Yunxia Wu,1,* Yue Zhang,1,* Zishan Wang,1 Yun Lu,1 Yabei Wang,1 Jie Pan,1 Chenxi Liu,1 Wen Zhu,2,* Yue Wang2,* 

1Academy of First Clinical Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, People’s Republic of China; 2Department of Rheumatology & Immunology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Yue Wang; Wen Zhu, Department of Rheumatology & Immunology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, People’s Republic of China, Email wangyue@njucm.edu.cn; zhuwen@njucm.edu.cn

Background: Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovitis and inflammatory cell infiltration. The traditional Chinese medicine prescription, Bitongqing (BTQ) exhibited significant efficacy in the clinical treatment of RA. However, the potential therapeutic mechanisms of BTQ in treating RA have not been fully investigated. This study aims to elucidate the effect of BTQ on collagen-induced arthritis (CIA) rat macrophage pyroptosis, providing a theoretical basis for treating RA.
Methods: This research employed liquid chromatography-mass spectrometry (LC-MS) to identify the primary components of BTQ. The therapeutic effects of BTQ were evaluated in a rat model of CIA. In vivo experiments were conducted using pathohistological staining, immunofluorescence, micro-CT, and Western blotting. Next, Mouse leukemia cells of monocyte macrophage cells (RAW264.7) were induced to undergo pyroptosis using lipopolysaccharide (LPS) and adenosine triphosphate (ATP), and the impact of BTQ on RAW264.7 macrophages was assessed through cell viability, immunofluorescence analysis, lactate dehydrogenase (LDH) secretion measurement, and Western blotting.
Results: BTQ had a therapeutic effect on CIA rats, which was mainly manifested as a reduction in joint inflammation, foot swelling, bone erosion, and amelioration of pathological changes in these rats. Further studies revealed that BTQ inhibited the levels of cytokine production interleukin-18 (IL-18) and interleukin-1β (IL-1β), and likewise, it inhibited the expression of key proteins in the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) mediated pyroptosis in the synovial tissues of CIA rats. The results of in vitro experiments demonstrated that BTQ attenuated LDH secretion, decreased IL-18 and IL-1β cytokine production, and downregulated expression of key proteins involved in the NLRP3-mediated pyroptosis on RAW264.7 macrophages.
Conclusion: The therapeutic potential of BTQ in CIA lies in its ability to inhibit NLRP3-mediated macrophage pyroptosis, thereby suggesting a promising strategy for the treatment of RA.

Keywords: rheumatoid arthritis, Bitongqing, pyroptosis, NLRP3 inflammasome, macrophage