Ganoderma lucidum spore powder alleviates rheumatoid arthritisassociated pain hypersensitivity through inhibiting accumulation, N1 polarization, and ROS production of neutrophils in mice

sen huang^1,2Zhaochun Zhan^2,3Fei Xu²Xiaolin Liu¹Zhenning Fang¹Wenbo Wu¹Zhile Liang^1,2Guoguo Liu¹Mengyuan Wang¹Helena Soares Da Silva¹Xin Luo^1,2,3,4*Kai Mo^1,4*

• ¹Zhujiang Hospital, Southern Medical University, Guangzhou, China
• ²Guangdong-Hong Kong-Macao Greater Bay Area Center for Brian Science and Brain-Inspired Intelligence, Guangzhou, China
• ³Shunde Hospital, Southern Medical University, Foshan, Guangdong Province, China
• ⁴Institute of Perioperative Medicine and Organ Protection, Zhujiang Hospital, Southern Medical University, Guangzhou, China

Introduction: Rheumatoid arthritis (RA) is a chronic condition characterized by joint pain that significantly impairs patients' work and daily lives. The limited understanding of the pathological mechanisms underlying RA-related pain poses challenges for effective clinical pain management. Ganoderma lucidum spore powder (GLSP) has demonstrated therapeutic benefits in various diseases, with no reported toxicity or adverse effects.Methods: This study investigates the role of neutrophils in the pathological mechanisms of RA-related pain using collagen-induced arthritis (CIA) mice and an ex vivo neutrophil model. A combination of techniques, including animal models, flow cytometry, behavioral testing, cell adoptive transfer, and network pharmacology analysis, was employed to evaluate the effects and targets of GLSP on pain symptoms and neutrophil activity in CIA mice.Results: Flow cytometric analysis revealed the accumulation and activation of neutrophils in the paws of CIA mice. Furthermore, the levels of pro-inflammatory CD95 + neutrophil subpopulations (N1 state) and ROS + cells in the affected paws were positively correlated with the severity of mechanical allodynia and heat hyperalgesia observed in these mice. Our findings indicate that oral administration of GLSP significantly alleviates joint destruction, paw swelling, and pain hypersensitivity in CIA mice. Notably, GLSP reversed CIA-induced neutrophil accumulation, N1 polarization, and reactive oxygen species (ROS) production. Both network pharmacology target prediction and in vivo/in vitro experimental validation indicated that GLSP inhibits N1 polarization and ROS production in neutrophils by modulating the TNF-α signaling pathway, thus exerting RA-specific analgesic effects.Discussion: In summary, this study offers new insights into the pathological mechanisms of RA-related pain and demonstrates that neutrophil accumulation, N1 polarization, and ROS production contribute to RA-related pain. GLSP alleviates RA-related pain by inhibiting the pro-inflammatory phenotype of neutrophils, highlighting its potential for clinical translation in the treatment of RA.