Integrating Network Pharmacology and Experimental Validation to Decipher the Mechanism of Action of Huanglian Jiedu Decoction in Treating Atherosclerosis

Abstract

Background: This study used network pharmacology, molecular docking and experimental validation to assess the effects of Huanglian Jiedu Decoction (HLJDD) on atherosclerosis (AS).

Methods: The components and targets of HLJDD were analyzed using the Traditional Chinese Medicine Systems Pharmacology database, and information on the genes associated with AS was retrieved from the GeneCards and OMIM platforms. Protein-protein interactions were analyzed using the STRING platform. A component-target-disease network was constructed using Cytoscape. GO and KEGG analyses were performed to identify molecular biological processes and signaling pathways, and the predictions were verified experimentally. Molecular docking was conducted with ChemOffice software, PyMOL software and Vina to verify the correlation of targets and compounds.

Results: HLJDD contained 31 active compounds, with quercetin, kaempferol, moupinamide and 5-hydroxy-7-methoxy-2-(3,4,5-trimethoxyphenyl)chromone as the core compounds. The most important biotargets of HLJDD in AS were ICAM-1, CD31 and RAM-11. The molecular docking results showed that the molecular docking interaction energy between the 3 key targets and the 4 high-degree components were much less than -5 kJ∙mol^-1. The experimental validation results showed that HLJDD might treat AS mainly by reducing TC, TG and LDL-C and increasing HDL-C, upregulating CD31 expression, reducing ICAM-1 and RAM-11 expression, and downregulating inflammatory factors, including CRP, IL-6 and TNF-α. These results support the network pharmacology data and demonstrate that HLJDD affects the expression of core genes and alters the leukocyte transendothelial migration signaling pathway.

Conclusion: Based on network pharmacology and experimental validation, our study indicated that HLJDD exerted anti-AS effect through upregulating CD31 expression and reducing the expression of ICAM-1 and RAM-11. HLJDD may be a potential therapeutic drug to the prevention of AS.

Keywords: Huanglian Jiedu Decoction; atherosclerosis; experimental validation; molecular docking; network pharmacology.

Figure 1

Network of HLJDD compound targets and AS targets.

Figure 2

PPI network of common targets. The yellow squares represent the core targets. The blue circulars represent other targets.

Figure 3

GO analysis. (A) The representative biological processes of therapeutic targets for the HLJDD against AS. The important terms in the group were tagged, with the related biological functional groups partially overlapped. (B) Percentage of each approach. (C) GO analysis of common targets of HLJDD and AS. The Y-axis represents significant GO biological function processes and the X-axis represents the counts of enriched targets.

Figure 4

KEGG analysis for common targets of HLJDD and AS. The Y-axis represents significant KEGG pathways and the X-axis represents the ratio of enriched targets in a pathway to all common targets. The size of the nodes shows count of targets, and gradient of color represents the adjusted p value.

Figure 5

Molecular docking charts of quercetin and ICAM-1 (A), kaempferol and ICAM-1 (B), moupinamide and ICAM-1 (C), 5-hydroxy-7-methoxy-2-(3,4,5-trimethoxyphenyl)chromone and ICAM-1 (D).

Figure 6

The TC, TG, LDL-C and HDL-C levels in rabbits.

Figure 7

(A) The serum CRP, IL-6 and TNF-α levels in rabbits. *p<0.05 versus the model group; **p< 0.01 versus the model group. Data are the means ± SD. (B) Representative Masson’s trichrome staining of rabbit aortic tissue (×40).

Figure 8

Effect of HLJDD on the protein expression levels of ICAM-1 (×40). (A) Control group (B) model group (C) HLJDD (1.5g/kg) group (D) HLJDD (3g/kg) group. (E) The integrated optical density of ICAM-1 was calculated by Ptps-2011 software.

Figure 9

Effect of HLJDD on the protein expression levels of CD31 (×40). (A) Control group (B) model group (C) HLJDD (1.5g/kg) group (D) HLJDD (3g/kg) group. (E) The integrated optical density of CD31 was calculated by Ptps-2011 software.

Figure 10

Effect of HLJDD on the protein expression levels of RAM-11 (×40). (A) Control group (B) model group (C) HLJDD (1.5g/kg) group (D) HLJDD (3g/kg) group. (E) The integrated optical density of RAM-11 was calculated by Ptps-2011 software.