Korean J Physiol Pharmacol.  2024 Jan;28(1):59-72. 10.4196/kjpp.2024.28.1.59.

Mechanism of Wenshen Xuanbi Decoction in the treatment of osteoarthritis based on network pharmacology and experimental verification

• You H
• Song S
• Liu D
• Ren T
• Yin SJ
• Wu P
• Mao J

• KMID: 2550289
• DOI: http://doi.org/10.4196/kjpp.2024.28.1.59

Abstract

To investigate the mechanism of Wenshen Xuanbi Decoction (WSXB) in treating osteoarthritis (OA) via network pharmacology, bioinformatics analysis, and experimental verification. The active components and prediction targets of WSXB were obtained from the TCMSP database and Swiss Target Prediction website, respectively. OA-related genes were retrieved from GeneCards and OMIM databases. Protein-protein interaction and functional enrichment analyses were performed, resulting in the construction of the Herb-Component-Target network. In addition, differential genes of OA were obtained from the GEO database to verify the potential mechanism of WSXB in OA treatment. Subsequently, potential active components were subjected to molecular verification with the hub targets. Finally, we selected the most crucial hub targets and pathways for experimental verification in vitro. The active components in the study included quercetin, linolenic acid, methyl linoleate, isobergapten, and beta-sitosterol. AKT1, tumor necrosis factor (TNF), interleukin (IL)-6, GAPDH, and CTNNB1 were identified as the most crucial hub targets. Molecular docking revealed that the active components and hub targets exhibited strong binding energy. Experimental verification demonstrated that the mRNA and protein expression levels of IL-6, IL-17, and TNF in the WSXB group were lower than those in the KOA group (p < 0.05). WSXB exhibits a chondroprotective effect on OA and delays disease progression. The mechanism is potentially related to the suppression of IL-17 and TNF signaling pathways and the down-regulation of IL-6.

Keyword

Inflammation; Molecular docking simulation; Network pharmacology; Osteoarthritis

Figure

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  Fig. 1 The protocol of our study procedures. WSXB, Wenshen Xuanbi Decoction; TCMSP, Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform; OA, osteoarthritis; DEGs, differential genes; PPI, protein-protein interaction; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; qRT-PCR: quantitative real-time polymerase chain reaction.

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  Fig. 2 Screening of targets and network construction. (A) Venn diagram of WSXB and OA targets. (B) Network of Drug-Component-Target. Circle nodes represent the drugs and components in WSXB, and the blue diamond represents the targets of WSXB. WSXB, Wenshen Xuanbi Decoction; OA, osteoarthritis.

• 

  Fig. 3 The PPI network. (A) The PPI network constructed in the STRING database. (B) The PPI network visualized by Cytoscape software. Node size and color were set to reflect the degree value. The greater the degree value is, the redder the color is. PPI, protein-protein interaction.

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  Fig. 4 Bubble diagram for GO and KEGG enrichment analysis. (A) Biological processes for the targets of WSXB in the treatment of OA. (B) Molecular function for the targets of WSXB in the treatment of OA. (C) Cellular components for the targets of WSXB in the treatment of OA. (D) represents KEGG for the targets of WSXB in the treatment of OA. The bubble size represents the Gene Count, and the bubble color represents the p-value. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; WSXB, Wenshen Xuanbi Decoction; OA, osteoarthritis.

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  Fig. 5 Circle diagram for KEGG enrichment analysis. (A) KEGG enrichment analysis of TOP10 (first 5). (B) KEGG enrichment analysis TOP10 (last 5). KEGG, Kyoto Encyclopedia of Genes and Genomes.

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  Fig. 6 Heatmap of binding affinity. The yellower the color is, the more stable the binding force. A represents AKT1, B represents TNF, C represents IL-6, D represents GAPDH, and E represents CTNNB1. 1 represents quercetin, 2 represents linolenic acid, 3 represents methyl linoleate, 4 represents isobergapten, and 5 represents beta-sitosterol.

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  Fig. 7 Schematic diagram of docking between quercetin and hub targets. Molecular models of quercetin binding to the targets (A) AKT1, (B) TNF, (C) IL-6, (D) GAPDH, (E) CTNNB1.

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  Fig. 8 Boxplot and UMAP plots before and after batch effect removal. (A, B) Boxplot plots before and after batch effect removal. (C, D) UMAP plots before and after batch effect removal.

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  Fig. 9 Heat map and volcano map of databases in the GEO. (A) Heat map and volcano map of GSE55235. (B) Heat map and volcano map of GSE55457. (C) Heat map and volcano map of GSE55584.

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  Fig. 10 Venn diagram of co-DEGs. DEGs, differential genes.

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  Fig. 11 Bubble diagram and bubble diagram for KEGG enrichment analysis of co-DEGs. (A) Biological processes of co-DEGs. (B) Cellular components of co-DEGs. (C) Molecular function of co-DEGs. (D) Bubble diagram for KEGG enrichment analysis of co-DEGs. KEGG, Kyoto Encyclopedia of Genes and Genomes; DEGs, differential genes.

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  Fig. 12 Results of experimental verification in vitro. (A) Effect of WSXB on the survival rate of FLS cells. (B) Effects of WSXB on expression of IL-17, IL-6, AKT1, TNF mRNA in FLS cells. (C) Effects of WSXB on expression of IL-17, IL-6, AKT1, TNF proteins in FLS cells. (a) Images of IL-17, IL-6, AKT1, and TNF proteins in FLS cells; (b) Relative protein expression of IL-17, IL-6, AKT1, and TNF in FLS cells. Values are presented as mean ± SD. WSXB, Wenshen Xuanbi Decoction; FLS, fibroblast-like synovial cells; KOA, knee osteoarthritis. Compared with the blank group, * represents p < 0.05; compared with the KOA group, # represents p < 0.05.

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