Rheumatoid arthritis (RA) is one of the most prevalent systemic chronic autoimmune diseases worldwide, affecting approximately 0.75% of the global population (Smolen et al., 2016). It is characterized by persistent inflammation of the synovial membranes, which leads to joint pain, stiffness, and swelling (Dong et al., 2020; Sun et al., 2023). The disease initiates within the first few years of visible arthritis and progresses through various stages, starting from asymptomatic immune dysfunction to being clinically recognized as RA. The etiology of RA is multifactorial, primarily associated with genetic polymorphisms, epigenetic modifications, immune dysregulation, gut microbiota dysfunction, and metabolic imbalances (Sparks, 2019). Despite extensive research, the complete pathophysiological mechanisms of RA remain elusive, contributing to the current absence of therapeutic options that can fully arrest the progression of RA.

Current treatments include biological agents, such as Disease-modifying antirheumatic drugs (DMARDs) and tumor necrosis factor (TNF) inhibitors (Lee and Weinblatt, 2001). However, these treatments were unable to attain immune balance or drug-free remission, thereby lacking the capability to repair the damaged tissues of RA patients (Alivernini et al., 2020; Kuo et al., 2019). The development of alternative therapies that can effectively halt the progression of RA is therefore a critical and urgent need (McInnes and Schett, 2011; Singh, 2022; Yu et al., 2022). Studies indicate that abnormalities in the immune system, including autoantibodies against rheumatoid factors (RF) and interleukin-1 beta (IL-1β), play a central role in the pathology of RA (Ding et al., 2023; Dong et al., 2020). Chronic synovial inflammation, a hallmark of RA, results from the persistent accumulation of inflammatory cells. Targeting these inflammatory pathways has proven effective in reducing RA-associated inflammation (Yu et al., 2022).

A number of studies have provided solid evidence that NLRP3 inflammasome activation has a deleterious role in RA development (Yin, H. et al., 2022). Consequently, appropriately managing the NLRP3 inflammasome is vital for the treatment of RA (Li et al., 2020; Yu et al., 2022). Macrophages, critical immune cells, function dually in inflammatory responses, shifting between anti-inflammatory M2 and pro-inflammatory M1 subtypes. Increased M1 macrophage levels are associated with heightened the production of inflammatory factors and aggravated RA pathology. Hence, inhibiting M1 macrophage polarization is beneficial for reducing inflammatory factors in RA (Alivernini et al., 2020; Gao et al., 2023; Liu, T. et al., 2021).

The incidence of RA in Tibet is notably higher than in other regions of China, a disparity influenced by factors such as plateau geography, climate, and diet (Zhang et al., 2020). Traditional Chinese medicine (TCM) is widely acknowledged for its cost-effectiveness, distinct curative effects, low toxicity, and minimal side effects in RA treatment (Li et al., 2023). In addition, TCM has a variety of pharmacological activities with significant health benefits, such as Saffron and Rhizoma polygonati (Abdalla et al., 2021; Xie et al., 2021). Tibetan medicine, a pivotal facet of TCM, boasts a lengthy history and extensive experience in RA treatment (Huang et al., 2021). With over 3800 years of heritage, Traditional Tibetan medicine (TTM) possesses a comprehensive grasp of the pathogenesis of RA and has actively explored various treatments, documenting more than 3000 types of herbal and mineral drugs (Li, Z. et al., 2022; Liu, C. et al., 2021). Ganlu Formula is a popular prescription with remarkable curative effect in the Tibetan medical classics "Four Medical Codes" and "Crystal Pearl Materia Medica".(Liu et al., 2016; Su et al., 2023; Wang et al., 2017). The basic formula consists of Ganlu Formula is Rhododendron anthopogonoides Maxim, Juniperus angosturana R.P.Adams, Ephedra sinica Stapf, Myricaria platyphylla Maxim, Artemisia sieversiana Ehrh.

Recent investigations into the Ganlu Formula have demonstrated that its aqueous extracts, particularly those derived from manna-based formulations, effectively alleviate joint swelling and mitigate RA symptoms in collagen-induced arthritic (CIA) rats(Wang et al., 2017; Wen et al., 2024). Despite these promising outcomes, the specific roles and underlying molecular mechanisms of the Ganlu Formula's ethyl acetate extract (GLEE) in RA treatment remain to be fully elucidated.

In the present study, our objectives were to investigate the effects of GLEE at the cellular level, assess their therapeutic efficacy in CIA rats, and analyze the active components to determine their effective material basis. Specifically, we aimed to elucidate whether the anti-arthritic effectiveness of GLEE is associated with the inhibition of NLRP3 inflammasome activation and the suppression of M1-type macrophage polarization.