Wutou decoction alleviates arthritis inflammation in CIA mice by regulating Treg cell stability and Treg/Th17 balance via the JAK2/STAT3 pathway

Ethnopharmacological relevance: Wutou Decoction (WTD) is a classic traditional Chinese medicine formula, which has shown clinical efficacy in treating rheumatoid arthritis (RA). The Treg stability and Th17/Treg imbalance is an important immunological mechanism in RA progression. Whether WTD regulates CD4⁺ T cell subsets has not been thoroughly investigated yet.

Aim of the study: This study aimed to explore the potential role and mechanisms of WTD in regulating the diminished stability of Treg cells and the imbalance of CD4⁺ T cell subsets via in vivo and in vitro experiments.

Materials and methods: Firstly, the therapeutic effects of WTD on the collagen-induced arthritis (CIA) mouse and its potential regulatory function on CD4⁺ T cell subsets were evaluated in vivo. Animal specimens were collected after 31 days of treatment with WTD. The anti-arthritic and anti-inflammatory effects of WTD were assessed through arthritis scoring, body weight, spleen index, serum IL-6 levels, and micro-PET/CT imaging. Gene enrichment analysis was performed to evaluate the activation T cell-related signaling pathway. Flow cytometry was used to determine the proportions of CD4⁺ T cell subsets in vitro and in vitro. Additionally, ELISA was used to assess the secretion of IL-10 and TGF-β by Treg cells under inflammatory conditions. The suppressive function of Treg cells on cell proliferation under inflammatory conditions was examined using CFSE labeling. Immunofluorescence staining was performed to detect the phosphorylation levels of STAT3 in CD4⁺ T cells from mouse spleen tissues. Western blotting was used to evaluate the phosphorylation levels of JAK2/STAT3 in Treg cells.

Results: WTD significantly alleviated joint inflammation in CIA mice. WTD reduced serum IL-6 levels in CIA mice, improved their body weight and spleen index. WTD treatment inhibited the activation of CD4⁺ T cell subgroup-related signaling in the joint tissues of CIA mice. In vitro and in vitro experiments showed that WTD increased the proportion of Treg cells and decreased the proportion of Th17 cells in CIA mice spleen. Furthermore, WTD promoted the secretion of IL-10 and TGF-β by Treg cells and enhanced the inhibitory capacity of Treg cells on cell proliferation under inflammatory conditions. Immunofluorescence detected decreased STAT3 phosphorylation levels in CD4⁺ T cells from CIA mice spleen, while western blotting revealed a decrease in JAK2/STAT3 phosphorylation levels in Treg cells in vitro.

Conclusions: Inhibiting JAK2/STAT3 phosphorylation is a potential mechanism through which WTD improves Treg cell stability, balances CD4⁺ T cell subsets, and attenuates RA joint inflammation.