Brazilian green propolis promotes the cytoprotective expression of heme oxygenase-1 against oxidative stress injury in murine myoblast cells

Background: Sarcopenia is a progressive skeletal muscle disorder characterized by the progressive loss of muscle mass and function, resulting in physical disability and mortality. Although sarcopenia impacts a large proportion of elderly individuals, no effective treatment for this disease has yet been identified. The excessive production of reactive oxygen species (ROS) can damage tissues and promote aging, and the daily use of dietary antioxidants can be effective for maintaining skeletal muscle health. Propolis, a natural substance that is collected by honey bees, has been used as traditional medicine, and many reports have described its antioxidative properties. However, how propolis exhibits cytoprotective effects and antioxidative effects in skeletal muscles remains unclear. The purpose of this study was to investigate the antioxidative effects of ethanol-extracted Brazilian green propolis (EEBP, from Baccharis dracunculifolia) and its three constituents using an in vitro myoblast cell model. Methods: Murine myoblast C2C12 cells were treated with either EEBP or its constituents, including caffeic acid, trans-ferulic acid, and p-coumaric acid, in the presence of 100 or 300 mM H2O2 to induce oxidative stress injury. The cell death ratio and cell viability were assessed by Hoechst 33342 and propidium iodide staining and the WST-8 assay, respectively. Simultaneously, intracellular ROS production was measured by CM-H2DCFDA [5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester] assay. Finally, immunoblotting was performed in myoblast cell lysates to assess the expression level of an antioxidative enzyme, heme oxygenase-1 (HO-1). Results: We demonstrated that EEBP significantly reduced H2O2-induced cell death at a concentration of 3 mu g/ml in myoblasts. Additionally, caffeic acid at 100 mu M improved cell viability under oxidative stress conditions, but not trans-ferulic acid or p-coumaric acid. Both EEBP and caffeic acid inhibited the H2O2-induced increase in ROS production. Finally, HO-1 expression was increased by treatment with either EEBP or caffeic acid. The increase in HO-1 expression induced by H2O2 was enhanced in the presence of EEBP and caffeic acid. Conclusions: These findings indicated that EEBP has protective effects against oxidative damage in C2C12 murine myoblast cell line. Caffeic acid is an EEBP constituent that contributes to cytoprotective activity. EEBP may act as an inducer of HO-1 to prevent oxidative stress-induced myoblast death.