Network Pharmacology-Based Strategy to Explore the Effect and Mechanism of Zhizhu Granule Improving Glucose-Lipid Metabolism in Rats with Metabolic Syndrome

Abstract

Objective: To explore the mechanism of the traditional Chinese medicine (TCM), Zhizhu granule (ZZG), in treating metabolic syndrome (MS) based on network pharmacology and pharmacodynamic experiment.

Materials and methods: Network pharmacology combined with a pharmacodynamic experiment was used to elucidate the therapeutic mechanism of ZZG in MS. Serum samples were collected from rats with MS, induced by a high-sugar-fat-salt diet (HSFSD) combined with streptozotocin (STZ), to measure the levels of biochemical markers. The glucose (GLU), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), systolic blood pressure (SBP), and diastolic blood pressure (DBP) were detected. The liver tissue of rats was used for histological examination and Western blot analysis.

Results: Network pharmacology analysis generated 69 drug-disease common targets and 10 hub genes closely related to ZZG against MS. KEGG pathway analysis revealed that the PI3K/AKT signaling pathway was the most potential pathway, which took part in the therapeutic mechanisms. In the animal experiments section, the therapeutic effect of ZZG on MS and the therapeutic pathway of ZZG on MS were verified. ZZG could significantly decrease the body weight, TC, TG, LDL-C and GLU levels in MS rats (all p<0.01), alleviate hepatocyte steatosis and decrease liver lipid droplet deposition. Western blot analysis indicated that compared with the model group, the expression levels of PI3K, AKT, and IRS-1 protein were significantly increased (all p<0.05), and the FOXO-1 was significantly decreased (all p<0.05) in the ZZG group.

Conclusion: ZZG can improve glucose-lipid metabolism disorder in rats with metabolic syndrome. The reported results provide experimental evidence for ZZG in the treatment of MS.

Keywords: PI3K/AKT pathway; Zhizhu granule; metabolic syndrome; network pharmacology; pharmacodynamic experiment.

Figure 1

Flow chart showing the detailed process of this study. 1–1. The main clinical symptoms of MS. 1–2. Venn diagram of the number of target proteins in four databases. 1–3. Venn diagram for the common targets of drug and MS. 1–4. PPI network of ZZG against MS. 1–5. The drug-component-target network of ZZG against MS. 1–6. The target-pathway network of ZZG against MS. 1–7. The enrichment analysis of the 69 potential therapeutic targets. 1–8. Analysis of body weight, Biochemical and blood pressure of rats. 1–9. HE staining and oil red O staining of the rats’ liver tissue. 1–10. Analysis of the protein expression levels on PI3K/AKT signaling pathway. See the corresponding figure in the manuscript for a detailed description.

Figure 2

Target proteins of ZZG and drug-disease common targets. (A) Venn diagram of the number of target proteins in four databases. The blue ovals represent disease targets obtained from the TTD database. The yellow ovals represent disease targets obtained from the GeneCards database; the green ovals represent disease targets obtained from the OMIM database; pink ovals represent disease targets obtained from the PharmGKB database; overlapping areas represent the overlapping targets of the four databases; unoverlapped areas represent targets that are individually owned by each database. (B) Venn diagram for the common targets of drug and MS. The blue circle represents the targets of the drug compounds; the yellow circle represents the targets of MS. The overlapping area represents the common targets of drugs and MS.

Figure 3

PPI network of ZZG against MS. (A) PPI network of protein targets obtained from the STRING database. (B) PPI network constructed by Cytoscape. The size of the nodes is proportional to the values of the degree. As the nodes’ color becomes lighter, the degree values become gradually smaller. (C) The top 10 hub genes cluster. (D) Sequencing of hub genes by degree values.

Figure 4

Analysis of key ingredients and signaling pathways of ZZG against MS. (A)The drug-component-target network of ZZG against MS. The green circle represents the three drugs of ZZG, with ZS representing Zhishi, BZ representing Baizhu, and HY representing Heye. The hexagon represents the active components of the three drugs, and the blue diamonds represent the target genes of ZZG against MS. The number of edges connected to the node represents the degree of freedom. (B)The target-pathway network of ZZG against MS. The red nodes represent the signaling pathways, and the green node represents the involved targets. The larger the shape and the more prominent the color of the targets, the more significant the enrichment. (C) The GO enrichment analysis of the 69 potential therapeutic targets. (D) The KEGG pathway enrichment analysis of the 69 potential therapeutic targets. The circle size represents the number of genes enriched in each KEGG pathway term, and the different colors represent the P-value.

Figure 5

Analysis of body weight, Biochemical and blood pressure of rats. (A) The figure of body weight change in rats. (B) Analysis of blood glucose in rats. (C) Analysis of blood pressure in rats. (D) Analysis of blood lipid in rats. *P < 0.05 vs control group, **P < 0.01 vs control group, ^#P < 0.05 vs model group, ^##P < 0.01 vs model group.

Figure 6

HE staining and oil red O staining of the rats’ liver tissue (200×). In H&E staining, the vacuoles indicate lipid droplets in the hepatocyte, and the more vacuoles, the more serious the hepatocyte steatosis. In Oil Red O staining, the more reddened area indicates more lipid droplets. (C) control group, (M) model group, (Z) ZZG group.

Figure 7

Analysis of the protein expression levels on PI3K/AKT signaling pathway. (A) Bar graph shows the relative protein expression level of p-PI3K, PI3K, p-AKT, AKT, FOXO-1, and IRS-1 in the liver tissue of rats. *P < 0.05 vs control group, **P < 0.01 vs control group, ^#P < 0.05 vs model group, ^##P < 0.01 vs model group. (B) The expression of the above proteins. (C) control group, (M) model group, (Z) ZZG group.