Abstract：Objective: To explore the mechanism of pinocembrin in reducing myocardial cell injury induced by hypoxia/reoxygenation by regulating autophagy mediated by hypoxia-inducible factor 1α (HIF-1α)/Bcl2/adenovirus E1B-interacting protein 3 (BNIP3) signaling pathway.Methods: Rat cardiomyocytes H9c2 were cultured in vitro, after establishing the H/R model, the mice were treated with 0, 25, 50, 100, 200 and 300 μg/mL pinocembrin for 24 hours, the cell viability of each treatment group was detected by MTT method, and the appropriate drug concentration was screened out. The H9c2 cells cultured in vitro were randomly separated into a control group, a model group, a low-dose pinocembrin group, a high-dose pinocembrin group, a HIF-1α siRNA negative control group, and a high-dose pinocembrin + HIF-1α siRNA group. Except for the control group, H/R models were established in the other groups, and then treated with pinocembrin, HIF-1α siRNA and their negative controls. The proliferation and apoptosis of cells in each group were detected by MTT method and flow cytometry respectively; enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of inflammatory factors interleukin (IL)-17, IL-1β and IL-18 in H9c2 cells in each group; kits were used to measure the levels of oxidative stress factors catalase (CAT), glutathione peroxidase (GSH-Px), reactive oxygen species (ROS) and malondialdehyde (MDA) in H9c2 cells in each group; monodansylpentanediamine (MDC) fluorescence staining was used to detect the formation of autophagosomes in H9c2 cells in each group; Western blotting was used to detect the expression of autophagy and HIF-1α/BNIP3 pathway-related proteins in H9c2 cells in each group. Results: Compared with the control group, the cell viability, the relative content of autophagosomes, the levels of CAT and GSH-Px, LC3-II/LC3-I, and the protein expressions of P62, HIF-1α and BNIP3 in the model group decreased (P<0.05), the apoptosis rate, the levels of IL-17, IL-1β and IL-18 in cell culture supernatant, and the levels of cellular ROS and MDA increased (P<0.05). Compared with the model group, the cell viability, the relative content of autophagosomes, the levels of CAT and GSH-Px, LC3-II/LC3-I, and the protein expressions of P62, HIF-1α and BNIP3 in the low-dose and high-dose pinocembrin groups increased (P<0.05), the apoptosis rate, the levels of IL-17, IL-1β and IL-18 in cell culture supernatant, and the levels of cellular ROS and MDA decreased (P<0.05); compared with those in the low-dose pinocembrin group, the cell viability, the relative content of autophagosomes, the levels of CAT and GSH-Px, LC3-II/LC3-I, and the protein expressions of P62, HIF-1α and BNIP3 in the high-dose pinocembrin group further increased (P<0.05), the apoptosis rate, the levels of IL-17, IL-1β and IL-18 in cell culture supernatant, and the levels of cellular ROS and MDA further decreased (P<0.05); there was no obvious difference in each index of cells in HIF-1α siRNA negative control group (P>0.05). Compared with the high-dose pinocembrin group, the cell viability, the relative content of autophagosomes, the levels of CAT and GSH-Px, LC3-II/LC3-I, and the protein expressions of P62, HIF-1α and BNIP3 in the high-dose pinocembrin+HIF-1α siRNA group decreased (P<0.05), the apoptosis rate, the levels of IL-17, IL-1β and IL-18 in cell culture supernatant, and the levels of cellular ROS and MDA increased (P<0.05). Conclusion: Pinocembrin promotes autophagy by up-regulating the HIF-1α/BNIP3 pathway, thereby reducing inflammation and oxidative stress, inhibiting hypoxia/reoxygenation-induced cardiomyocyte apoptosis and reducing cell injury.
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2022, Vol. 28 
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