Interpreting the pharmacological mechanisms of Juanbi recipe on rheumatoid arthritis through network pharmacology, molecular docking

Abstract

Background: Traditional Chinese medicine (TCM) offers the advantage of effectively relieving rheumatoid arthritis (RA) with minimal side effects. The Juanbi recipe is a commonly utilized TCM treatment for RA, yet its pharmacological mechanism remains unclear. Network pharmacology serves as an effective tool for identifying pharmaceutical ingredients and potential therapeutic targets of TCM, thereby uncovering its mechanisms. This study aimed to identify the core target genes and explore the mechanisms underlying the treatment of RA with the Juanbi recipe.

Methods: This study adopted the method of network pharmacology to filter key gene targets of Juanbi recipe in RA treatment. Single-cell ribonucleic acid (RNA) sequencing data was used to screen the key genes to form the core genes of Juanbi recipe in RA treatment. The molecular docking technique was used to verify the core target genes and explore the mechanisms of Juanbi recipe in RA treatment. The RA model of mice was induced by the collagen-induced arthritis and the effect of Juanbi recipe was evaluated by intragastric administrating of extraction of Juanbi recipe. Enzyme linked immunosorbent assay was used to analysis serum inflammatory factors. Hematoxylin and eosin staining was used to evaluate inflammation and immunohistochemical (IHC) staining was used to evaluate core target genes and pathways in synovium of ankle.

Results: This study screened out 281 active molecules in Juanbi recipe, found 105 key target genes of Juanbi recipe in RA treatment, and drew an "ingredient - molecule - gene" diagram. Juanbi recipe reduced the levels of serum interleukin (IL)-1 and IL-6, the inflammatory infiltration in synovium, demonstration that Juanbi recipe reduced both systemic and synovial inflammatory response. Single cell RNA sequencing data were used to select six core target genes and six core active molecules of Juanbi recipe in RA treatment. The pathways of Juanbi recipe in RA treatment involved in activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB) pathway. Results of western blot and IHC staining showed that Juanbi recipe decreased the expressions of c-jun and p65, which demonstrated that Juanbi recipe inhibited the expression of AP-1 and NF-κB pathway in RA.

Conclusions: The core active molecules of Juanbi recipe could inhibit key factors of AP-1 and NF-κB pathway to inhibit the inflammation, which played a protective role in RA.

Keywords: Juanbi recipe; Rheumatoid arthritis (RA); activator protein-1 (AP-1); network pharmacology; nuclear factor kappa B (NF-κB).

Figure 1

Hematoxylin and eosin staining of ankle joints. RA, rheumatoid arthritis.

Figure 2

Serum interleukin-1 and -6 levels. *, P<0.05; **, P<0.01; ns, not significant. RA, rheumatoid arthritis.

Figure 3

Key target genes of Juanbi recipe in treating rheumatoid arthritis. (A) There are 661 genes related to rheumatoid arthritis, 439 genes targeted by Juanbi recipe, and 105 key target genes of Juanbi recipe in the treatment of rheumatoid arthritis. (B) Diagram of “drug molecule target gene” of Juanbi recipe on the treatment of rheumatoid arthritis.

Figure 4

GO and KEGG analysis of key target genes of Juanbi recipe in RA treatment. GO analysis contained biological processes (A), cellular components (B), and molecular functions (C). (D) Pathways involved analyzed by KEGG. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; RA, rheumatoid arthritis; IL-17, interleukin 17; TNF, tumour necrosis factor.

Figure 5

Clustering and differential analysis of PBMCs and synovium from single cell RNA sequencing data. PBMCs were classified into 5 groups of cells (A) and 263 differential genes were found (B). Synovial cells were classified into 4 groups of cells (C) and 76 differential genes were found (D). The key target genes of Juanbi recipe were intersected with differential genes from the two single cell RNA sequencing data (E). UMAP, uniform manifold approximation and projection; DEG, differential expression gene; FC, fold change; NS, not significant; RA, rheumatoid arthritis; scRNA-seq, single-cell RNA sequencing; PBMCs, peripheral blood mononuclear cells; DC, dendritic cells; NK, natural killer cells.

Figure 6

The conformations of the stable binding of the core active molecules and the core target proteins. (A) Beta-sitosterol binding to JUN; (B) daidzein binding to FOS; (C) quercetin binding to FOS; (D) quercetin binding to MMP3; (E) quercetin binding to NFKBIA; (F) quercetin binding to SPP1.

Figure 7

The expression of c-jun and p65 in synovium examined by Western blot. RA, rheumatoid arthritis.

Figure 8

The expression of c-jun in ankle joint examined by immunohistochemical staining. RA, rheumatoid arthritis.

Figure 9

The expression of p65 in ankle joint examined by immunohistochemical staining. RA, rheumatoid arthritis.