Gut microbiota/sphingomyelin-mediated necroptosis was associated with low doses of aflatoxin B1 driving NASH-associated fibrosis

Non-alcoholic steatohepatitis (NASH) is commonly associated with liver fibrosis. Aflatoxin B1 (AFB1) is a potent hepatotoxin. Our previous study indicated that AFB1 at 40 μg/kg.bw for 2 weeks aggravated NASH instead of fibrosis. This study aims to investigate whether extended AFB1 processing time could aggravate hepatic fibrosis and their underlying mechanisms. The results showed that AFB1 at 20, 40 and 80 μg/kg.bw for 4 weeks aggaravated NASH-associated fibrosis in CDAHFD-fed mice as demonstrated by increasing serum and liver lipid accumulation, inflammation and fibrosis. AFB1 at 40 μg/kg.bw induced gut microbiota disorders and intestinal barrier damage. AFB1 significantly elevated serum sphingomyelin (SM) levels and necroptosis was significantly enriched with differential metabolites. Also, AFB1 markedly increased liver SM levels and necroptosis. Fecal microbiota from AFB1-treated mice aggravated hepatic fibrosis, SM accumulation and necroptosis in NASH mice. SM treatment promoted hepatic fibrosis and necroptosis as same as AFB1 treatment. Myriocin alleviated the AFB1-aggravated fibrosis and necroptosis. In addition, GSK-872 mitigated the exacerbating effects of AFB1 on NASH- associated hepatic fibrosis. These data indicated that low doses of AFB1 for 4 weeks aggravated NASH-associated fibrosis via SM-mediated necroptosis by gut microbiota disorders, suggesting low doses of AFB1 is also a risk for NAFLD.