Yiqi Huoxue Decoction modifies the expression of myocardial cytoskeleton-associated proteins by regulating the AMPK signaling pathway in H9c2 cells exposed to hypoxic conditions

Abstract

Background: In myocardial ischemia, hypoxia leads to destruction of the cytoskeleton, and especially the imbalance of microtubule polymerization-depolymerization, which seriously affects the structure and function of cardiomyocytes. We previously showed that a Yiqi Huoxue Decoction (YQHX) improves mitochondrial function and decreases anti-oxidative effects in hypoxia-induced H9c2 cell injury. Therefore, in this study we investigated whether YQHX protects against hypoxia-induced damage by decreasing damage to the cardiac cytoskeleton. Methods: After reaching 70%–80% confluence, H9c2 cells were synchronized in serum-free Dulbecco's Modified Eagle Medium for 6 hours, then divided into control, model, and YQHX (100, 200, 400 μg/mL) groups, which were then grown in a hypoxic atmosphere for 12 hours. Cardiac cell viability was assessed using an xCELLigence system. The levels of lactate dehydrogenase, maleic dialdehyde, and superoxide dismutase in H9c2 cell supernatants were measured. Hoechst 33258 staining was employed to observe cardiac cell apoptosis. Confocal microscopy, immunofluorescence, and western blot analysis were performed to evaluate the protective effects of the YQHX against hypoxia-induced injury in the H9c2 cell line. Results: Cells that were pretreated with YQHX were more able to maintain their microtubule structure in the early stages of hypoxia and had better myocardial fitness in response to hypoxia compared with cells that were not pretreated. However, hypoxia-induced upregulation of α-tubulin and β-tubulin expression antagonized the protective effect of YQHX (100 μg/mL). In addition, YQHX (100 μg/mL) treatment significantly upregulated MAP4 protein expression (P = .003) and downregulated p-AMPKα protein expression (P < .001) compared with the model group. Conclusion: The results indicate that YQHX plays a role in protecting against oxidative stress injury and apoptosis in H9c2 cells. Notably, our results suggested that the YQHX could mitigate the damage to the cardiac cytoskeleton and the dysregulation of AMPK-related protein signaling pathways that are induced by hypoxia. Keywords: Yiqi huoxue Decoction, Hypoxia, Myocardial cytoskeleton, Microtubules, AMPK