Abstract: Objective: To investigate the protective effect of luteolin (LUT) and folic acid (FA) on aflatoxin B1 (AFB1)-induced injury of human normal esophageal epithelial cells (HEECs) as well as on the methylation of the MTHFR gene. Methods: HEECs were exposed to different concentrations (0, 13, 25, 50, 100 and 200 μmol/L) of AFB1 for 24, 48 or 72 h, and cell viability was assessed by cell counting kit-8 (CCK-8) assay. Subsequently, HEECs were randomly divided into several groups including negative control, AFB1 poisoning, LUT intervention (160 μmol/L), FA intervention (20 and 200 μmol/L) and joint intervention (160 μmol/L LUT + 20 μmol/L FA, and 160 μmol/L LUT + 200 μmol/L FA). After 24 h of treatment, cell viability was assessed by CCK-8 assay, cell cycle and apoptosis were analyzed by flow cytometry, the protein expression of MTHFR was detected by Western blot, and the methylation of the MTHFR gene promoter was detected by the MassARRAY method. Results: Cell proliferation was inhibited after being exposed to each AFB1 concentration. Compared with AFB1 poisoning group, cell cycle arrest and inhibitory and apoptotic rates were significantly reduced and the up-regulation of MTHFR protein expression was decreased in LUT and joint intervention groups (P < 0.05). Moreover, LUT and/or FA interventions reduced the hypermethylation level of the MTHFR gene promoter (P < 0.05). Conclusion: AFB1 has toxic effects on HEECs as indicated by the inhibition of proliferation, cell cycle arrest, the promotion of apoptosis, and the up-regulation of MTHFR protein expression. LUT ameliorates these injuries and protects against the toxic effects of AFB1. Moreover, AFB1 can increase the methylation level of the MTHFR gene promoter, resulting in epigenetic changes. LUT and/or FA intervention can reduce the methylation level and reverse the epigenetic changes.

Key words: luteolin, folic acid, aflatoxin B1, human normal esophageal epithelial cells, MTHFR, methylation, epigenetic