Prepared radix polygoni multiflori and emodin alleviate lipid droplet accumulation in nonalcoholic fatty liver disease through MAPK signaling pathway inhibition

Figures

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  Figure 1. Enrichment analysis of the anti-NAFLD mechanisms of PRPM. (A) Sixty-six common targets of NAFLD and PRPM. (B) The top 10 significantly enriched (p < 0.05) terms in BP, CC and MF of GO analysis. The Y-axis represents the enrichment count of the target, and the X-axis represents the GO category of the target gene. (C) The top 15 pathways with significant enrichment (p < 0.05) were selected. The Y-axis represents the pathway, while the colors represent the significance of differential enrichment, and the size of the circles represents the number of genes. (D) Ingredient-target-pathway network of PRPM. The red rhombuses indicate ingredients, the green circles indicate targets, and the orange rounded rectangle indicates pathways.

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  Figure 2. Volcano plot and heatmap of differentially expressed genes and the functional enrichment of KEGG and GO. (A) Volcano plot was constructed using the fold change values and P-adjust; red indicates upregulation, while blue indicates downregulation. (B) Heatmap of the differential gene expression; different colors represent the trend of gene expression in different tissues. The top 50 upregulated genes and the top 50 downregulated genes are shown in this figure. (C–F) The enriched KEGG signaling pathways and GO functions were selected to demonstrate the primary biological actions of major potential mRNAs. The abscissa indicates the gene ratio, and the enriched pathways are presented in the ordinate. Colors indicate the significance of differential enrichment, and the size of the circles indicates the number of genes. In the enrichment results, p < 0.05 and FDR <0.05 were considered meaningful pathways (enrichment score with −log10 (P) of more than 1.3).

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  Figure 3. PRPM improves nonalcoholic fatty liver disease. (A) H&E staining, ×100, ×200, ×400; Oil red staining, ×200, ×400. (B) Quantitative analysis of the Oil red O staining-positive area (%). (C–G) ELISA analysis of the insulin index, serum triglycerides (TG), alanine aminotransferase (ALT), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). *indicates a comparison with NC, P < 0.05. #indicates a comparison with NAFLD, P < 0.05. The white line indicates 50 μm.

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  Figure 4. PRPM mediates the MAPK signaling pathway in NAFLD. (A) IHC staining of P38, phosphorylated P38, ERK1/2 and phosphorylated ERK1/2 in NAFLD mouse models, ×200, ×400. (B–E) Correlation analysis of H-scores of P38, phosphorylated P38, ERK1/2 and phosphorylated ERK1/2. (F) Representative Western blot of P38, phosphorylated P38, ERK1/2 and phosphorylated ERK1/2. (G–J) Western blot analysis. *indicates comparison with NC, P < 0.05. #indicates a comparison with NAFLD, P < 0.05. The white line indicates 50 μm.

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  Figure 5. Emodin mediates the MAPK signaling pathway in HepG2 cells. (A) IF staining of P38, phosphorylated P38, ERK1/2 and phosphorylated ERK1/2 (200×) in HepG2 cells. (B–E) Florescence intensity analysis of P38, phosphorylated P38, ERK1/2 and phosphorylated ERK1/2. (F) Representative Western blot of P38, phosphorylated P38, ERK1/2 and phosphorylated ERK1/2. (G–J) Western blot analysis. *indicates a comparison with NC, P < 0.05. The white line indicates 50 μm.

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  Figure 6. PRPM mediates the AMPK signaling pathway in NAFLD. (A) IHC staining of SREBP-1, ×200, ×400. (B) A correlation analysis of the H-scores of SREBP-1. (C) Representative Western blot of SREBP-1, ACC1, FASN, and ACLY. (D–G) Western blot analysis. (H–K) Relative mRNA levels of SREBP-1, ACC1, FASN, and ACLY (ratio: measured mRNA/GAPDH mRNA). *indicates comparison with NC, P < 0.05. #indicates a comparison with NAFLD, P < 0.05. The white line indicates 50 μm.

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  Figure 7. Emodin mediates the AMPK signaling pathway in HepG2 cells. (A) IF staining of SREBP-1. (B) Florescence intensity analysis of SREBP-1, ×200. (C) Representative Western blot of SREBP-1, ACC1, FASN, and ACLY. (D–G) Western blot analysis. (H–K) Relative mRNA levels of SREBP-1, ACC1, FASN, and ACLY (ratio: measured mRNA/GAPDH mRNA). (L–N) Nile red and Oil red O staining and analysis. *indicates comparison with NC, P < 0.05. A indicates a comparison with FFA Medium, P < 0.05. The white line indicates 50 μm.

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  Figure 8. The MAPK activator diprovocim restored the inhibitory effect of emodin on the AMPK signaling pathway. (A) Representative Western blot of SREBP-1, ACC1, FASN, and ACLY. (B–E) Western blot analysis. (F) Representative Western blot of SREBP-1, ACC1, FASN, ACLY, and P-P38. (G–K) Western blot analysis. (L–N) Nile red and Oil red O staining and analysis. *indicates comparison with NC, P < 0.05. A indicates a comparison with FFA Medium, P < 0.05. The white line indicates 50 μm.