Study on the Mechanism of Improving HIV/AIDS Immune Function with Jian Aikang Concentrated Pill Based on Network Pharmacology Combined with Experimental Validation

Abstract

Purpose: This study was the first to screen the active compounds of Jian Aikang Concentrated Pill (JAKCP) with network pharmacology, predict its potential targets, screen the signaling pathways, and combine with cellular experimental validation to explore the potential mechanism of JAKCP for the treatment of acquired immunodeficiency syndrome (AIDS).

Methods: The main compounds and targets of Chinese herbs in JAKCP were identified by TCMSP; the targets of AIDS were collected from Genecards, Online Mendelian Inheritance in Man (OMIM), Disgenet, Therapeutic Target Database (TTD) and Drugbank; the network of "Chinese herbs-active compounds-targets" for JAKCP was constructed by Cytoscape, and protein-protein interaction (PPI) network was constructed using STRING to generate the intersection targets, Metascape was conducted to analyze the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), and the network of "main active compounds-core targets-pathways" was constructed by Cytoscape. Finally, the effect of JAKCP on the survival rate of HIV pseudovirus-infected MT-4 cells was investigated by CCK-8 assay, and the predicted targets were verified by ELISA, qPCR and Western blot.

Results: A total of 147 active compounds of JAKCP were screened covering 351 targets and 416 AIDS disease targets were obtained, besides 140 intersection targets and 321 KEGG pathways were collected. Ultimately, quercetin, kaempferol, stigmasterol, beta-sitosterol, epigallocatechin gallate were identified as the important compounds, the core targets are HSP90AA1, IL-10, IL-6, TNF, IL-1β, TP53, and IL-1ɑ, and the biological pathways and processes mainly include T cell activation, regulation of DNA-binding transcription factor activity and apoptotic signaling pathway. Experiments on the targets of "T cell activation" demonstrated that JAKCP promotes the survival of HIV pseudovirus-infected MT-4 cells. Also, JAKCP down-regulated mRNA and protein levels of IL-1ɑ, IL-1β, and IL-6 while up-regulated mRNA and protein levels of IL-2, IL-6ST, and IL-10 in vitro.

Conclusion: JAKCP exerted regulatory immune functions through multi-component, multi-target and multi-pathway, thereby providing novel ideas and clues for the treatment of AIDS.

Keywords: HIV/AIDS; Jian Aikang Concentrated Pill; mechanism; multi-pathway; network pharmacology.

Figure 1

Crossover active compounds of RS (Renshen), LZ (Lingzhi), AY (Aiye), HJT (Hongjingtian) and GQZ (Gouqizi) herbs in JAKCP. In this figure, the blue area represent candidate compounds of RS, the red area represent candidate compounds of LZ, the green area represent candidate compounds of AY, the yellow area represent candidate compounds of HJT, the Orange area represent candidate compounds of GQZ. Finally, Quercetin, Kaempferol, Stigmasterol, Beta-sitosterol and Mandenol were the active compounds crossed by more than two herbal medicines.

Figure 2

Network of the relationship between five kinds of Chinese herbs, active compounds and targets in JAKCP. All nodes were visualized in degree value, the larger the node, the darker the color and the higher the degree value. The five kinds of Chinese herbs are RS (Renshen), LZ (Lingzhi), AY (Aiye), HJT (Hongjingtian) and GQZ (Gouqizi), among them, the hexagons in the bright yellow area marked with RS represent main compounds contained in RS; the hexagons in the green area marked with LZ represent main compounds contained in LZ; the hexagons in the purple area marked with AY represent main compounds contained in AY; the hexagons in the blue area marked with HJT represent main compounds contained in HJT; the hexagons in the dark yellow area marked with GQZ represent main compounds contained in GQZ; The red diamonds in the middle area represent the targets that all compounds intersect. A1, A2, B1, B2, C1 are the crossover active compounds of five kinds of Chinese herbs in JAKCP.

Figure 3

The venn diagram of 140 intersection targets of AIDS (Acquired immunodeficiency syndrome) and JAKCP (Jian Aikang Concentrated Pill).

