Tongxin formula protects H9c2 cardiomyocytes from cobalt chloride-induced hypoxic injury via inhibition of apoptosis.

Abstract

In this study, the effect of the Tongxin formula (TXF) on the apoptosis of H9c2 cardiomyocytes induced by cobalt chloride (CoCl2) was investigated, and the potential mechanism was explored. A hypoxic injury model of H9c2 cardiomyocytes was established using CoCl2. The cell viability was measured using a Cell Counting Kit-8 assay. The lactate dehydrogenase (LDH) release and caspase-3 activity were measured using spectrophotometry. The apoptosis was measured via Annexin V-FITC/PI staining and flow cytometry. The changes in the mitochondrial membrane potential were examined using immunofluorescence microscopy following the loading of JC-1 probes. The expressions of apoptosis-related proteins and key proteins in the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway were examined via immunoblotting. The different TXF concentrations studied significantly improved the percentage of viability of cardiomyocytes with hypoxic injury, and the LDH release, apoptotic rate, caspase-3 activity, and levels of cleaved caspase-3 protein were reduced in the injured cells. Additionally, the TXF group had increased mitochondrial membrane potential, upregulated expression of Bcl-2 and p-Akt proteins, and significantly reduced expression of cleaved caspase-3 protein in the cells with hypoxic injury. Moreover, in the TXF group, the treatment significantly reduced the BAX protein expression, but the difference was not statistically significant compared with the CoCl2 group. In this study, TXF regulated the expression of apoptosis-related proteins, inhibited apoptosis, increased the mitochondrial membrane potential, and alleviated damage to the mitochondrial membrane, thereby protecting the cardiomyocytes from hypoxic injury. The underlying mechanism could be related to activation of the PI3K/Akt signaling pathway and upregulation of the Bcl-2 protein.