Therapeutic effects of shaogan fuzi decoction in rheumatoid arthritis: Network pharmacology and experimental validation

Abstract

Shaogan Fuzi Decoction (SGFD), one of the classical prescriptions of Chinese Medicine, has a long history in the treatment of rheumatoid arthritis (RA), but definitive studies on its efficacy and mechanism of action are lacking. This study aims to elucidate the pharmacodynamic role of SGFD against RA and the potential mechanisms based on a combination of network pharmacology and experimental verification. The RA model in rats was induced by intradermal injection of bovine type Ⅱ collagen and incomplete Freund's adjuvant at the tail root. SGFD was administered once a day by oral gavage for 4 weeks. After SGFD administration, rat's arthritis index (AI) score and paw swelling decreased to some extent, and synovial inflammation, vascular hyperplasia, and cartilage destruction of the ankle joint were improved. Simultaneously, thymus and spleen index and serum levels of C-reactive protein (CRP) were lowered. Network pharmacology revealed that quercetin, kaempferol, naringenin, formononetin isorhamnetin and licochalcone A were the potentialiy active components, and IL6, TP53, TNF, PTGS2, MAPK3 and IL-1β were potential key targets for SGFD in the treatment of RA. Ingredients-targets molecular docking showed that the components had the high binding activity to these target proteins. The mechanism of SGFD for RA involves various biological functions and is closely correlated with TNF signaling pathway, Osteoclast differentiation, T cell receptor signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, NF-κB signaling pathway, toll-like receptor signaling pathway, and so on. Western blot and ELISA showed that the expression of toll-like receptor 4 (TLR4), nuclear factor kappa-B (NF-κB) p65, phosphorylated c-Jun N-terminal kinase (p-JNK), p-p38, phosphorylated extracellular regulated kinase (p-ERK) and TNF-α was significantly upregulated in the synovium of RA rats, and the levels of serum inflammatory factors were significantly increased. SGFD inhibits the activation of the TLR4/NF-κB/MAPK pathway and the expression/production of pro-inflammatory cytokines. In summary, SGFD could improve the symptoms and inflammatory response in collagen-induced arthritis (CIA) rat model. The mechanism might be related to the regulation of TLR4/MAPKs/NF-κB signaling pathway and the reduction of inflammatory factor release, which partially confirms the results predicted by network pharmacology.

Keywords: TLR4/MAPKs/NF-κB signaling pathway; inflammatory; network pharmacology; rheumatoid arthritis; shaogan fuzi Decoction.

FIGURE 1

The flowchart of this study based on an integration strategy of network pharmacology and experimental verification.

FIGURE 2

Example of arthritis index score in rats. (A) score 0. (B) score 1. (C) score 2. (D) score 3. (E) score 4.

FIGURE 3

Effect of SGFD on joint swelling in RA rats. (A). Images of rats’ paw swelling in each group (a1-f1: before treatment; a2-f2: after treatment). (B). The results of right hind paw swelling rate. (C). The results of left hind paw swelling rate. (D). The results of AI scores. Data were shown as mean ± SD (Normal, n = 13; Model, TG, SGFD-L, SGFD-M, n = 12; SGFD-H, n = 10). ^* p < 0.05, ^** p < 0.01 vs. normal group; ^# p < 0.05, ^## p < 0.01 vs. model group.

FIGURE 4

Effect of SGFD on pathological changes of the ankle joint in RA rats.

FIGURE 5

Effects of SGFD on the spleen and thymus index and the serum levels of RF and CRP. (A). Spleen index. (B). Thymus index. (C). Serum RF concentration. (D). Serum CRP concentration. Data were shown as mean ± SD (Normal, n = 13; Model, TG, SGFD-L, SGFD-M, n = 12; SGFD-H, n = 10). ^* p < 0.05, ^** p < 0.01 vs. normal group; ^# p < 0.05, ^## p < 0.01 vs. model group.

FIGURE 6

Network pharmacology analysis for screening targets and pathways of SGFD. Venn diagram of (A). Active compounds in SGFD and (B). Common target of SGFD and RA. (C). PPI network of common targets. The target is represented by a circle node. The color of node changed from green to red corresponds to a degree from small to bigger. The thickness of the edge and the combined score value between the protein have a positive correlation. (D). GO enrichment analysis for common targets. (E). KEGG pathway analysis for common targets. (F). Herb-Compound-Target-Pathway network. The red triangles represent the herbal medicines; the yellow squares represent the active chemical compounds of SGFD; the blue dots represent the key targets in the treatment of RA with SGFD; the green dots represent the pathways based on enrichment analysis of key targets.

FIGURE 7

Ingredients-Targets Molecular Docking (A). The binding energy of the main active components of SGFD and the key targets. (B). The binding site of the main active components of SGFD and the key targets. The molecular docking poses of Quercetin—TNF (a), Formononetin—TNF (b), Licochalcone A—IL1β (c), Kaempferol—TNF (d), Licochalcone A—TNF (e), Isorhamnetin—TNF (f), Quercetin—IL1β (g), Isorhamnetin—IL1β (h), Formononetin—PTGS2 (i), Naringenin—TNF (j), Kaempferol—IL1β (k), Formononetin—IL1β (l), Naringenin—IL1β(m), Kaempferol—PTGS2 (n), Isorhamnetin—PTGS2 (o), Kaempferol—MAPK3 (p), Formononetin—MAPK3 (q), Quercetin—PTGS2 (r), Naringenin—IL6 (s), Quercetin—IL6 (t), Licochalcone A—PTGS2 (u), Kaempferol—IL6 (v), Isorhamnetin—IL6 (w), Naringenin—PTGS2 (x), Formononetin—IL6 (y), Naringenin—TP53 (z), Isorhamnetin—MAPK3 (aa), Licochalcone A—MAPK3 (ab), Quercetin—MAPK3 (ac), Licochalcone A—IL6 (ad), Naringenin—MAPK3 (ae), Formononetin—TP53 (af), Licochalcone A—TP53 (ag), Kaempferol—TP53 (ah), Quercetin—TP53 (ai), Isorhamnetin—TP53 (aj).

FIGURE 8

Effects of SGFD on the serum levels of inflammatory cytokines. (A). Serum IFN-γ concentration. (B). Serum IL-17 concentration. (C). Serum TNF-α concentration. (D). Serum IL-1β concentration. (E). Serum IL-6 concentration. Data were shown as mean ± SD (n = 8). ^* p < 0.05, ^** p < 0.01 vs. normal group; ^# p < 0.05, ^## p < 0.01 vs. model group.

FIGURE 9

SGFD treatment suppressed TLR4/NF-κB/MAPKs pathway. (A). Western blotting assay was performed to measure the expression of TLR4, NF-κB p65, JNK, p-JNK, ERK, p-ERK, p38, p-p38 and TNF-α in synovial tissues of knee joints (B–G). Relative expression of TLR4, NF-κB p65, p-p38/p38, p-ERK/ERK, p-JNK/JNK and TNF-α were quantified. Data were shown as mean ± SD (n = 3). ^* p < 0.05, ^** p < 0.01 vs. normal group; ^# p < 0.05, ^## p < 0.01 vs. model group.

FIGURE 10

A model representing SGFD molecular targets in TLR4/MAPK/NF-κB pathway. SGFD exerts its anti-inflammatory effect by inhibiting TLR4 and NF-κB expression, the phosphorylation of JNK and p38 in synovial tissues. The action of SGFD is indicated by red lines.