Elaborate the Mechanism of Ancient Classic Prescriptions (Erzhi Formula) in Reversing GIOP by Network Pharmacology Coupled with Zebrafish Verification

Abstract

Osteoporosis is a degenerative disease that endangers human health. At present, chemical drugs used for osteoporosis have serious side effects. Therefore, it is valuable to search herbs with high safety and good curative effect in antiosteoporosis. Erzhi formula (EZF), an ancient classic compound, has been reported to have a beneficial effect in antiosteoporosis, but its mechanism is unclear. In this paper, the active compounds of EZF were found in Systems Pharmacology Database, and gene targets related to osteoporosis were obtained in GeneCards. The GO functional and KEGG pathway enrichment analysis were performed by Metascape. The network of "components-targets-signal pathway" was constructed by Cytoscape. Next, molecular docking between the active components and hub genes related to the PI3K-Akt signaling pathway was conducted by Autodock. In the verification experiment, the zebrafish induced by prednisolone (PNSL) was used to reproduce glucocorticoid-induced osteoporosis (GIOP) model, and then the reversal effects of EZF were systematically evaluated according to the behavior, skull staining area, bone mineralization area (BMA), average optical density (AOD), and cumulative optical density (COD). Finally, it was shown that 24 components in EZF could regulate 39 common gene targets to exert antiosteoporosis effect. Besides, the main regulatory mechanisms of EZF were 4 signaling pathways: PI3K-Akt, JAK-STAT, AGE-RAGE, and cancer pathway. In PI3K-Akt signaling pathway, wedelolactone, dimethyl wedelolactone, specnuezhenide, ursolic acid, acacetin, beta-sitosterol, apigenin, and kaempferol can bind tightly with EGF, IL-2, and IL-4 genes. Compared with the model group, the moving distance, swimming speed, and cumulative swimming time of zebrafish in EZF group were significantly increased (P < 0.05). Meanwhile, the BMA and COD of zebrafish were significantly improved after the intervention of EZF (P < 0.05). In summary, the 24 components of EZF exert their antiosteoporosis effects by regulating 39 related gene targets, among which the PI3K signaling pathway is crucial. EZF can promote bone formation and reversed GIOP through "multicomponent/multitarget/multipathway" and the medium dose of EZF may be the most suitable concentration for the treatment of GIOP in zebrafish model.

Figure 1

Venn diagram of drug targets and disease common targets.

Figure 2

Protein-protein interaction (PPI) network and top 10 hub genes. (a) Protein-protein interaction (PPI) network; (b) top 10 hub genes selected by MCC method.

Figure 3

KEGG enrichment analysis for signal pathways. The y-axis shows top significantly enriched KEGG categories, and the x-axis displays the number of enrichment genes of these terms (P < 0.05), and the color represents the adjusted P value; the redder, the more significant the enrichment. The height of the column is related to P value. The higher the column is, the more significant the enrichment is.

Figure 4

GO-BP biological process of enrichment analysis. The y-axis shows top significantly enriched GO-BP categories, and the x-axis displays the number of enrichment genes of these terms (P < 0.05), and the color represents the adjusted P value; the redder, the more significant the enrichment. The height of the column is related to P value. The higher the column is, the more significant the enrichment is.

Figure 5

GO-MF molecular function of enrichment analysis. The y-axis shows top significantly enriched GO-MF categories, and the x-axis displays the number of enrichment genes of these terms (P < 0.05), and the color represents the adjusted P value; the redder, the more significant the enrichment. The height of the column is related to P value. The higher the column is, the more significant the enrichment is.

Figure 6

GO-CC cellular component of enrichment analysis. The y-axis shows top significantly enriched GO-CC categories, and the x-axis displays the number of enrichment genes of these terms (P < 0.05), and the color represents the adjusted P value; the redder, the more significant the enrichment. The height of the column is related to P value. The higher the column is, the more significant the enrichment is.

Figure 7

The “chemical components-target genes-signal pathway” network of EZF in treating osteoporosis. FLL: Fructus Ligustri Lucidi; EP: Ecliptae herba.

Figure 8

PI3K-Akt pathway in preventing osteoporosis mediated by EZF.

Figure 9

Top ten molecular dockings diagram of EZF for treating GIOP. (a) IL4, wedelolactone; (b) EGF, ursolic-acid; (c) IL2, ursolic acid; (d) IL4-, acacetin; (e) IL2, beta-sitosterol; (f) IL4, apigenin; (g) IL4, kaempferol; (h) EGF, specnuezhenide; (i) IL4, dimethyl wedelolactone; (j) IL4, specnuezhenide. “a” shows the amino acid residues and hydrogen bond lengths attached to the active component (ligand), where the colored rainbow represents the ligands and the pure color represents the amino acid residues. “b” is the complete docking of protein and ligand.

Figure 10

Effect of EZF extract on the behavior of GIOP zebrafish (n = 15). (a) The moving distance detected by animal behavior analyzer in each group. (b) The swimming speed detected by animal behavior analyzer in each group. (c) Cumulative swimming time detected by animal behavior analyzer in each group. Compared with the blank group, ^##P < 0.01; compared with the model group, ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 11

Reversal effect of EZF on bone mineralization in GIOP zebrafish (n = 15). (a) Blank control group. (b) Blank DMSO group. (c) Model group. (d) EZF high-dose group. (e) EZF medium-dose group. (f) EZF low-dose group. (g) Positive control group.

Figure 12

Quantitative analysis of the effect of EZF in reversing GIOP in zebrafish (n = 15). (a) The average optical density in different groups. (b) Cumulative optical density in different groups. (c) Staining area in different groups. Compared with the blank group, ^###P < 0.001; compared with model group, ^∗P < 0.05 and ^∗∗P < 0.01.