Gentiana scabra Restrains Hepatic Pro-Inflammatory Macrophages to Ameliorate Non-Alcoholic Fatty Liver Disease

Figures

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  FIGURE 1. Bioinformatics analysis identified pharmacodynamic targets of GS. (A) A compound-target network was constructed for the seven candidate compounds of GS and 76 corresponding targets. (B) PPI networks were separately constructed for 76 potential targets of GS and 112 known NAFLD-related targets. (C) PPI networks were intersected to identify the putative targets of GS against NAFLD, which were screened by DC value. (D) An inflammation-related signaling network including TBK1 was achieved from the intersected network.

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  FIGURE 2. GS improved inflammation and insulin resistance in high-fat-fed mice. (A) The body weights were recorded weekly, n = 8. *p < 0.05 between the HFD group and the GS group. (B, C) The liver weights and liver-to-body weight ratio were measured and calculated at the end of this experiment, n = 8. (D–F) The hepatic and serum concentrations of triglyceride and cholesterol were detected by ELISA, n = 6–8. (G, H) IPGTT and ITT, n = 7–8. **p < 0.01, ***p < 0.001 between the HFD group and the GS group. # p < 0.05, ## p < 0.01, ### p < 0.001 between the HFD group and the AML group. (I) HE and oil red O staining, while the NAS score was computed by steatosis, intralobular inflammation and hepatocyte ballooning to assess disease severity, n = 6. Results are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 3. GS regulated TBK1 to restrain inflammatory cascade. (A) The relative mRNA expressions in liver tissues were determined by PCR, n = 4–5. (B) Western blot analyses of p-TBK1, TBK1, p-NF-κB and NF-κB in liver tissues and statistical graphs, n = 3. (C) Immunohistochemical staining of TBK1 and NF-κB and statistical graphs, n = 4–6. Results are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 4. GS inhibited pro-inflammatory macrophage recruitment. (A) Western blot analysis of CCR2 in liver tissues and statistical graph, n = 3. (B) Immunohistochemical staining of CCR and statistical graph, n = 4–6. (C) The phenotype of macrophages in liver tissues was measured by flow cytometry, n = 3. F4/80+CD11b+ was labeled to represent the macrophages, while CD86+CD206- macrophages were recognized as M1 phenotype, and CD86−CD206+ macrophages were recognized as M2 phenotype. (D) Double immuno-fluorescence staining of iNOS and CD11b. Results are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 5. GS regulated TBK1 to improve oxidative stress in PA-stimulated hepatocytes. (A) Western blot analyses of p-TBK1, TBK1, p-NF-κB and NF-κB in hepatocytes and statistical graphs, n = 3. (B) Immuno-fluorescence staining of TBK1. (C) Double immuno-fluorescence staining of NF-κB and MCP1. (D) The mitochondrial membrane potential assay was detected by JC-1, three samples were collected together for testing, and the experiment was repeated three times. Aggregates with red fluorescence in normal cells were distributed in the second quadrant, while monomers with green fluorescence delivered in the third quadrant represented decreased mitochondrial membrane potential. (E) The ROS assay was detected with DCFH-DA, three samples were collected together for testing, and the experiment was repeated three times. Results are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 6. TBK1 promoted NFκB-MCP1 signaling pathway. (A) The schematic diagram of co-culture system for hepatocytes and BMDMs. (B) Western blot analyses of p-TBK1, TBK1, p-NF-κB and NF-κB in hepatocytes and statistical graphs, n = 3. (C) Immuno-fluorescence staining of MCP1. (D) The mitochondrial membrane potential assay was detected by JC-1, three samples were collected together for testing, and the experiment was repeated three times. (E) The ROS assay was detected with DCFH-DA, three samples were collected together for testing, and the experiment was repeated three times. Results are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 7. The schematic diagram.