Systems pharmacology-based exploration reveals mechanisms of anti-steatotic effects of Jiang Zhi Granule on non-alcoholic fatty liver disease

Figure 1. Pharmacodynamics investigation. The levels of ALT in the cell supernatants. (A) Hepatocytes were treated with various concentrations of PA for 24 h; (B) Hepatocytes were treated with 0.4 mM PA in the presence or absence of JZG (100 μg/mL) for different time periods; (C) Hepatocytes were treated with 0.4 mM PA in the presence or absence of JZG (100 μg/mL) for 24 h, and fluorescence staining with Oil Red O and BODIPY was performed to detect lipid droplets. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. Candidate compound screening and target prediction for JZG. (A) A compound-target network was constructed to comprehend the complex interactions between all the candidate compounds in JZG and their corresponding targets; (B) Enrichment analysis was performed by ClueGO to investigate the biological processes of JZG; (C) Enrichment analysis was performed by ClueGO to investigate the signalling pathways of JZG.

Figure 3. PPI network generation and putative target identification for JZG against NAFLD. (A) PPI networks were constructed for the putative targets of JZG as well as for known NAFLD-related targets, and the intersection of these two networks was used to identify candidate targets of JZG against NAFLD based on the DC value; (B) Enrichment analysis was performed using ClueGO to investigate the biological processes of JZG against NAFLD; (C) Enrichment analysis was performed using ClueGO to investigate the signalling pathways of JZG against NAFLD.

Figure 4. JZG could activate autophagy in hepatocytes. Hepatocytes were treated with PA (0.4 mM) in the presence or absence of either JZG (100 μg/mL) or rapamycin (2 μM) for 24 h. (A) Western blot analyses, the groupings were cropped from different gels subjected to identical conditions; (B) Autophagosomes were observed by TEM; (C) The progression of autophagosome formation was visualized by a cell line stably expressing mRFP-GFP-LC3. Data are expressed as the mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. JZG could regulate the mTOR signalling. Hepatocytes were treated with PA (0.4 mM) in the presence or absence of either JZG (100 μg/mL) or rapamycin (2 μM) for 24 h. (A) Western blot analyses, the groupings were cropped from different gels subjected to identical conditions; (B) The progression of autophagic flux was visualized by a cell line stably expressing mCherry-p62. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6. JZG protected mitochondrial integrity by ameliorating hepatic lipid accumulation. Hepatocytes were treated with PA (0.4 mM) in the presence or absence of either JZG (100 μg/mL) or rapamycin (2 μM) for 24 h. (A) Fluorescence staining with BODIPY and Lyso-Tracker was performed to demonstrate the co-localization of lipid droplets and lysosomes; (B): The mitochondrial integrity and ROS generation was detected by JC-1 and DCFH-DA, respectively.

Figure 7. JZG could activate autophagy to improve NAFLD in vivo. Male C57BL/6 mice were fed either a standard or MCD diet for three weeks. Mice in the JZG group were administered JZG (994 mg/kg) once daily via oral gavage, whereas mice in the other two groups were administered normal saline (vehicle). (A) Western blot analyses, the expression of autophagy-related proteins in liver tissues of the mice and the groupings were cropped from different gels subjected to identical conditions; (B) Inflammation and lipid droplet in the liver was examined by Haematoxylin and Eosin (H&E) staining and Oil Red O staining, respectively; (C) Liver TG levels. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8. The schematic diagram of the systems pharmacology approach and the potential mechanisms of the anti-steatotic effects of JZG on NAFLD.