Fig. 4

Riboflavin supplementation suppresses hepatic injury and genes associated with inflammation in etfa+/− livers. Adult zebrafish were exposed to riboflavin containing water (0.6 mg/L) for 1 week, with water replacement (with riboflavin) once per day. Whole livers were harvested, and relative amounts of (A) FAD levels and (B) serum ALT levels were measured in etfa+/− with (white bars) or without (blue bars) riboflavin treatment compared to the wild type (etfa+/+) (see Materials and Methods) (n = 4 to 6). (C) Representative images of H&E-stained liver sections in the wild type and etfa+/− mutants with or without riboflavin treatment (n > 6 per each); scale bar, 25 µm. (D) Total RNA was isolated from whole livers as above, and mRNA expression of genes associated with inflammation (tnfa, nfkb, il1b, and mmp9), cell death (casp3a and dffa), injury (a1at), and fibrosis (col1a1a) from pooled RNA were measured by qRT-PCR (see Materials and Methods). (E) Changes in relative mRNA expression levels of flad1 were also measured. An equal amount of total RNA from three livers were pooled and used for cDNA synthesis; the same amount of cDNA was used for qPCR analyses. Error bars show mean ± SD; *P < 0.05, **P < 0.005. Abbreviation: n.s., not significant.