八味沉香散含药血清对H9c2细胞氧糖剥夺损伤的保护机制研究

Study on the protective mechanism of Bawei chenxiang powder containing serum on H9c2 cells injured by oxygen-glucose deprivation

目的研究八味沉香散含药血清对H9c2细胞氧糖剥夺损伤的保护机制。方法将H9c2细胞分为空白组、模型组和八味沉香散低、中、高剂量组(含药血清剂量依次为2.5、8、12 g/kg)。体外培养H9c2细胞并建立氧糖剥夺损伤模型,含药血清干预后检测细胞存活率,观察细胞形态,检测生化指标乳酸脱氢酶(LDH)、肌酸激酶(CK)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、呼吸链复合酶Ⅰ(ComplexⅠ)、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)水平,检测细胞内活性氧(ROS)含量、线粒体膜电位和细胞凋亡情况,检测氧化应激相关蛋白[Kelch样ECH关联蛋白1(Keap1)、核转录因子E2相关因子2(Nrf2)、血红素加氧酶1(HO-1)、NADH氧化还原酶辅酶10(Ndufa10)、硫氧还蛋白(Trx)]、凋亡相关蛋白[B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、胱天蛋白酶3(Caspase-3)和细胞色素C(Cytc)]的表达。结果与空白组比较,模型组细胞形态破损,LDH、CK和MDA水平均显著升高(P<0.01),CAT、ComplexⅠ、SOD、GSH-Px水平和线粒体膜电位均显著降低(P<0.01),细胞内ROS含量和凋亡率均显著升高(P<0.01),氧化应激相关蛋白(Keap1、Nrf2、HO-1、Ndufa10和Trx)和促凋亡相关蛋白(Bax、Caspase-3、Cytc)表达水平均显著升高(P<0.05),抗凋亡蛋白Bcl-2表达水平显著降低(P<0.05)。给予八味沉香散含药血清后,细胞形态改善,以上指标大部分显著逆转(P<0.05或P<0.01)。结论八味沉香散含药血清对H9c2细胞氧糖剥夺损伤有较好的保护作用,其机制与降低细胞氧化损伤、抑制细胞凋亡相关。

OBJECTIVE To study the protective mechanism of Bawei chenxiang powder containing serum on H9c2 cells injured by oxygen-glucose deprivation(OGD). METHODS H9c2 cells were divided into blank group, model group and Bawei chenxiang powder low-dose, medium-dose and high-dose groups(the dose of drug containing serum 2.5, 8, 12 g/kg). H9c2 cells were cultured in vitro to establish OGD model. After intervention with drug-containing serum, survival rate of cell was detected.The cell morphology was observed;the levels of lactate dehydrogenase(LDH), creatine kinase(CK), superoxide dismutase(SOD), catalase(CAT), respiratory chain complexⅠ(ComplexⅠ), glutathione peroxidase(GSH-Px) and malondialdehyde(MDA) were detected. The contents of reactive oxygen species(ROS), mitochondrial membrane potential and apoptosis were also detected. The expressions of oxidative stress-related proteins [Kelch ECH association protein 1(Keap1), nuclear factor erythroid 2-related factor 2(Nrf2), heme oxygenase 1(HO-1), NADH oxidoreductase coenzyme 10(Ndufa10), thioredoxin(Trx)] and apoptosis-related proteins [B-cell lymphoma 2(Bcl-2), Bcl-2 associated X protein(Bax), Caspase-3 and cytochrome C(Cytc)]were detected. RESULTS Compared with blank group, the cell morphology of model group was damaged;the levels of LDH, CK and MDA were significantly increased(P<0.01), while the levels of CAT, Complex Ⅰ, SOD and GSH-Px and mitochondrial membrane potential were significantly decreased(P<0.01). The content of intracellular ROS and apoptotic rate were significantly increased(P<0.01);the expressions of oxidative stress-related proteins(Keap1, Nrf2, HO-1, Ndufa10 and Trx) and proapoptosis proteins(Bax, Caspase-3 and Cytc) were significantly increased(P<0.05), while the expression of anti-apoptotic protein Bcl-2 was significantly decreased(P<0.05). After administration of Bawei chenxiang powder containing serum, the cell morphology improved, and most of the above indexes were significantly reversed(P<0.05 or P<0.01). CONCLUSIONS Bawei chenxiang powder containing serum has a good protective effect on H9c2 cells damaged by OGD, the mechanism of which is related to the reduction of oxidative damage and inhibition of cell apoptosis.