Ficus hirta Vahl. Ameliorates Nonalcoholic Fatty Liver Disease through Regulating Lipid Metabolism and Gut Microbiota

Figures

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  Figure 1. UPLC-Q/TOF-MS/MS analysis. The total ion chromatogram (TIC) of FV of by UPLC-Q/TOF-MS/MS in the (a) negative and (b) positive ion modes.

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  Figure 2. Amelioration of lipid accumulation in PA-induced HepG2 cells by FV. (a) Oil red O was used to measure the level of lipid accumulation (magnification 100x, scale bar = 250 μm). (b) The oil red O-positive area was analyzed and quantified. (c–j) The relative mRNA expression levels of FABP1, SCD1, CD36, HMGCR, ACACA, CCL5, IL-1β, and TNF-α were determined by qRT-PCR. (k) The proinflammatory factor protein levels of IL-1β, IL-6, and TNF-α. (l) The protein levels related to lipid metabolism of SREBP-1, ACOX1, CD36, CPT1α, and HMGCR were analyzed by western blotting, and the relative ratios were calculated and expressed as the mean ± SD; n = 3. #P < 0.05 means that the difference between the NC group and the PA group is significant. ∗P < 0.05 means that the difference between the PA group and the FV (30 mg/mL) group or the FV (15 mg/mL) group is significant.

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  Figure 3. Effects of FV on accumulation of liver fat and lipid levels in serum in HFD-fed mice. (a) Body weight, (b) macroscopic observation of livers in subjects in different groups, (c) weekly food intake per mouse in each group, (d) liver index, (e) light microscopic H&E image and oil red O staining image of liver tissues of subjects in different groups (x100 original magnification, scale bar = 250 μm; 400x original magnification, scale bar = 50 μm), H&E score and oil red O score, (f) serum TC, (g) liver TC, (h) liver TG, (i) serum AST, (j) ALT, and (k) serum LDL-C. The data are expressed in the format of mean ± SD (n = 8, 10, 10). #P < 0.05 means there is a significant difference between the NFD group and the HFD group. ∗P < 0.05 means the difference between the HFD group and the FV-L group or the FV-H group is significant. NFD: normal fat diet group; HFD: high-fat diet group; FV-L group: high-fat diet with FV administration at dose of 5 g medicinal materials/kg body weight; FV-H group: high-fat diet with FV at dose of 10 g medicinal materials/kg body weight.

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  Figure 4. Effects of FV on liver lipogenesis-related markers in the mice fed with HFD. (a–h) The relative mRNA expression levels of Acaca, Cd36, Cpt1α, Srebp-1, Hmgcr, Pparα, Pparγ, and Scd1 were determined by qRT-PCR. (i) The lipid metabolism relevant protein levels of SREBP-1, ACOX1, CD36, CPT1α, and HMGCR were analyzed by western blotting, and the relative ratios were calculated. (j–l) Serum IL-6, IL-1β, and TNF-α were identified by ELISA kits. (m, n) The relative mRNA expression levels of IL-1β, TNF-α, and Ccl5 were identified by qRT-PCR. (p) The proinflammatory factor protein levels of IL-1β, IL-6, and TNF-α were analyzed by western blotting, and their relative ratios were calculated. The densitometry was obtained by averaging repeated experiments results, normalized to GAPDH. The data are expressed as the mean ± SD, n = 3. #P < 0.05 means there is significant difference between the NFD group and the HFD group. ∗P < 0.05 means the difference between the HFD group and the FV-L group or the FV-H group is significant.

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  Figure 5. Key targets and pathway between the NFD group, the HFD group, and the FV-H group revealed by RNA-seq analyses. (a) PCA of the RNA-seq data from the NFD group, the HFD group, and the FV-H group. (b) Volcano plot indicating the DEGs (red, upregulated genes; green, downregulated genes) from three groups (HFD group vs. NFD group or FV-H group). (c) The KEGG pathway enrichment analysis of crossover DEGs. (d) The GO enrichment analysis of crossover DEGs. (e, f) GSEA pathway enrichment analysis of pathways that are related to inflammation, lipid metabolism, and fibrosis. (g) Heat map of gene expression profiles related to lipid metabolism, fibrosis, and inflammation based on RNA-seq dataset. n = 3 in each group.

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  Figure 6. Alleviation of lipid accumulation and inflammation by FV through regulating CD36 in HepG2 cells with PA inducement. (a, c) CD36 protein levels after CD36 knockdown or CD36 overexpression and quantitative analysis. (b, d) TG contents of HepG2 cells in different treatment groups. (e, f) Oil red O to examine the accumulation level of lipid in HepG2 cells and their quantified scores (magnification 100x, scale bar = 250 μm). (g) The mRNA levels of SCD1, PPAR γ, ACACA, SREBP-1, IL-1β, and CCL5 in siCD36 HepG2 cells with or without FV administration under the condition of PA inducement. (h) The mRNA levels of PPAR γ, SREBP-1, IL-1β, and TNF-α in CD36 OE HepG2 cells with or without FV administration under the condition of PA inducement. (i) The protein levels of CD36, HMGCR, ACOX1, and SREBP-1 in siCD36 HepG2 cells. (j) The protein levels of CD36, HMGCR, ACOX1, and SREBP-1 in CD36 OE HepG2 cells. n = 3, ∗P < 0.05 vs. NC group for (a, c); #P < 0.05 vs. NC group, ∗P < 0.05 vs. PA group or PA+siCD36 group/PA+CD36 OE group (b–j).

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  Figure 7. Diversity of gut microbiota in mouse models induced by different diets. (a) PCoA result structure of each group. (b) Taxonomic distribution of bacterial communities of NAFLD mouse fecal samples at the phylum level. (c) Firmicutes/Bacteroidetes ratio at the phylum level. (d) LDA score of differentially abundant taxa between the NFD group and the HFD group. (e) LDA score of differentially abundant taxa between the HFD group and the FV-H group. (f) Relative abundance of g_Allobaculum, g_Faecalibaculum, g_Lactococcus, g_norank_f_Muribaculaceae, g_Ileibacterium, g_Lachnospiraceae_UCG-006, g_Desulfovibrio, and g_Ruminococcus_torques_group at the genus levels. n = 8, 10, 10; #P < 0.05 vs. the NFD group, ∗P < 0.05 vs. the HFD group.

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  Figure 8. Correlation analyses between metabolic parameters and genera microbiota. (a) Spearman correlation heat map between 8 genera microbiota and 10 metabolic parameters; Spearman correlation heat map between 8 genera microbiota and 4 signaling pathways. (b–e) Linear regression analyses between metabolic parameters and genera microbiota. Significant correlations were marked by ∗P < 0.05.

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  Figure 9. The speculative mechanism of Ficus hirta Vahl. for treatment of NAFLD in HFD-fed mice. FV alleviates nonalcoholic fatty liver disease through adjusting lipid metabolism and inflammation via downregulation of CD36 and regulating the structure of gut microbiota.