The effects and mechanisms of Xiaoyao San on nonalcoholic fatty liver disease rat based on transcriptomics and proteomics analysis

Figures

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  Fig. 1. Total ion current diagrams of XYS mass spectrometry analysis in (A) negative ion mode, and (B) positive ion mode, and of XYS drug-containing serum in (C) negative ion mode, and (D) positive ion mode.

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  Fig. 2. Growth curves (A), water intake (B), food intake (C), body weight (D), liver index (E), and Lee’s index (F) of rats in different groups. *P < 0.05, **P < 0.01, ***P < 0.001, vs NC group; #P < 0.05, ##P < 0.01, ###P < 0.001, vs NAFLD group.

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  Fig. 3. Morphological changes of liver in each group (A); H&E staining results on pathological changes in liver (B); Lipid accumulation in the liver observed by Oil red O staining (C). ▲: Lipid droplet distribution. NAS score (D), and Oil red O area ratio (E) in each group. *P < 0.05, **P < 0.01, ***P < 0.001, vs NC group; #P < 0.05, ##P < 0.01, ###P < 0.001, vs NAFLD group.

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  Fig. 4. The levels of ALT (A), AST (B), GGT (C), TC (D), TG (E), HDL-C (F), LDL-C (G), FBG (H), ROS (I), IL-1β (J), IL-6 (K), and TNF-α (L) in serum of rats in each group. *P < 0.05, **P < 0.01, ***P < 0.001, vs NC group; #P < 0.05, ##P < 0.01, ###P < 0.001, vs NAFLD group.

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  Fig. 5. Analysis of the difference of mRNA expression level in liver tissue between NC group or XYS group and NAFLD group. (A): PCA results of RNA-seq data. (B): Histogram of differential gene expression among different groups. (C, D): Volcano maps depicting the relative abundance of transcripts. The abscissa represented the change of gene expression multiples, and the ordinate marked the significance level of differential genes. The red and green dots indicated the up-regulated and downregulated differential genes, respectively; and the gray dots referred to non-DEGs. (E): Heat maps of gene expression among different groups. The abscissa represented the sample name and hierarchical clustering results, and the ordinate was the differential genes and hierarchical clustering results. Red indicated high expression while blue signified low expression.

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  Fig. 6. Difference analysis of protein expression levels in liver tissues between NC group or XYS group and NAFLD group. (A): PCA results of proteome data. (B): Histogram of differential protein expression. (C, D): Volcanic maps displaying the relative abundance of proteins. The abscissa represented the change of protein expression multiples, and the ordinate showed the significance level of differential proteins. The red and green dots marked the upregulated and downregulated DEPs, respectively, and the gray ones were non-DEPs. (E): Heat maps for protein expression. The abscissa represented the sample name and hierarchical clustering results, and the ordinate signified the differential protein and hierarchical clustering results, with red and green indicating high and low expression, respectively.

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  Fig. 7. Venny diagram of DEPs and DEGs between the NC group and the NAFLD group (A) and that between the NAFLD group and the XYS group (B). Note: all_tran and diff_tran represented all genes and DEGs identified by the transcriptome, respectively; all_prot and diff_prot signified all proteins and DEPs identified by the proteome, respectively. Protein and gene differential fold clustering heat map between the NC group and the NAFLD group (C) and that between the NAFLD group and the XYS group (D). Note: Each row in the figure represented a protein or a corresponding gene, and the two columns were the difference multiples of the proteome and transcriptome data, respectively. The red part marked the up-regulated protein or gene, while the downregulated protein or gene was labelled in green. GO enrichment analysis results of DEGs and DEPs in each group based on transcriptome and proteome (E, F). Note: The abscissa represented the secondary GO item, and the ordinate represented the number of genes or proteins enriched to the GO item. Bar charts based on the results of KEGG pathway enrichment analysis (G, H). Note: The abscissa represented the number of differential proteins and differential…

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  Fig. 8. ELISA results of cAMP content (A) in liver tissue of rats in each group. Quantitative analysis of p-PKA/PKA (B), PPARα (C), FOXO1 (D), p-NF-κB/NF-κB (E), RI3P (F), p-MLKL (G) and TGF-β (H) protein in liver tissue of rats in each group. *P < 0.05, **P < 0.01, ***P < 0.001, vs NC group; #P < 0.05, ##P < 0.01, ###P < 0.001, vs NAFLD group.

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  Fig. 9. Based on the integration of transcriptomics and proteomics analyses, the mechanism of XYS in the treatment of NAFLD. Schematic representation of the pathway retrieved from the KEGG database19 (https://www.kegg.jp/). (KEGG Pathway ID: rno03320, rno04024, rno04068, rno04064).

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  Fig. 10. The chemical analysis of XYS and its mechanism of action in NAFLD treatment.