Investigation of the Active Ingredients and Mechanism of Polygonum cuspidatum in Asthma Based on Network Pharmacology and Experimental Verification

Abstract

Background: Polygonum cuspidatum is a Chinese medicine commonly used to treat phlegm-heat asthma. However, its anti-asthmatic active ingredients and mechanism are still unknown. The aim of this study was to predict the active ingredients and pathways of Polygonum cuspidatum and to further explore the potential molecular mechanism in asthma by using network pharmacology.

Methods: The active ingredients and their targets related to Polygonum cuspidatum were seeked out with the TCM systematic pharmacology analysis platform (TCMSP), and the ingredient-target network was constructed. The GeneCards, DrugBank and OMIM databases were used to collect and screen asthma targets, and then the drug-target-disease interaction network was constructed with Cytoscape software. A target protein-protein interaction (PPI) network was constructed using the STRING database to screen key targets. Finally, GO and KEGG analyses were used to identify biological processes and signaling pathways. The anti-asthmatic effects of Polygonum cuspidatum and its active ingredients were tested in vitro for regulating airway smooth muscle (ASM) cells proliferation and MUC5AC expression, two main symptoms of asthma, by using Real-time PCR, Western blotting, CCK-8 assays and annexin V-FITC staining.

Results: Twelve active ingredients in Polygonum cuspidatum and 479 related target proteins were screened in the relevant databases. Among these target proteins, 191 genes had been found to be differentially expressed in asthma. PPI network analysis and KEGG pathway enrichment analysis predicted that the Polygonum cuspidatum could regulate the AKT, MAPK and apoptosis signaling pathways. Consistently, further in vitro experiments demonstrated that Polygonum cuspidatum and resveratrol (one active ingredient of Polygonum cuspidatum) were shown to inhibit ASM cells proliferation and promoted apoptosis of ASM cells. Furthermore, Polygonum cuspidatum and resveratrol inhibited PDGF-induced AKT/mTOR activation in ASM cells. In addition, Polygonum cuspidatum decreased H₂O₂ induced MUC5AC overexpression in airway epithelial NCI-H292 cells.

Conclusion: Polygonum cuspidatum could alleviate the symptoms of asthma including ASM cells proliferation and MUC5AC expression through the mechanisms predicted by network pharmacology, which provides a basis for further understanding of Polygonum cuspidatum in the treatment of asthma.

Keywords: ASM cells; MUC5AC; Polygonum cuspidatum; asthma; network pharmacology.

Figure 1

Entire study design based on network pharmacology and experimental verification.

Figure 2

Disease and active ingredient intersection targets. (A) Intersection target Venn diagram. (B) “Drug-target-disease” network.

Figure 3

The key therapeutic targets of Polygonum cuspidatum in the treatment of asthma. (A) PPI visualization network. (B) Thirty core target selections. High value and high betweenness proteins are considered to occupy the core positions in the PPI network and are more likely to serve as key core targets.

Figure 4

Enrichment analysis of KEGG pathways. (A) According to the p.adjust value of targets, the first 20 signaling pathways were involved in the treatment of asthma with Polygonum cuspidatum. In the bubble chart, the p.adjust value indicates significance, and the larger circles indicate a greater number of enrichment targets and the redder circles indicate greater significant targets. (B) According to the number of targets, the first 20 signaling pathways were involved.

Figure 5

The effect of Polygonum cuspidatum extraction on ASM cells proliferation and apoptosis. (A) ASM cells were treated with PDGF (10 ng/mL) and Polygonum cuspidatum extraction (0.25 mg/mL, 0.5 mg/mL, 1 mg/mL and 2 mg/mL) at different concentrations alone or in combination for 24h. Cell proliferation was detected by CCK-8. The data were expressed as mean ± SD (n=6) of each group. (B, C) ASM cells were treated with Polygonum cuspidatum extraction (0.25 mg/mL, 0.5 mg/mL, 1 mg/mL and 2 mg/mL) for 24h. The apoptosis of ASM cells were detected by Annexin-V staining. The data were expressed as mean ± SD (n=3) of each group. ^ns P>0.05 vs PDGF group or control group and ** P<0.01 vs PDGF group or control group, #P<0.01vs control group.

Figure 6

PDGF induced ASM cells proliferation by activating AKT/mTOR pathway. (A–C) ASM cells were treated with PDGF (10ng/mL) for 1h, 5h, and 12h. The expression levels of p-AKT and p-mTOR were analyzed by Western blotting. The relative protein levels of P-AKT and P-mTOR were calculated by Image J, respectively and expressed as mean ± SD (n=3) of each group. * P < 0.05 and ** P < 0.01 vs control group. (D) ASM cells were treated with PDGF (10ng/mL) and LY294002 (10 μM), Rapamycin (500 nM) for 5h, respectively. Cell proliferation was detected by CCK-8. The data were expressed as mean ± SD (n=8) of each group, ** P<0.01 vs PDGF group, ^# P<0.01vs control group.

Figure 7

Polygonum cuspidatum extraction inhibited PDGF-induced AKT/mTOR activation. (A–C) ASM cells were treated with PDGF (10ng/mL) and Polygonum cuspidatum extraction (0.25 mg/mL, 0.5 mg/mL, 1 mg/mL and 2 mg/mL) for 5h, respectively. The expression levels of p-AKT and p-mTOR were analyzed by Western blotting. The relative protein levels of p-AKT and p-mTOR were calculated by Image J and expressed as mean ± SD (n=3) of each group. ^ns P>0.05 vs PDGF group, * P<0.05 and ** P<0.01 vs PDGF group, ^# P<0.01vs control group.

Figure 8

Polygonum cuspidatum extraction inhibited H₂O₂-induced MUC5AC expression. ASM cells were treated with H₂O₂ (80 μM) and Polygonum cuspidatum extraction (1 mg/mL and 2 mg/mL) at different concentrations alone or in combination for 24h. The expression levels of MUC5AC were analyzed by Real-time PCR. The data were expressed as mean ± SD (n=3) of each group. ** P<0.01 vs H₂O₂ group, ^# P<0.01vs control group.

Figure 9

The effects of resveratrol on ASM cells proliferation and apoptosis. (A) HPLC chromatograms of resveratrol in Polygonum cuspidatum extraction. (B, C) ASM cells were treated with PDGF (10 ng/mL) and resveratrol (6.25 μM, 12.5 μM, 25 μM, 50 μM) at different concentrations alone or in combination for 24h and 48h. Cell proliferation was detected by CCK-8. The data were expressed as mean ± SD (n=6) of each group, ^ns P>0.05 vs PDGF group, ** P<0.01 vs PDGF group, ^#P<0.01vs DMSO group (control group). (D, E) The apoptosis of ASM cells were detected by Annexin-V staining after treatment with resveratrol (25 μM, 50 μM) for 48 h. The data were expressed as mean ± SD (n=3) of each group, ** P<0.01 vs DMSO group (control group).

Figure 10

Resveratrol inhibited PDGF-induced AKT/mTOR activation. (A–C) ASM cells were treated with PDGF (10 ng/mL) and Resveratrol (25 μM, 50 μM) for 5h, respectively. The expression levels of p-AKT and p-mTOR were analyzed by Western blotting. The relative protein levels of p-AKT and p-mTOR were calculated by Image J and expressed as mean ± SD (n=3) of each group. * P<0.05 and ** P<0.01  vs PDGF group, ^#P<0.01vs DMSO group (control group).