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

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.