Activation of TGR5 receptor reduces damage of ET-1 on H9C2 cardiomyocytes by activating Nrf2

Yan Hao, Haitao Yuan, Houzhi Yu
Panminerva Medica 2020 May 14

BACKGROUND: To investigate the effect of G protein-coupled bile acid receptor 1 (TGR5) on oxidative stress injury of H9C2 cardiomyocytes induced by endothelin-1 (ET-1), and to explore the possible mechanism.

METHODS: H9C2 cardiomyocytes were treated with ET-1 at concentrations of 10-8, 10-7 and 10-6 mmol/L for 12, 24, 36, and 48 h, respectively. At the same time, oxidative stress injury models were established. After the oxidative stress injury model was established, INT-777 (TGR5 agonist), siRNA-TGR5 (the virus interfered with TGR5 expression) and TGR5 empty virus were treated for 48 h. CCK-8 method was used to observe the survival rate of cardiomyocytes. Biochemical kit method was used to detect malonaldehyde (MDA), superoxide dismutase (SOD), Creatine Kinase (CK) and lactic dehydrogenase (LDH) contents. Western blot was used to detect the expression of nuclear factor erythroid 2-related factor -2 (Nrf2) protein in the nucleus. And real-time PCR was used to detect Haem oxygenase (HO-1) mRNA, quinone oxidoreductase-1 (NQO-1) mRNA, thioprotein reductase-1 (Txnrd-1) mRNA expression levels.

RESULTS: At the concentrations of 10-8, 10-7 and 10-6 mmol/L, ET-1 could induce the decrease of SOD activity and increase of LDH activity, and increase of CK and MDA production in H9C2 cardiomyocytes. Moreover, with the increase of ET-1 concentration and the extension of culture time, the oxidative stress damage of cardiomyocytes became more serious. After ET-1 intervention, the expressions of Nrf2 protein, HO-1 mRNA, NQO-1 mRNA and Txnrd-1 mRNA were decreased. The 30 μmol/L INT-777 could effectively improve the oxidative stress induced by ET-1, and compared with the ET-1 group, the survival rate of H9C2 cardiomyocytes increased obviously. In addition, the MDA or CK content and LDH activity were all decreased. On the contrary, the SOD activity increased significantly, and the expressions of Nrf2 protein, HO-1 mRNA, NQO-1 mRNA and Txnrd-1 mRNA increased obviously. However, siRNA-TGR5 can partially block the improvement effect of TGR5 agonist on cardiomyocytes injury.

CONCLUSIONS: Activation of TGR5 receptor may reduce the damage of ET-1 on H9C2 cardiomyocytes by activating Nrf2 and its downstream antioxidant genes HO-1, NQO-1 and Txnrd-1.

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