Figure 4

PPI network diagram of intersection targets for JAKCP and AIDS. Edges represent protein–protein interactions, edge thickness indicates the strength of data support, the larger the node, the higher the degree value. The circles with different colors in the figure represent the target names with different degree values.

Figure 5

The strategy of key targets in the treatment of AIDS by JAKCP. DC is Degree Centrality, BC is Betweenness Centrality, CC is Closeness Centrality and LAC is Local Average Connectivity-based method, they are four important indicators used in the strategy of screening effective compounds, 122 key targets were finally obtained after three screenings of the above indicators.

Figure 6

Potential Module network within the core targets-PPI network. Different colored circles represent different target categories. The size of the circular shape represents the magnitude of the degree value, and the lines between them represent the tightness of the connection between different targets.

Figure 7

GO and KEGG analysis for the major targets of JAKCP, (A) lists the top 20 significantly enriched biological processes in the GO analysis, namely GO-BP; (B) lists the top 20 significantly enriched cellular component in the GO analysis, namely GO-CC; (C) lists the top 20 significantly enriched molecular function in the GO analysis, namely GO-MF; (D) shows the top 20 signaling pathways in the KEGG pathway enrichment analysis. The Y-axis in the figure represents the top 20 biological functions, such as BP, CC, MF and KEGG, the X-axis represents the ratio of the number of target pathway genes to the total number of genes, the size of the bubble area represents the number of enriched genes, and the color of the bubble Represents the significance of enrichment, that is, the magnitude of the q-value value.

Figure 8

The network of “main active compounds-core targets-pathways”. All nodes were visualized in degree value, the larger the node, the darker the color and the higher the degree value. A2, B1, C1 are the crossover active compounds of five kinds of Chinese herbs in JAKCP, A2 is Beta-sitosterol, B1 is Kaempferol, C1 is Quercetin, the other red circles represent the important effective compounds finally screened out, the blue diamonds in the middle represent the corresponding targets, and the green arrows around represent the corresponding pathways.

Figure 9

Cell viability effects of JAKCP treatment on MT-4 cells with HIV pseudovirus, Drug concentration-cell viability curves were generated based on the cell viability assay. (A) The OD value of JAKCP treatment on MT-4 cells with HIV pseudovirus. (B) The cell survival rate of JAKCP treatment on MT-4 cells with HIV pseudovirus. All data was presented as mean ± standard deviation, ^###:p<0.001, compared with control group; **:p < 0.01, compared with model group.

Figure 10

Expression levels of IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10 in serum from JAKCP among groups Con (Control), Mod (Model), P (Positive), H (High), M (Medium) and L (Low). Each histogram is marked with its corresponding target name, which is IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10. All data was presented as mean ± standard deviation, ^#:p < 0.05,^###:p<0.001, compared with control group;*:p < 0.05,**:p < 0.01, ***:p<0.001, compared with model group.

Figure 11

The expression of IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10 mRNA levels were examined by qPCR treated with JAKCP among groups Con (Control), Mod (Model), P (Positive), H (High), M (Medium) and L (Low). Each histogram is marked with its corresponding target name, which is IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10. All data was presented as mean ± standard deviation,^#:p < 0.05, ^##:p<0.01, ^###:p<0.001, compared with control group; *:p < 0.05,**:p < 0.01, compared with model group.

Figure 12

The expression of IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10 protein levels were examined by Western blot among groups Con (Control), Mod (Model), P (Positive), H (High), M (Medium) and L (Low).(A) Western blot analysis showed the optical density ratio of IL-1ɑ/Tubulin, IL-1β/Tubulin, IL-2/Tubulin, IL-6/Tubulin, IL-6ST/Tubulin and IL-10/Tubulin on MT-4 cells with HIV pseudovirus among different groups.(B) The expression of IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10 protein levels was analyzed by images of the Western-blot results in different groups. All data was presented as mean ± standard deviation, ^#:p < 0.05, ^##:p<0.01, ^###:p<0.001, compared with control group;*:p < 0.05,**:p < 0.01,***:p<0.001,****:p<0.0001, compared with model group.