Fig. 1. RBN inhibited lipid accumulation by inhibiting lipogenesis-related mRNA expression and improved NAFLD pathological features. (A) AML-12 cells were treated with a combination of oleic acid and palmitic acid (OPA) with or without (w/wo) RBN for 18 h, and hepatic TG level was measured. The values presented are the means ± SD from two independent experiments. The means with different superscript letters are significantly different, p<0.05 (one-way ANOVA). (B) AML-12 cells were treated with OPA w/wo RBN for 24 h, and cytotoxicity was measured using MTT assays. (C) AML-12 cells were incubated with OPA in the presence or absence of RBN for 18 h. Total RNA was extracted from the cells, and cDNA was synthesized for qRT-PCR. The results are demonstrated as the relative fold change of the control value, and the values presented are the means ± SD from three independent experiments. The means with different superscript letters are significantly different, p<0.05 (one-way ANOVA). (D) Body weight gain was calculated by subtracting the weight at the start of the experiment from that at the end of experiment. The values presented are the means ± SE. The means with different superscript letters are significantly different, p<0.05 (one-way ANOVA). (E) Representative images of the liver are demonstrated (left panel), and the average values of liver mass after 12 weeks of RBN supplementation are measured (right panel). The data demonstrated are the means ± SE (n=6/each group). The means with superscript letters are significantly different, p<0.05 (one-way ANOVA). (F) After a 12-week RBN supplementation, the mouse liver specimens were stained with H&E, and representative images are shown (left panel). The livers were used for measuring hepatic TG level (right panel). The results are demonstrated as the means ± SE (n=6/each group). The means with superscript letters are significantly different, p<0.05 (one-way ANOVA). (G) An oral glucose tolerance test (OGTT) was performed following an overnight fast at week 11. Blood glucose concentrations in mice (left panel) during an OGTT at the fifth time point. The under curve (AUC) (right panel) was calculated from OGTT. The data were expressed as the means ± SE (n=6/each group). The means with superscript letters are significantly different, p<0.05 (one-way ANOVA). (H) The level of serum insulin was measured after a 12-week RBN supplementation (left panel). HOMA-IR was determined by the formula indicated in the Materials and Method section (right panel). The data is presented as the means ± SE (n=6/each group). The means with superscript letters are significantly different, p<0.05 (one-way ANOVA).
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