Shengmai injection alleviates H2O2‑induced oxidative stress through activation of AKT and inhibition of ERK pathways in neonatal rat cardiomyocytes

Abstract

Ethnopharmacological relevance: Shengmai injection (SMI) is a classical traditional Chinese medicine (TCM) officially recorded in Pharmacopoeia of the People's Republic of China (version 2015) and has long been used to treat heart failure in China. However scientific evidence for the anti-oxidative stress potential of SMI used in traditional medicine is lacking.

Aim of study: The present study aimed to evaluate the efficacy of SMI in alleviating H₂O₂‑induced Oxidative Stress the underlying mechanisms MATERIALS AND METHODS: H₂O₂-induced oxidative stress model of cardiomyocytes was established with primary cultured neonatal rat cardiomyocytes. CCK8 cell viability assay and lacatate dehydrogenase cytotoxicity assay were performed to ensure the safety dose and lowest effective dose for the mode employing CCK-8 cell viability assay kit and lactate dehydrogenase cytotoxicity assay kit. ROS levels were determined using CM-H2DCFDA fluorescent probe in cardiomyocytes with H₂O₂-induced oxidative stress. The change of NAD(P)H level in cardiomyocytes was evaluated during the process of oxidative stress. The content of myocardial cytosolic Ca²⁺ and Ca²⁺ was determined using Fura-2/AM and Rhod 2-AM fluorescent probe in mitochondrial in the process of oxidative stress. Annexin V-FITC/PI double staining was applied to examine the apoptotic cells in cardiomyocytes with oxidative stress. To identify the apoptosis after oxidative stress myocardial cells with the application of Annexin V-FITC/PI double staining apoptosis detection kit. Quantitative polymerase chain reaction (RT-PCR) was applied to measure the expression of antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GSR). Western blot was performed to observe the phosphorylation of AKT and ERK1/2.

Results: SMI was shown to significantly attenuate oxidative stress-induced cell proliferation arrest and apoptosis in neonatal rat cardiomyocytes. In addition, SMI treatment could decrease the production of reactive oxygen species (ROS), nicotinamide adenine dinucleotide (NADH) and malondialdehyde (MDA), and reduce the overloads of cytoplasmic Ca²⁺ and mitochondrial Ca²⁺ induced by H₂O₂. SMI could also restore the mRNA expression and activities of SOD, GSR, and CAT suppressed by H₂O₂. Mechanistically, SMI upregulated intracellular AKT phosphorylation and downregulate ERK1/2 phosphorylation in H₂O₂-treated cardiomyocytes. Pretreatment with LY294002, an AKT phosphorylation inhibitor, suppressed the protective role of SMI in cardiomyocytes, while pretreatment with PD98059, an ERK1/2 phosphorylation inhibitor, enhanced the effect of SMI.

Conclusions: In conclusion, SMI may attenuate oxidative stress-induced damage in cardiomyocytes potentially through the AKT and ERK1/2 pathway and can function as a promising injectable traditional Chinese medicine to treat oxidative stress-induced injury.

Keywords: AKT; ERK1/2; Neonatal rat cardiomyocytes; Oxidative stress; Shengmai injection.