激活TGR5受体减轻ET-1致乳小鼠心肌细胞氧化应激损伤的作用及机制

Attenuation of endothelin-1-induced oxidative stress in mouse cardiomyocytes by activation of G protein-coupled bile acid receptor-1 and its mechanism

摘要:

目的：观察G蛋白偶联胆汁酸受体-1（G protein-coupled bile acid receptor 1，TGR5）激活后对内皮素-1（endothelin-1，ET-1）所致的乳鼠心肌细胞氧化应激损伤的作用，并探讨其可能的机制。方法：原代培养心肌细胞，ET-1浓度分别为10-8、10-7、10-6 mmol/L，分别作用12、24、36、48 h，建立心肌细胞氧化应激损伤模型。建立氧化应激损伤模型后，分别给予 INT-777（TGR5激动剂）、TGR5 siRNA（病毒干扰TGR5表达）及TGR5空病毒处理48 h。采用CCK-8法观察心肌细胞的存活率，生化试剂盒法检测丙二醛（malonaldehyde，MDA）、超氧化物歧化酶（superoxide dismutase，SOD）、乳酸脱氢酶（lactic dehydrogenase，LDH）含量，Western blot检测细胞核中核因子相关因子-2（nuclear factor erythroid 2-related factor -2，Nrf2）蛋白的表达，Real-time PCR检测Nrf2下游抗氧化基因血红素加氧酶（heme oxygenase，HO-1）mRNA、醌氧化还原酶（quinone oxidoreductase，NQO-1）mRNA、硫氧化蛋白还原酶-1（thioredoxin reductase-1，Txnrd-1）mRNA的表达水平。结果：浓度为10-8、10-7、10-6 mmol/L的ET-1均可诱导SOD活力下降（P=0.000），MDA生成增加（P=0.000）；且随着ET-1浓度增加和培养时间延长，心肌细胞氧化应激损伤程度越严重。ET-1（10-6 mmol/L）组MDA及LDH明显增加（均P=0.000），SOD活性明显下降（P=0.000），Nrf2、HO-1 mRNA、Txnrd-1 mRNA表达增加（均P=0.000）。予以30 μmol/L INT-777可有效改善ET-1诱导的氧化应激损伤，与ET-1组相比，心肌细胞存活率明显增加（P=0.000），MDA及LDH减少（均P=0.000），SOD活性增加（P=0.000），Nrf2、HO-1 mRNA、NQO-1 mRNA、Txnrd-1 mRNA表达明显增加（均P=0.000）；而病毒干扰TGR5组可部分阻断TGR5激动剂对心肌细胞损伤的改善作用，Nrf2、HO-1 mRNA、NQO-1 mRNA、Txnrd-1 mRNA表达下降（均P=0.000）。结论：激活TGR5可能通过激活Nrf2其下游抗氧化基因HO-1、NQO-1及Txnrd-1减轻ET-1对心肌细胞的损伤作用。

Abstract:

Objective：To observe the effect of G protein-coupled bile acid receptor-1（TGR5） activation on endothelin-1（ET-1）-induced oxidative stress in neonatal mouse cardiomyocytes，and to explore its possible mechanism. Methods：Primary cultured car－diomyocytes were treated with ET-1 at concentrations of 10-8，10-7，and 10-6 mmol/L for 12，24，36，and 48 hours to establish a car－diomyocyte model of oxidative stress. After model establishment，cells were treated with INT-777（TGR5 agonist），TGR5 siRNA （TGR5 expression inhibition），or empty virus for 48 hours. The survival rate of cardiomyocytes was observed by CCK-8 assay. The content of malondialdehyde（MDA），superoxide dismutase（SOD），and lac－tate dehydrogenase（LDH） was determined by biochemical kits. The protein expression of nuclear factor erythroid 2-related factor－2（Nrf2） in the nucleus was measured by Western blot. Real-time PCR was used to determine the mRNA expression levels of Nrf2 downstream antioxidant genes such as heme oxy－genase（HO-1），quinone oxidoreductase（NQO-1），and thioredoxin reductase-1（Txnrd-1）. Results：ET-1 at concentrations of 10-8，10-7，and 10-6 mmol/L significantly reduced SOD activity and increased MDA expression（all P=0.000）；the oxidative stress of car－diomyocytes was enhanced with the increases in ET-1 concentration and culture time. In the cells treated with 10-6 mmol/L ET-1，the content of MDA and LDH was significantly increased，SOD activity was significantly reduced，and the protein expression of Nrf2 and the mRNA expression of HO-1 and Txnrd-1 were significantly increased（all P=0.000）. The treatment with 30 μmol/L INT-777 effec－tively improved ET-1-induced oxidative stress：compared with the ET-1 group，the cardiomyocytes treated with INT-777 had a sig－nificantly higher survival rate（P=0.000），significantly reduced MDA and LDH，a significantly higher SOD activity，and significantly higher protein expression of Nrf2 and mRNA expression of HO-1，NQO-1，and Txnrd-1（all P=0.000）. Inhibition of TGR5 expression by virus partially blocked the improvement of cardiomyocyte injury by the TGR5 agonist，suggested by significantly reduced protein expression of Nrf2 and mRNA expression of HO-1，NQO-1，and Txnrd-1（all P=0.000）. Conclusion：TGR5 activation attenuates ET-1-induced oxidative stress of cardiomyocytes probably by activating Nrf2 downstream antioxidant genes such as HO-1，NQO-1，and Txnrd-1.