Hei-Gu-Teng Zhuifenghuoluo Granule Modulates IL-12 Signal Pathway to Inhibit the Inflammatory Response in Rheumatoid Arthritis

Abstract

Rheumatoid arthritis (RA) is a type of chronic systemic inflammatory disease; it has a very complicated pathogenesis, and multiple pathological changes are implicated. Traditional Chinese medicine (TCM) like Tripterygium wilfordii Hook. F. or Sinomenium acutum (Thunb.) Rehd et Wils. has been extensively used for centuries in the treatment of arthritic diseases and been reported effective for relieving the severity of RA. Hei-Gu-Teng Zhuifenghuoluo granule (HGT) which contains Periploca forrestii Schltr., Sinomenium acutum (Thunb.) Rehd et Wils., and Lysimachia paridiformis Franch. var. stenophylla Franch. was a representative natural rattan herb formula for the treatment of RA in China, but the mechanism has not been elucidated. This study aimed at exploring the mechanism of HGT on RA using the bioinformatics analysis with in vivo and in vitro experiment validation. The potential action mechanism was first investigated by bioinformatics analysis via Ingenuity Pathway Analysis (IPA) software. After that, we use experimental validation such as collagen-induced arthritis (CIA) mice model in vivo and U937 cell model in vitro. The bioinformatics results suggested that HGT may have anti-inflammatory characteristic on RA and IL-12 signaling pathway could be the potential key trigger. In vivo experiments demonstrated that HGT ameliorated the symptoms in CIA mice and decreased the production of inflammatory cytokines in both mice ankle joints and serum. Furthermore, HGT effectively inhibited the activation of IL-12R and STAT4 on IL-12 signaling pathway. In vitro experiments showed that HGT inhibited the production of IL-12R and STAT4 induced by IL-12 in lipopolysaccharide- (LPS-) stimulated U937 cells. Moreover, IL-12R knockdown was able to interfere with the inhibition effects of HGT on the production of these cytokines. Our results confirmed the anti-inflammatory property of HGT, which was attributed to its inhibition on IL-12 signaling pathway.

Figure 1

Bioinformatics analysis results. (a) The relationship between compound-related genes and RA disease-related genes; (b) shared signaling pathways between gene molecular networks related with rheumatoid arthritis (RA) and protein target molecular network of HGT performed using the Ingenuity Pathway Analysis (IPA) compare module. The signaling pathways of HGT were represented as dark blue, while the signaling pathways of RA were represented as light blue. The results showed that IL-12 signaling pathway was the top one shared signaling pathway.

Figure 2

Effects of HGT on CIA mice. (a) The morphological characteristics of the representative of the CIA mice joint; (b) arthritic score in different days after the treatment of HGT; MTX represented the methotrexate group; HGT represented the HGT group. Date are represented as the mean ± SD (n = 7); ^∗∗ p < 0.01, comparison with model group.

Figure 3

Effects of HGT on histopathological of CIA mice joints. (a) Histopathological characteristics of representative CIA mice joints; (b) inflammation score in the different group after treatment with HGT; MTX represented the methotrexate group, HGT represented the HGT group. Date are represented as the mean ± SD (n = 7); ^∗∗ p < 0.01, comparison with the model group.

Figure 4

The effect of the expression of cytokines in mice. (a–c) IL-6, IL-1β, and TNF-α in the CIA mice with the treatment of HGT and also the mRNA expression in CIA mice joints with treatment. The data are represented as the mean ± SD (n = 7); ^∗∗ p < 0.01, when compared with the model group.

Figure 5

The effects of HGT on IL-12 and STAT4 expression in mice. (a) Immunohistochemistry of IL-12 in joints of mice with the treatment of HGT; (b) the levels of IL-12 mRNA in mouse joints after the treatment of HGT; (c) the expression of IL-12Rβ1 and STAT4 protein in CIA mice joints; (d) IL-12 in serum of CIA mice with the treatment of HGT. The data are represented as the mean ± SD (n = 7); ^∗∗ p < 0.01, when compared with the model group.

Figure 6

Effects of HGT on the expression of cytokines in U937 cells. (a–c) IL-6 levels, TNF-α levels, and IL-1β levels in LPS-induced U937 cells after the treatment of IL-12 siRNA and HGT; (d–f) IL-6, TNF-α, and IL-1β mRNA levels in LPS-induced U937 cells after treatment with IL-12 siRNA and HGT. The data are represented as the mean ± SD (n = 7); ^∗ p < 0.05 and ^∗∗ p < 0.01, when compared with the LPS group.

Figure 7

The effects of HGT on cytokine expression after IL-12Rβ1 knockdown. Firstly, the U937 cells were first treated with LPS and the other without LPS, and then all cells were transfected with IL-12Rβ1 siRNA. 48 h later, the U937 cells were then treated with HGT or without HGT for 48 h. (b) Western blot of the protein of IL-12Rβ1 and STAT4; (a, c, d, e, f) IL-12, IFN-γ, IL-4, IL-13, and IL-5 productions were determined by ELISA. The data are represented as the mean ± SD (n = 7); ^∗∗ p < 0.01, when compared with the LPS group.

Figure 8

Schematic diagram depicting how HGT modulates the IL-12 signaling pathway to inhibit the inflammatory response in rheumatoid arthritis. By inhibiting IL-12R, HGT could inhibit STAT4 and then inhibit the inflammation, meanwhile, decreasing TNF-α and IFN-γ.