Preventive Effects of Hydroxysafflor Yellow A on Hypoxia/Reoxygenation-Induced Injury in Rat Cardiomyocytes by Promoting the HIF-1α/BNIP3 Signaling Pathway-Mediated Mitophagy

Abstract

Background: Our preliminary research has suggested that hydroxysafflor yellow A (HSYA) has an alleviative effect on myocardial ischemia/reperfusion (I/R) injury. Here, we investigate the possible mechanism implicated. Methods: A hypoxia/reoxygenation (H/R) model, which mimicked the I/R model in vitro, was constructed in rat cardiomyocytes H9c2. Following the intervention with HSYA and transfection, the viability and apoptosis of cardiomyocytes were evaluated by the cell counting kit-8 (CCK-8) and flow cytometry assays. The mitochondrial membrane potential (∆Ψm) was determined with JC-1 analysis with both a confocal microscope and flow cytometry. The content of adenosine triphosphate (ATP) was quantified with a commercial kit. Reverse-transcription quantitative PCR (RT-qPCR) and western blot were employed to quantify the levels of hypoxia inducible factor-1α/BCL2 Interacting Protein 3 (HIF-1α/BNIP3) pathway-, mitochondrial fission- and mitophagy-related factors. Results: HSYA enhanced the viability, prevented apoptosis, restored the ∆Ψm loss, and promoted the ATP content in H/R-treated cardiomyocytes. HSYA inhibited the expressions of mitochondrial fission factor (Mff), Fission, Mitochondrial 1 (Fis1), and P62 yet promoted those of HIF-1α, BNIP3, and Beclin1 and increased LC3II/LC3I ratio in H/R-treated cardiomyocytes. BNIP3 silencing was associated with the suppression of viability and ATP levels, and the promotion of apoptosis and ∆Ψm loss in H/R-modeled cardiomyocytes, in addition to the observation that the modulatory effects of HSYA on both factors related to mitochondrial fission- and HIF-1α/BNIP3 pathway-mediated mitophagy were reversed after BNIP3 silencing. Conclusions: HSYA exerts a preventive effect on H/R-modeled cardiomyocytes by enhancing the HIF-1α/BNIP3 signaling pathway-mediated mitophagy.