Naringin protects H9C2 cardiomyocytes from chemical hypoxia-induced injury by promoting the autophagic flux via the activation of the HIF-1α/BNIP3 signaling pathway

Abstract

Naringin, a natural bioflavonoid, has been shown to exert protective effects in multiple cardiovascular diseases; however, the protective effects of naringin against hypoxic/ischemia-induced myocardial are not yet fully understood. Autophagy is a vital factor involved in the pathogenesis of myocardial injury. The aim of the present study was to investigate the protective effects of naringin on H9c2 cells against chemical hypoxia [cobalt chloride (CoCl2)]-induced injury. The role of autophagy and the hypoxia-inducible factor-1α (HIF-1α)/Bcl-2/BCL2 interacting protein 3 (BNIP3) signaling pathway in the protective effects of naringin were also assessed. The results revealed that naringin pre-treatment significantly attenuated the CoCl2-induced cytotoxicity and apoptosis, and also decreased caspase-3 activity, which had been increased by CoCl2. In addition, CoCl2 increased Beclin-1 expression, enhanced the IL3B-II/IL3B-I ratio and increased p62 expression in the H9C2 cells. Treatment with 3-methyladenine (3-MA), a selective inhibitor of autophagy, also blocked CoCl2-induced cytotoxicity and apoptosis. Notably, treatment with bafilomycin A1 (Baf A1), an inhibitor of the vacuolar H+ ATPase of lysosomes, resulted in an increase in the upregulation of the LC3B-II/LC3B-I ratio, but did not further increase the LC3B-II/LC3B-I ratio compared with CoCl2 treatment. These results suggested that CoCl2 inhibited the autophagic flux, which resulted in myocardial cell damage. Furthermore, naringin pre-treatment exacerbated Beclin 1 expression and the increased IL3B-II/IL3B-I ratio, and reduced p62 expression in CoCl2-treated H9C2 cells. 3-MA and Baf A1 both reversed the protective effects of naringin against CoCl2-induced injury, indicating that naringin attenuated CoCl2-induced myocardial cell injury by the increasing autophagic flux. Moreover, naringin treatment resulted in upregulated expression levels of HIF-1α and BNIP3 in the CoCl2-treated H9C2 cells. The inhibition of the HIF-1α/BNIP3 signaling pathway using 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (an inhibitor of HIF-1α) prevented the effects of naringin on the autophagic flux and reversed its protective effects against CoCl2-induced injury. Taken together, these results suggest that naringin protects the H9C2 cells against CoCl2-induced injury by enhancing the autophagic flux via the activation of the HIF-1α/BNIP3 signaling pathway.