The Curative Effects of Vitamins D3&K2 on the Expressions of p53, Bax, Bcl-2, and Oxidative Stress Induced by Methotrexate in ZFL Epithelial Cells

Methotrexate (MTX), a folate antimetabolite, is a cytotoxic drug known to cause cytotoxicity associated with free oxygen radicals. This study investigated the effect of vitamins D3 and K2 on MTX-induced liver cell injury using the zebrafish liver cell line (CRL-2643). Observed effects, levels of antioxidant enzymes, lipid peroxidation marker, and total antioxidant/oxidant status were evaluated by spectrophotometric methods. The mRNA expressions of p53, Bax, and Bcl-2 were measured using RT-PCR. In addition, acridine orange/ethidium bromide staining was performed to analyze the apoptosis status of the cells. The IC₅₀ value of MTX at 48 h was calculated as 442 µg/mL with an MTT assay. The doses of D3&K2 are determined based on the recommended dose to be taken daily. As a result, this study suggests that MTX treatment induced oxidative damage on the liver cell, as assessed by increased reactive oxygen levels, lipid peroxidation, and decreased glutathione levels. In addition, while MTX increased the expression of the p53 and proapoptotic marker Bax, it decreased the expression of the anti-apoptotic factor Bcl-2. In conclusion, D3&K2 treatment protects against MTX-induced liver cell toxicity. It is thought that they can be used as a potential agent in clinical applications with MTX in treatment.