Methyl ferulic acid exerts anti-apoptotic effects on L-02 cells via the ROS-mediated signaling pathway

The present study aimed to investigate the anti-apoptotic effects of methyl ferulic acid (MFA) on L-02 cell apoptosis induced by ethanol, and to elucidate the possible underlying mechanisms. L-02 cells were examined after being soaked in ethanol (400 mM) to allow the ethanol to permeate into the cells for 24 h. Cell survival was measured by MTT assay. Cell apoptosis was assessed by both flow cytometry and single-stranded DNA assays. Intracellular reactive oxygen species (ROS) production was determined using the 2,7-dichlorofluorescein-diacetate dye. The protein expression levels of p38, p-p38, JNK, p-JNK, NADPH oxidase 4 (NOX4), p22, Bax and Bcl-2 were measured by western blot analysis. The mRNA expression levels of NOX4 and p22 were measured by RT-PCR. It was identified that MFA markedly suppressed the ethanol-induced apoptosis and necrosis of L-02 cells. In addition, MFA decreased the expression levels of superoxide dismutase, catalase and phospholipid hydroperoxide gluthione peroxidase, and downregulated the levels of Bax/Bcl-2 and the cleaved forms of caspase-3 in a dose- and time-dependent manner. This indicated that MFA attenuated the apoptosis of L-02 cells. MFA also decreased the elevated mRNA and protein expression levels of Nox4 and p22(phox), and the production of intracellular ROS triggered by ethanol. Further analysis demonstrated that MFA significantly attenuated the phosphorylation of JNK and p38, which are major components of the mitogen-activated protein kinase (MAPK) pathways. On the whole, the findings of this study demonstrated that MFA attenuated the apoptotic cell death of L-02 cells by reducing the generation of ROS and inactivating the MAPK pathways.