织病理学损伤有明显修复作用。细胞损伤模型中,模型组细胞存活率显著下降(P<0.001),细胞周期阻滞于G1期(P<0.01),细胞凋亡显著升高(P<0.01),ROS水平显著升高(P<0.01),线粒体膜电位显著下降(P<0.01),Sirt3、Caspase-3、B淋巴细胞瘤-2(B cell lymphoma-2,Bcl-2)蛋白表达水平显著降低(P<0.01),Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、细胞色素C(cytochrome C,Cyt C)、c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)、p38、细胞外调节蛋白激酶1/2(extracellular regulated protein kinases 1/2,ERK1/2)蛋白表达水平显著升高(P<0.05、0.01);与模型组比较,人参糖蛋白组细胞存活率显著升高(P<0.05、0.01),细胞增殖周期恢复,细胞凋亡率显著降低(P<0.05),ROS水平显著降低(P<0.05),线粒体膜电位显著升高(P<0.05),Sirt3、Caspase-3、Bcl-2蛋白表达水平显著升高(P<0.05),Bax、Cyt C、JNK、p38、ERK1/2蛋白表达水平显著降低(P<0.05)。结论人参糖蛋白能够通过抗氧化应激、降低线粒体膜电位、提高H9c2细胞内ROS水平并调控MAPK信号通路抑制细胞凋亡,从而保护阿霉素诱导的心肌损伤。

Objective In vitro and in vivo experiments were conducted to investigate the protective effect and mechanism of ginseng glycoproteins on adriamycin-induced cardiotoxicity. Methods SD rats myocardial injury model was established. After intervention with ginseng glycoprotein, activities of lactate dehydrogenase(LDH), creatine kinase isoenzymes-MB(CK-MB), and superoxide dismutase(SOD) and glutathione(GSH) levels in serum were detected;Hematoxylin-eosin(HE) staining was used to observe the pathological changes of rat myocardial tissue. H9 c2 injury model of cardiomyocytes was established, and viability of H9 c2 cells was detected by CCK-8 method;H9 c2 cells cycle, apoptosis, reactive oxygen species(ROS) levels and mitochondrial membrane potential changes in mitochondrial membrane potential were detected by flow cytometry;Western blotting was used to detect expressions of apoptosis-related proteins, mitogen-activated protein kinase(MAPK) signaling pathway related proteins and silent information regulator 2 related enzymes 3(Sirt3) in H9 c2 cells. Results In myocardial injury rats model, the myocardial fiber arrangement of model group was disordered, the muscle fiber was severely degenerated, the activities of LDH and CK-MB were significantly increased(P < 0.01), and the SOD activity and GSH level were significantly decreased(P < 0.05, 0.01);Compared with model group, the myocardial muscle bundles in high-dose ginseng glycoprotein group were arranged more neatly, activities of LDH and CK-MB were significantly decreased(P < 0.05), and level of GSH was significantly increased(P < 0.05). The pathological damage of myocardial tissue caused by adriamycin had obvious repairing effect. In cell injury model, cell viability of model group was significantly decreased(P < 0.001), cell cycle was arrested in G1 phase(P < 0.01), apoptosis was significantly increased(P < 0.01), and level of ROS was significantly increased(P < 0.01), mitochondrial membrane potential was significantly decreased(P < 0.01), expressions of Sirt3, Caspase-3, B cell lymphoma-2(Bcl-2) were significantly decreased(P < 0.01), expressions of Bcl-2 associated X protein(Bax), cytochrome C(Cyt C), c-Jun N-terminal kinase(JNK), p38, extracellular regulated protein kinases 1/2(ERK1/2) were significantly increased(P < 0.05, 0.01);Compared with model group, cell viability of ginseng glycoprotein group was significantly increased(P < 0.05, 0.01), cell proliferation cycle was restored, and apoptosis rate was significantly reduced(P < 0.05), ROS level was significantly reduced(P < 0.05), and mitochondrial membrane potential was significantly increased(P < 0.05), expressions of Sirt3, Caspase-3 and Bcl-2 in ginseng glycoprotein group were significantly increased(P < 0.05), expressions of Bax, Cyt C, JNK, p38, ERK1/2 were significantly reduced(P < 0.05). Conclusion Ginseng glycoprotein can prevent oxidative stress, reduce mitochondrial membrane potential, increase ROS level in H9c2 cells, and regulate MAPK signaling pathway to inhibit cell apoptosis, thereby protecting adriamycin-induced myocardial injury.