Qinlian Hongqu Decoction Modulates FXR/TGR5/GLP-1 Pathway to Improve Insulin Resistance in NAFLD Mice: Bioinformatics and Experimental Study

Figures

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  Figure 1. Impact of QLHQD on obesity and glycolipid metabolism in NAFLD mouse models. (A) Description of the animal experimental protocol. (B) Presentation of a line graph depicting mouse weight changes. (C) Analysis of body weight variations in the mice. (D) Evaluation of the influence of QLHQD on blood lipids in the NAFLD mouse model. (E) Assessment of the effects of QLHQD on blood glucose levels, insulin levels, and the insulin resistance index in the NAFLD mouse model. (F) Illustration of the oral glucose tolerance test (OGTT) curve along with the calculation of the AUC. (G,H) Impact of QLHQD on the serum AST and ALT levels in the NAFLD mouse model. Results are presented as the means ± SDs (n = 6). *p < 0.05, **p < 0.01, ns: not significant compared to the model control.

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  Figure 2. Impact of QLHQD on liver lipid accumulation in NAFLD mouse models. (A) Abdominal images of each experimental group. (B) Evaluation of the influence of the QLHQDs on the liver weight of the mice, n = 6. (C) Assessment of the effect of QLHQD on liver weight in mice, n = 6. (D,E) Examination of the impact of QLHQD on histopathological alterations in the livers of NAFLD mice, n = 3. (F) Analysis of the effects of QLHQD on liver TG, TC, and FFA levels in NAFLD mice, n = 6. (G) Effects of QLHQD on the expression levels of CYP7A1, SPEBP1C, and HGMCR in NAFLD mice (×200) were examined, n = 3. (H) Relative mRNA levels of CYP7A1, SPEBP1C, and HGMCR were analyzed, n = 6. Results are presented as the means ± SDs, *p < 0.05, **p < 0.01, ns: not statistically significant compared with the model control.

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  Figure 3. Network pharmacology analysis. (A) Venn diagram was used to illustrate the overlap between the QLHQD and NAFLD targets. (B) Drug-component-target-disease network was constructed, where triangles represent QLHQD, yellow nodes represent drugs for QLHQD, purple nodes represent targets, and diamond-shaped nodes represent hyperlipidemia. (C) Top 10 BPs, MFs, and CCs in the GO analysis. (D) Top 20 lipid metabolism-related pathways were determined via KEGG analysis and labeled at the pathway level. (E) Top 20 pathways related to lipid metabolism from KEGG analysis.

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  Figure 4. Bioinformatics analysis. (A) PCA of GSE147562. (B) Volcano map of DEGs in the data set. (C) Heatmap of DEGs in the data set. (D–I) GSEA results for DEGs.

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  Figure 5. Validation of molecular docking. (A) Common signaling pathways identified via KEGG and GSEA. (B) Molecular docking heatmaps. (C) Binding pattern of the FXR protein with luteolin. (D) Binding mode of the FXR protein to wogonin. (E) Binding mode of the FXR protein to berberine. (F) Binding mode of the FXR protein with hesperetin. (G) Binding mode of the FXR protein with baicalin. (H) Binding mode of the FXR protein with nobiletin. (I) Binding mode of the TGR5 protein with Luteolin. (J) Binding mode of the TGR5 protein with baicalin.

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  Figure 6. Impacts of QLHQD on intestinal tissue FXR, TGR5 and GLP1 in the NAFLD mouse model. (A) Identification of FXR protein expression through immunofluorescence detection (scale bar: 200 μm). (B) Identification of TGR5 protein expression through immunofluorescence detection (scale bar: 200 μm). (C) Identification of GLP1 protein expression through immunofluorescence detection (scale bar: 200 μm). (D) Relative mRNA levels of FXR, TGR5, and GLP-1, n = 6. (E) Protein expression of FXR, TGR5, and GLP-1 was detected via protein blotting. (F) Quantitative analysis of the FXR, TGR5, and GLP-1 band intensities. n = 3. Results are presented as the means ± standard deviations. Statistical significance is denoted as *p < 0.05, **p < 0.01, and ns indicates no significance compared with the model control.

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  Figure 7. Effects of the QLHQDs on insulin resistance in the liver tissues of NAFLD model mice. (A) PI3K protein expression was detected via immunofluorescence (scale bar: 200 μm). (B) Identification of AKT protein expression through immunofluorescence detection (scale bar: 200 μm). (C–H) Relative mRNA levels of PI3K, AKT, INSR, GSK3B, GCk, and GYS2, n = 6; (I,J) PAS staining of livers and semiquantitative analysis, n = 3. Results are presented as the means ± standard deviations. Statistical significance is denoted as *p < 0.05, **p < 0.01, and ns indicates no significance compared with the model control.