Uncovering pharmacological mechanisms of Wu-tou decoction acting on rheumatoid arthritis through systems approaches: drug-target prediction, network analysis and experimental validation

Abstract

Wu-tou decoction (WTD) has been extensively used for the treatment of rheumatoid arthritis (RA). Due to lack of appropriate methods, pharmacological mechanisms of WTD acting on RA have not been fully elucidated. In this study, a list of putative targets for compositive compounds containing in WTD were predicted by drugCIPHER-CS. Then, the interaction network of the putative targets of WTD and known RA-related targets was constructed and hub nodes were identified. After constructing the interaction network of hubs, four topological features of each hub, including degree, node betweenness, closeness and k-coreness, were calculated and 79 major hubs were identified as candidate targets of WTD, which were implicated into the imbalance of the nervous, endocrine and immune (NEI) systems, leading to the main pathological changes during the RA progression. Further experimental validation also demonstrated the preventive effects of WTD on inflammation and joint destruction in collagen-induced arthritis (CIA) rats and its regulatory effects on candidate targets both in vitro and in vivo systems. In conclusion, we performed an integrative analysis to offer the convincing evidence that WTD may attenuate RA partially by restoring the balance of NEI system and subsequently reversing the pathological events during RA progression.

Figure 1. A schematic diagram of the systematic strategies for uncovering the pharmacological mechanisms of herbal formula Wu-tou decoction acting on RA.

Figure 2. Interaction network of hubs selected from network of putative targets of Wu-tou decoction (WTD) and known RA-related targets.

According to their associated biological processes or pathways, these hub nodes were implicated into the imbalance of nervous, endocrine and immune (NEI) system, leading to the main pathological changes during the RA progression.

Figure 3

(A) Rheumatoid arthritis pathway (KEGG ID: hsa05323) downloaded from KEGG database. (B) Interaction network of Wu-tou decoction (WTD) candidate targets which are involved into Rheumatoid arthritis pathway and related pathways during the progression of RA.

Figure 4. Effects of Wu-tou decoction (WTD) on severity of arthritis in collagen-induced arthritis (CIA) rats.

(A) macroscopic evidence of arthritis such as erythema or swelling was markedly observed in vehicle-treated CIA rats, while dose of 3.8 g/(kg·day) WTD significantly attenuated arthritis severity in CIA mice; (B) Doses of 0.95 ~ 3.8 g/(kg·day) WTD significantly decreased the mean arthritis score in a dose-dependent manner compared with vehicle-treated CIA mice; (C) Doses of 0.95 ~ 3.8 g/(kg·day) WTD significantly decreased the arthritis incidence in a dose-dependent manner compared with vehicle-treated CIA rats; (D) Doses of 0.95 ~ 3.8 g/(kg·day) WTD significantly decreased the percentage of arthritis limbs in a dose-dependent manner compared with vehicle-treated CIA rats; (E) Doses of 0.95 ~ 3.8 g/(kg·day) WTD significantly increased the time of arthritis first appeared compared with vehicle-treated CIA rats. Data are represented as the mean ± S.D. (n = 12). ‘#', P <0.05, comparison with the normal control.‘*’, ‘**', and ‘***', P <0.05, P <0.01, and P <0.001, respectively, comparison with the vehicle control.

Figure 5. Effect of Wu-tou decoction (WTD) on radiological changes and histologic lesions of CIA rats.

(A) shows the clinical manifestation of CIA rats on day 21 after immunization, red swelling in the paws was obviously improved in the WTD-treated group. (B) displays histological observations of the joints in rats (HE staining). (C) shows the results of safranin-O staining in cartilage of joints. (D), (E) and (F) show the inflammation scores, bone destruction score and the content of proteoglycan in joints respectively, as described in methods. Data are represented as the mean ± SD (n = 12). ‘*’, ‘**’, and ‘***', P <0.05, P <0.01, and P <0.001, respectively, comparison with the vehicle control.

Figure 6. Effect of Wu-tou decoction (WTD) on the expression of three acetylcholine receptors CHRM1, CHRM3 and CHRNA2, and one glucocorticoid receptor NR3C1 in the joint of CIA rats detected by Western blot analysis.

Treatment of the rats was the same as the description in Figure 3. (A) Representative blots of CHRM1, CHRM3, CHRNA2 and NR3C1 proteins; (B)~(E) Relative expression levels of CHRM1, CHRM3, CHRNA2 and NR3C1 proteins in different groups. Data are represented as the mean ± S.D. ‘^###', P <0.001, comparison with the normal control. ‘*’, ‘**’, and ‘***', P <0.05, P <0.01, and P <0.001, respectively, comparison with the vehicle control.

Figure 7. Effect of Wu-tou decoction (WTD) on the expression of MMP-1 and MMP-13 in the joint of CIA rats and in human fibroblast-likesynoviocytes of rheumatoid arthritis (HFLS-RA).

Treatment of the rats was the same as the description in Figure 3. (A) and (B) respectively showed the few positive signals for MMP-1 and MMP-13 in normal control rats, while MMP-1 and MMP-13 were strongly expressed in cartilage of the CIA rats. (C) and (D) Doses of 0.95 ~ 3.8 g/(kg·day) WTD significantly decreased the expression of MMP-1 and MMP-13 compared with vehicle-treated CIA rats. Data are represented as the mean ± S.D. (n = 12). '^###', P <0.001, comparison with the normal control. ‘*', ‘**', and ‘***', P <0.05, P <0.01, and P <0.001, respectively, comparison with the vehicle control. (E) Representative blots of MMP-1 and MMP-13 proteins detected by western blot analysis; (F)~(G) Relative expression levels of MMP-1 and MMP-13 proteins in different groups. Data are represented as the mean ± S.D. ‘^##', P <0.01, comparison with the control cells.‘*' and ‘**', P <0.05 and P <0.01, respectively, comparison with the IL-1β-induced vehicle control.

Figure 8. Effect of Wu-tou decoction (WTD) on IL-1β, TNFα, HIF-1α and VEGF in sera of CIA rats and in human fibroblast-likesynoviocytes of rheumatoid arthritis (HFLS-RA).

Treatment of the rats was the same as the description in Figure 3. (A-D). Data are represented as the mean ± SD (n = 12). ‘^###', P <0.001, comparison with the normal control. ‘*', ‘**', and ‘***', P <0.05, P <0.01, and P <0.001, respectively, comparison with the vehicle control. (E)~(F) Representative blots of IL-1β, TNFα, HIF-1α and VEGF proteins detected by western blot analysis; (G)~(J) Relative expression levels of IL-1β, TNFα, HIF-1α and VEGF proteins in different groups. Data are represented as the mean ± S.D. ‘^##', P <0.01, comparison with the control cells. ‘*’ and ‘**', P <0.05 and P <0.01, respectively, comparison with the IL-1β-induced vehicle control.