Mechanism Investigation of Wuwei Shexiang Pills on Gouty Arthritis via Network Pharmacology, Molecule Docking, and Pharmacological Verification

Abstract

Background: Gout is a common crystal-related arthritis caused by the deposition of monosodium urates (MSU). Tibetan medicine Wuwei Shexiang Pills (WSP) has been demonstrated to exhibit anti-inflammatory, antihyperuricemia, and antigout activities. However, the underlying mechanism is unknown.

Objectives: To explore the mechanisms of Wuwei Shexiang Pills on gouty arthritis via network pharmacology, molecule docking, and pharmacological verification.

Methods: The ingredients and targets of WSP were obtained by searching and screening in BATMAN-TCM and SwissADME. The targets involving the gout were acquired from public databases. The shared targets were put onto STRING to construct a PPI network. Furthermore, Metascape was applied for the GO and KEGG enrichment analysis to predict the biological processes and signaling pathways. And molecular docking was performed to validate the binding association between the key ingredients and the relative proteins of TNF signaling. Based on the serum pharmacology, the predicted antigout mechanism of WSP was validated in MSU-induced THP-1 macrophages. The levels of inflammatory cytokines and mRNA were measured by ELISA and qRT-PCR, respectively, and MAPK, NF-κB, and NLRP3 signaling-associated proteins were determined by western blot and immunofluorescence staining.

Results: 48 bioactive ingredients and 165 common targets were found in WSP. The data showed that 5-Cis-Cyclopentadecen-1-One, 5-Cis-Cyclotetradecen-1-One, (-)-isoshyobunone, etc. were potential active ingredients. TNF signaling, HIF-1 signaling, and Jak-STAT signaling were predicted to be the potential pathways against gout. The molecule docking analysis found that most ingredients had a high affinity for p65, NLRP3, IL-1β, TNF-α, and p38. The data from in vitro experiment showed that WSP suppressed the production and gene expression of inflammatory cytokines. Furthermore, WSP could inhibit the activation of MAPK, NF-κB, and NLRP3 signaling pathways.

Conclusion: Our finding suggested that the antigout effect of WSP could be achieved by inhibiting MAPK, NF-κB, and NLRP3 signaling pathways. WSP might be a candidate drug for gouty treatment.

Figure 1

Workflow graph of this study.

Figure 2

In vitro experiment protocol.

Figure 3

(a) Venn diagram. The value demonstrated the number of shared and unique targets of WSP and gout. (b) A total of 8 modules have been found in the PPI network. The color depths of nodes are positively correlated with their degrees. The size and color depths of edges are positively correlated with the interaction degree of the two nodes.

Figure 4

(a) GO biological processes of WSP against gout. (b) KEGG enrichment analysis results. The color scale represented the –Log10 (p value), and the dot size indicated the gene count in each term. (c) The ingredient-target-pathway network. The different colors indicated the different ingredients of herbs. The size of nodes was positively correlated with their degrees. The bigger the nodes are, the more important they were.

Figure 5

Molecule docking results of the main active components and the key TNF-associated targets. (a) The number in the grid represents the binding energy between ligands and macromolecules. The darker the color is, the stronger the affinity is. (b) 4 ingredients (SX10 (5-Cis-Cyclopentadecen-1-One), ZCP8 ((-)-isoshyobunone), ZCP6 (Acoronene), and SX5 (Estradiol)) and NF-κB P65. (c) 7 ingredients (SX26 (5-Cis-Cyclotetradecen-1-One, ZCP8 ((-)-isoshyobunone), MX30 (Beta-Ionone), SX1 (Androstenedione), ZCP12 (Acolamone), ZCP23 (Isoacolamone), and SX5 (Estradiol)) and NLRP3. (d) 9 ingredients (SX10 (5-Cis-Cyclopentadecen-1-One, SX26 (5-Cis-Cyclotetradecen-1-One), ZCP8 ((-)-isoshyobunone), MX30 (Beta-Ionone), SX1 (Androstenedione), ZCP12 (Acolamone), ZCP4 (Shyobunone), ZCP6 (Acoronene), and SX5 (Estradiol)) and IL-1β. (e) 13 ingredients (SX10 (5-Cis-Cyclopentadecen-1-One), ZCP8 ((-)-isoshyobunone), MX1 (Artemisia Ketone), MX30 (Beta-Ionone), MX5 ((E)-6,10-Dimethyl-9-methylene-5-undecen-2-one), SX1 (Androstenedione), ZCP12 (Acolamone), ZCP20 (Calacone), ZCP23 (Isoacolamone), ZCP4 (Shyobunone), ZCP5 (Acoragermacrone), ZCP6 (Acoronene), and SX5 (Estradiol)) and TNF-α. (f) 9 ingredients (SX10 (5-Cis-Cyclopentadecen-1-One), ZCP8 ((-)-isoshyobunone), MX30 (Beta-Ionone), SX1 (Androstenedione), ZCP20 (Calacone), ZCP23 (Isoacolamone), ZCP4 (Shyobunone), ZCP6, and SX5 (Estradiol)) and MAPK P38.

Figure 6

Effect of containing serum on cell viability and WSP attenuated the production of inflammatory cytokines in MSU-activated THP-1 macrophages. (a) The viability of MSU-activated THP-1 macrophages was evaluated by CCK-8 assay. Values are mean ± SD (n = 6). ##p < 0.01 vs. CON group; ^∗∗p < 0.01 vs. MOD group. (b–e) Levels of IL-1β, TNF-α, IL-18, and IL-6 in the supernatant of MSU-activated THP-1 macrophages measured by ELISA. Values are mean ± SD (n = 4∼5). (f–i) Relative mRNA levels of IL-1β, TNF-α, IL-18, and IL-6 of MSU-activated THP-1 macrophages measured by qRT-PCR. Values are mean ± SD (n = 3). #p < 0.05, ##p < 0.01 vs. CON group; ∗p < 0.05, ^∗∗p < 0.01 vs. MOD group.

Figure 7

WSP downregulated the protein expression of MAPK/NF-κB signaling pathways in MSU-activated THP-1 macrophages. (a, b) Levels of p-P38, p-ERK, p-JNK, P38, ERK, and JNK in MSU-activated THP-1 macrophages were analyzed by western blot. Values are mean ± SD (n = 3). (c) The nuclear translocation activity of NF-κB p65 in MSU-activated THP-1 macrophages was analyzed by immunofluorescence staining. Values are mean ± SD (n = 3). (d, e) Levels of p-p65, p-IKKα/β, p65, IKKα, and IKKβ in MSU-activated THP-1 macrophages were analyzed by western blot. Values are mean ± SD (n = 3). #p < 0.05, ##p < 0.01 vs. CON group; ∗p < 0.05, ^∗∗p < 0.01 vs. MOD group.

Figure 8

WSP suppressed NLRP3 inflammasome activation in MSU-activated THP-1 macrophages. (a, b) Levels of NLRP3, ASC, and cleaved Caspase-1 in MSU-activated THP-1 macrophages were analyzed by western blot. Values are mean ± SD (n = 3). (c) Relative mRNA levels of NLRP3 of MSU-activated THP-1 macrophages measured by qRT-PCR. Values are mean ± SD (n = 3). #p < 0.05, ##p < 0.01 vs. CON group; ∗p < 0.05, ^∗∗p < 0.01 vs. MOD group.