Integrative lipidomic and transcriptomic study unravels the therapeutic effects of saikosaponins A and D on non-alcoholic fatty liver disease

Figure 1. SSa and SSd ameliorated HFSW-induced hepatic steatosis and liver injury. Mice were fed chow diet or HFSW diet for 8 weeks, and administered with either SSa or SSd as described in the Material and methods. (A) Body weights of mice in different groups. (B) The serum ALT, AST, and ALP activity levels. (C) The serum triglycerides (TG) levels and low-density lipoprotein-cholesterol (LDL-C) levels. (D) The intrahepatic TG (IHTG) levels. (E) Representative images of hematoxylin and eosin staining of liver paraffin sections. Scale bar: 50 μm. Results are presented as mean ± SEM (n ≥ 8 mice in each group). ∗∗∗P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. HFSW group.

Figure 2. SSa and SSd extensively modulated hepatic lipid metabolism. Lipidomic analysis was performed on mice liver tissue samples (n = 8). (A) Significant differentially altered lipids (DALs) were clustered and plotted as a heatmap. (B) Four clusters (clusters 1–4) were highlighted, and the trend lines indicating the change of intrahepatic levels of these DALs were shown. (C) DALs in each cluster were subcategorized based on their lipid classes. (D) and (E) Fold changes of DALs belonging to different lipid classes upon SSa and SSd challenges (SSa vs. HFSW and SSd vs. HFSW) were plotted in panels (D) and (E), respectively. The size of each dot represents −log10 (P value).

Figure 3. SSa and SSd regulated hepatic transcriptome in a large scale. RNA-seq analysis was performed on mice liver tissue samples (n = 4). (A) Heatmap represented the expression profile of differentially expressed genes (DEGs) (P < 0.05) identified in the comparison of SSa treatment groups (SSa-L, SSa-M, and SSa-H) vs. HFSW group, and SSd (SSd-L, SSd-M, and SSd-H) treatment groups vs. HFSW group. (B) Gene set enrichment analysis (GSEA) plot for KEGG entry: NAFLD based on DEGs in SSa and SSd groups vs. HFSW group. NES, normalized enrichment score. (C) and (D) GO pathway enrichment analysis (BP: biological process) based on DEGs in SSa and SSd groups vs. HFSW group. Padj, adjusted P value.

Figure 4. Effects of SSa and SSd on glycerollipid metabolism. (A) The expression profile of DEGs implicated in the metabolism of glycerolipid was shown as heatmap. (B) GSEA plot for KEGG entry: glycerollipid metabolism based on DEGs. (C) and (D) The relative mRNA levels of Pnpla3 and Lpin1 (C) and Ces1d and Lipg (D) were determined by real-time PCR and normalized using Hprt1 as an internal control. Results are presented as mean ± SEM (n ≥ 6 mice in each group). ∗∗∗P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. HFSW group. (E) The changes of intrahepatic levels of diacylglycerols (DAG) and phosphatidic acids (PHA) in the treatment group (SSa and SSd) and model group (HFSW), when compared to the control group. (F) The biological process of glycerolipid metabolism. The orange and green dots represented increased and decreased lipids after SSa and SSd treatment, respectively. The red and blue squares represented upregulated and downregulated genes upon SSa and SSd challenges, respectively.

Figure 5. Effects of SSa and SSd on the regulation of fatty acid biogenesis. (A) The expression profile of DEGs implicated in the biosynthesis of fatty acid was shown as heatmap. (B) The relative mRNA levels of Acaca and Fasn were determined by real-time PCR and normalized using Hprt1 as an internal control. (C) The relative protein levels of ACCα and FASN were determined by Western blot analysis and using β-actin as a loading control. Representative images of immunoblots were shown (n = 6 mice, pooled samples). (D) The relative mRNA level of Srebf1 was determined by real-time PCR and normalized using Hprt1. (E) Representative immunoblot images of SREBP-1 and mature SREBP-1 were shown and β-actin was used as a loading control (n = 6 mice, pooled samples). (F) The expression profile of SREBP-1 target genes was presented as heatmap. Results were presented as mean ± SEM (n ≥ 6 mice in each group). ∗∗∗P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. HFSW group.

Figure 6. The effect of SSa and SSd on fatty acid degradation. (A) The expression profile of DEGs implicated in the fatty acid catabolism was shown as heatmap. (B) GSEA plot for KEGG entry: fatty acid degradation based on DEGs. (C) The relative mRNA levels of Cpt1a, Acsl1, and Acox1 were determined by real-time PCR and normalized with Hprt1. (D) The protein level of CPT-1α was determined by Western blot analysis using β-actin as a loading control. Representative immunoblot images were shown (n = 6 mice, pooled samples). (E) The changes of intrahepatic levels of acyl-carnitines (ACARs) in the treatment group (SSa and SSd) and model group (HFSW), when compared to the control group. (F) The biological process of fatty acid biosynthesis and degradation. The orange and green dots represented increased and decreased lipids after SSa and SSd treatment, respectively. The red and blue squares represented upregulated and downregulated genes upon SSa and SSd challenges, respectively. Results were presented as mean ± SEM (n ≥ 6 mice in each group). ∗∗P < 0.01 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. HFSW group.

Figure 7. The effect of SSa and SSd on fatty acid metabolism in primary liver cells. Mouse primary hepatocytes (MPH) were incubated with OA:PA (500 μmol/L:250 μmol/L) and treated with SSa and SSd as indicated for 24 h. (A) Representative images (scale bar: 100 μm) of oil red O staining of MPH and magnified images (scale bar: 50 μm). (B) The intracellular TG levels were determined as described in the method. (C) and (D) The relative mRNA levels of Acaca and Fasn (C), and Ppara, Acox1, and Cpt1a (D) were determined by real-time PCR and normalized using Hprt1 as an internal control. Results were presented as mean ± SEM (n = 3). #P < 0.05, ##P < 0.01 vs. control group; ∗P < 0.05, ∗∗P < 0.01 vs. OA:PA group.

Figure 8. Regulative effects of SSa and SSd on transcription factors (TFs) involved in lipid metabolism. (A) Transcription factors enrichment analysis (TFEA) plot based on the upregulated DEGs enriched in lipid metabolism pathways. (B) The expression profile of predicted TFs involved in the regulation of lipid metabolism. (C) The in vivo biotransformation and metabolism of SSa and SSd. (D) Molecular docking results of SSs and metabolites binding to indicated TFs. Binding free energy (BFE) and relative BFE (RBFE) indicating the potential binding affinity were calculated as instructed in the method. (E) The representative images for the binding mode of SSa (purple), SSd (pink), and synthetic PPARα agonist TIPP703 (white) with the crystal structure of PPARα (PDB ID: 2ZNN, grey). (F) The expression profile of PPARα and its coactivators. (G) The protein levels of PPARα and RXRα in the liver were determined by Western blot using β-actin as a loading control (n = 6 mice, pooled samples). (H) The expression profile of PPARα target genes was presented as a clustered heatmap.