Explore the Mechanism of Astragalus mongholicus Bunge against Nonalcoholic Fatty Liver Disease Based on Network Pharmacology and Experimental Verification

Abstract

Objective: Astragalus mongholicus Bunge [Fabaceae] (AMB), a traditional Chinese medicine (TCM), has been widely used to treat liver diseases in the clinic. However, the efficacy and mechanism of AMB in the treatment of nonalcoholic fatty liver disease (NAFLD) remain unclear. The purpose of this study was to systematically investigate the active components and mechanisms of AMB against NAFLD based on network pharmacology, molecular docking, and experimental verification.

Methods: First, the bioactive components and relevant targets of AMB were screened from the Traditional Chinese Medicine Systematic Pharmacology (TCMSP) database, and NAFLD-related targets were obtained from the GeneCards database. Then, the AMB-NAFLD protein target interaction network was built by the STRING database. GO and KEGG pathway enrichment analyses were performed using the DAVID database. The component targets were visualized using Cytoscape software. Finally, molecular docking and experiments were used to verify the results of network pharmacological prediction.

Results: Network pharmacology predicted that quercetin may be the main active component in AMB, and the TNF and MAPK signaling pathways may be the key targets of AMB against NAFLD. Molecular docking validation results demonstrated that quercetin, as the main active component of AMB, had the highest binding affinity with TNF. Furthermore, quercetin played a distinct role in alleviating NAFLD through in vitro experiments. Quercetin upregulated the phosphorylation levels of AMPK and inhibited the expression of p-MAPK and TNF-α. In addition, we further discovered that quercetin could increase ACC phosphorylation and CPT1α expression in PA-induced HepG2 cells.

Conclusions: Our results indicated that quercetin, as the main active component in AMB, exerts an anti-NAFLD effect by regulating the AMPK/MAPK/TNF-α and AMPK/ACC/CPT1α signaling pathways to inhibit inflammation and alleviate lipid accumulation.

Figure 1

Flow chart of the pharmacological mechanisms of AMB against NAFLD.

Figure 2

Compound-target network construction. (a) The component-target network of AMB. The orange triangles represent active compounds, and the green nodes represent component-related targets. (b) Venn diagram. The orange part represents the number of AMB targets, and the green part represents the number of NAFLD targets. (c) Compound-NAFLD targets network. The circular nodes represent potential active components in AMB, and the square represents the 99 common targets of AMB and NAFLD. (d) Degree of potential active compounds.

Figure 3

The protein-protein interaction (PPI) network analyze of overlapping targets. The color and depth of the nodes (orange → yellow → green) are in descending order of degree values, and node sizes are proportional to their degree.

Figure 4

Overlapped term-based analysis. (a) GO enrichment analysis for the major targets of AMB against NAFLD. The green, orange, and purple color rectangles represent biological process (BP), cellular component (CC), and molecular function (MF), respectively. (b) The top 20 KEGG pathway analysis for the major targets of AMB against NAFLD.

Figure 5

The component-target-pathway network constructed by Cytoscape. The yellow arrows represent active components of AMB, the green nodes represent the targets, and the orange triangles represent signaling pathways. Node sizes are proportional to their degree.

Figure 6

Molecular docking model of the top 3 key target proteins with the highest docking scores docked with quercetin. (a) TNF, (b) IL6, and (c) PTGS2.

Figure 7

Anti-NAFLD effect of quercetin in PA-induced HepG2 cells. (a) TG contents in HepG2 cells treated with DMSO or 25 μM quercetin in response to BSA or 0.4 mM PA stimulation for 24 h (n = 12). Data are presented as the mean ± S.E.M. ^∗P < 0.05 and ^∗∗P < 0.01. (b) Oil red O staining showing that PA induced lipid accumulation, which was suppressed after quercetin treatment for 24 h in HepG2 cells (n = 4).

Figure 8

Quercetin suppresses inflammation by regulating the AMPK/MAPK/TNF-α signaling pathway in PA-induced HepG2 cells. (a) qPCR analyses of TNF-α mRNA levels in HepG2 cells stimulated by 0.4 mM PA with DMSO or 25 μM quercetin for 24 h (n = 7 − 8). (b) Western blot analysis of p-AMPK, AMPK, p-p38, p38, p-ERK, ERK, p-JNK, and JNK levels in HepG2 cells stimulated by PA with quercetin (n = 3). (c) Gray value analysis of p-AMPK/AMPK, p-p38/p38, p-ERK/ERK, and p-JNK/JNK. Data are presented as the mean ± S.E.M. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 9

Quercetin increases fatty acid β-oxidation by regulating AMPK/ACC/CPT1α signaling in PA-induced HepG2 cells. (a) The protein expression levels of p-ACC, ACC, and CPT1α were examined by Western blot analysis in HepG2 cells (n = 3). (b) The gray value analysis of p-ACC, ACC, and CPT1α. Data are presented as the mean ± S.E.M. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 10

Quercetin exerts an anti-NAFLD effect by regulating the AMPK/MAPK/TNF-α and AMPK/ACC/CPT1α signaling pathways to inhibit inflammation and enhance fatty acid β-oxidation. Activation of AMPK by quercetin leads to downregulation of MAPK signaling pathways and decreases the expression of TNF-α, which may contribute to relieve inflammation. In addition, phospho-AMPK phosphorylates ACC, the rate-limiting enzyme of de novo lipogenesis. This decrease in ACC activity limits malonyl CoA production, relieving the inhibition of CPT-1α activity and enhancing fatty acid β-oxidation.