Metabolomics combined with network pharmacology reveals a role for astragaloside IV in inhibiting enterovirus 71 replication via PI3K-AKT signaling

Abstract

Background: Astragaloside IV (AST-IV), as an effective active ingredient of Astragalus membranaceus (Fisch.) Bunge. It has been found that AST-IV inhibits the replication of dengue virus, hepatitis B virus, adenovirus, and coxsackievirus B3. Enterovirus 71 (EV71) serves as the main pathogen in severe hand-foot-mouth disease (HFMD), but there are no specific drugs available. In this study, we focus on investigating whether AST-IV can inhibit EV71 replication and explore the potential underlying mechanisms.

Methods: The GES-1 or RD cells were infected with EV71, treated with AST-IV, or co-treated with both EV71 and AST-IV. The EV71 structural protein VP1 levels, the viral titers in the supernatant were measured using western blot and 50% tissue culture infective dose (TCID₅₀), respectively. Network pharmacology was used to predict possible pathways and targets for AST-IV to inhibit EV71 replication. Additionally, ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was used to investigate the potential targeted metabolites of AST-IV. Associations between metabolites and apparent indicators were performed via Spearman's algorithm.

Results: This study illustrated that AST-IV effectively inhibited EV71 replication. Network pharmacology suggested that AST-IV inhibits EV71 replication by targeting PI3K-AKT. Metabolomics results showed that AST-IV achieved these effects by elevating the levels of hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-hydroxy-1 H-indole-3- acetamide, oxypurinol, while reducing the levels of PC (14:0/15:0). Furthermore, AST-IV also mitigated EV71-induced oxidative stress by reducing the levels of MDA, ROS, while increasing the activity of T-AOC, CAT, GSH-Px. The inhibition of EV71 replication was also observed when using the ROS inhibitor N-Acetylcysteine (NAC). Additionally, AST-IV exhibited the ability to activate the PI3K-AKT signaling pathway and suppress EV71-induced apoptosis.

Conclusion: This study suggests that AST-IV may activate the cAMP and the antioxidant stress response by targeting eight key metabolites, including hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-Hydroxy-1 H-indole-3-acetamide, oxypurinol and PC (14:0/15:0). This activation can further stimulate the PI3K-AKT signaling to inhibit EV71-induced apoptosis and EV71 replication.

Keywords: Astragaloside IV; Enterovirus 71; Metabolomics; Network pharmacology; Oxidative stress response; PI3K-AKT signaling.

Fig. 1

AST-IV inhibits EV71 replication. (A) Chemical structure of AST-IV. (B, C) A CCK-8 assay was performed to measure the viability of GES-1 cells in different treatment groups. (D, E) Western blotting was used to analyze the levels of the viral structural protein VP1 in the different treatment groups. (F) The viral titers in the supernatants of different treatment groups were determined by TCID50 assay. The data are presented as the mean ± SD (n = 3) of at least three independent experiments. ****P < 0.0001 compared with the control group; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared with the EV71-infected group

Fig. 2

Effects of AST-IV on EV71-induced apoptosis. (A, B) Fluorescence images of EV71-infected GES-1 cells were obtained in the presence of AST-IV for 2 h-prior. The cells were stained with PI. (C-E) Western blot analysis of the apoptosis marker proteins cleaved PARP and cleaved caspase-3 in EV71-infected GES-1 cells in the presence of AST-IV. The data are presented as the mean ± SD (n = 3) of at least three independent experiments. ****P < 0.0001 compared to thecontrol group; ##P < 0.01, ###P < 0.001, ####P < 0.01 compared to the EV71-infected group

Fig. 3

OPLS-DA and PCA analysis of LC-MS/MS of GES-1 cells. (A) OPLS-DA and (B) PCA score plots of the control group and EV71 group. (C) OPLS-DA and (D) PCA score plots of the control group and AST-IV group. (E) OPLS-DA and (F) PCA score plots of the EV71 group and EV71 + AST-IV group

Fig. 4

Analysis of differential metabolites in the four groups. (A) The volcano plot shows the differential metabolite levels between the control group and the EV71 group. (B) The volcano plot shows the differential metabolite levels between the control group and the AST-IV group. (C) The volcano plot shows the differential metabolite levels between the EV71 group and the EV71 + AST-IV group. (D) Venn diagram showing the overlapping metabolites from the control group, EV71 group, AST-IV group and EV71 + AST-IV group

Fig. 5

The relative peak regions of possible GES-1 cell indicators associated with EV71 infection that could be regulated by AST-IV. The data are shown as the mean ± SEM for GES-1 cells from each group (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the control group; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 compared to the EV71-infected group

Fig. 6

Effects of AST-IV on antioxidative activity. (A, B) ROS levels, (C) CAT activity, (D) GSH-Px activity, (E) T-AOC activity, (F) SOD activity, and (G) MDA levels were measured in different treatment groups in GES-1 cells. (H, I) Western blotting was used to analyze VP1 levels in EV71-infected and NAC-treated GES-1 cells. (J) The TCID50 assay was performed to determine the viral titers in the supernatant of EV71-infected and NAC-treated GES-1 cells. The data are presented as the mean ± SD (n = 3) of at least three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the control group; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.01 compared with the EV71-infected group

Fig. 7

Targets of AST-IV and HFMD. (A) Venn diagram of HFMD-related targets. (B) Venn diagram of AST-IV-related targets. (C) Venn diagram of the common targets between AST-IV and HFMD. (D) Visualization of the AST-IV-target network. (E) Visualization of the AST-IV-common targets-HFMD network

Fig. 8

Network construction and analysis. (A) The PPI network was constructed using Cytoscape 3.9.0. (B) Venn diagram showing the 6 overlapping hub genes screened by seven algorithms. (C) The PPI network of the 6 overlapping hub genes. (D) GO enrichment analysis. (E) KEGG enrichment analysis. (F) The most significant pathways were screened by Metascape

Fig. 9

The effects of AST-IV on the PI3K-AKT pathway in EV71-infected and AST-IV-treated GES-1 cells. (A-C) Western blot analysis of p-PI3K and p-AKT levels in EV71-infected GES-1 cells. (D-F) The protein levels of p-PI3K, t-PI3K, p-AKT, and t-AKT in AST-IV-treated GES-1 cells were analyzed by western blot. (G-I) The protein levels of p-PI3K and p-AKT in EV71-infected and AST-IV-treated GES-1 cells were analyzed by Western blot. (J, K) The conformation of the molecular docking between AST-IV and AKT1 or PIK3R1. The data are presented as the mean ± SD (n = 3) of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group; #P < 0.05, ##P < 0.01 compared with the EV71-infected group

Fig. 10

Correlation between targeted metabolites and apparent indicators. (A) Association analysis of metabolites with apparent indicators by Spearman’s method. Differences were considered statistically significant at P < 0.05. (B) Network (only correlations with |R| > 0.6 with P < 0.05 were presented) among metabolites and apparent indicators. Red nodes represent differential metabolites, and purple nodes represent apparent indicators. Red lines indicate positive correlations, while purple lines indicate negative correlations