In the last decade, there has been significant advancement in the studies examining the impact of the immune system on the development of Juvenile Idiopathic Arthritis (JIA). The latest research on the pathogenesis of JIA is centered around immune cells and cytokines. Several cytokines and cytokine-receptor loci that associate with different populations to varying degrees have been identified. These proinflammatory factors are critical in JIA and major contributors to disease progression and bone destruction [39]. This study aimed to investigate the impact of electroacupuncture treatment on CIA rats and explore the underlying mechanism. Our results showed that electroacupuncture treatment effectively suppressed bone destruction in CIA rats in vivo and reduced inflammation in vivo and in vitro.
Acupuncture has been used for many years to treat joint-related diseases [40], and electroacupuncture is a recent application for conditions like osteoarthritis and Rheumatoid Arthritis. Many clinical studies have reported that electroacupuncture can alleviate Rheumatoid Arthritis symptoms and enhance the patients' motor function [41].
Electroacupuncture is an alternative therapy that uses small needles and a low-level electrical current to stimulate specific points in the body. This therapy has been found to have immune-enhancing, antioxidant, and anti-tumor properties [42]. It has gained popularity in China for its ability to regulate immune function and use it in the anti-tumor field [43], [44].
Given these previous outcomes related to EA, we established a CIA rats model and found that EA treatment has a significant positive effect in reducing inflammation and bone damage in the joints of rats with CIA as confirmed by histomorphological analysis and Micro-CT. CIA rats experienced considerable loss of bone mass and cortical thickness, along with deterioration of trabecular bone microstructure. EA treatment not only alleviated symptoms of arthritis and subchondral bone breakage but also decreased bone loss. This was likely due to the suppression of inflammatory cytokine-mediated bone destruction, which occurs when inflammatory cytokines such as IL-1β, IL-6, and TNF-α promote osteoclast formation and bone resorption [45], [46].
Previous research has substantiated the effectiveness of EA in reducing cartilage damage and abnormal inflammatory responses linked to osteoarthritis [47]; however, the underlying cellular and molecular mechanisms are not yet fully understood. Recent findings suggest that macrophages can activate cells in the immediate environment to release inflammatory cytokines [48]. During an immune system malfunction, synovial tissue has an increased number of macrophages that produce inflammatory cytokines [49]. Macrophage polarization, influenced by various cytokines and microbial products, is vital in regulating inflammatory reactions. IL-1β, IL-6, TNF-α, and iNOS are the primary markers and products of M1 macrophages, whereas M2 macrophages, which express Arg-1 and IL-10, are deemed anti-inflammatory effector cells as they secrete a plethora of anti-inflammatory cytokines [50], [51].
Recent studies have demonstrated that alterations in the M1/M2 ratio can significantly impact the development of various inflammatory-related ailments, including JIA [52]. We wondered whether EA could modify the macrophage phenotype in CIA rats and hence alleviate arthritis symptoms. Our study showed that the CIA group had a high M1/M2 macrophage ratio. However, EA was observed to decrease iNOS levels in the synovial tissue and increase Arg-1 expression, as well as the qPCR test results showed that the CIA rats displayed significantly elevated TNF-α, IL-1β, and IL-6 levels when compared to the control group, indicating that EA could effectively transform macrophages from the M1 to the M2 phenotype in an inflammatory milieu. These results suggest that EA can modify macrophages from M1 to M2 phenotype and suppress the secretion of proinflammatory cytokines during pathological conditions. That phenotype switching may be a productive therapeutic approach not only for arthritis but also for other inflammatory diseases.
In addition, the pyroptosis of innate immune cells, including monocytes and macrophages, has been identified as a crucial source of the proinflammatory cytokines IL-1β and IL-18, and has been observed to play a role in the progression of RA pathology. [28]. In addition, there is a positive correlation between IL-18 levels in both serum and synovial fluid and C-reactive protein, joint involvement, and IL-6 levels in children diagnosed with SoJIA. Elevated levels of IL-18 in serum serve as an indicator of active SoJIA disease, while mild to moderate elevation of IL-18 levels correlates with a stable SoJIA state [53]. Even though IL-18 levels were high in the serum of all children diagnosed with JIA, the elevation of IL-18 levels was more significant in children with SoJIA in particular [54]. Some research suggests that children with SoJIA and high levels of IL-6 may experience more joint involvement, while those with high levels of IL-18 may develop macrophage activation syndrome.
TNF-α is a crucial factor in the immune system's pathogenesis and inflammation in JIA [28]. The heightened presence of TNF-α in both the serum and synovial fluid leads to increased systemic and local inflammatory responses [55]. TNF-α not only contributes to the inflammatory reaction within the synovial tissues but also triggers joint degradation. Specifically, Anti-TNF-α biological agents that target this cytokine can effectively alleviate the disease, rapidly reduce inflammation, control arthritis symptoms, and prevent joint structure damage [56]. During disease activity in patients with JIA, there is an abnormal elevation of IL-18 and TNF-α cytokines [57]. Similarly, in this experiment, there was a significant increase in the pro-inflammatory cytokines IL-18 and TNF-α in the CIA rats, indicating their involvement in the inflammatory process.
Pyroptosis is a GSDMD-dependent programmed cell death mediated by caspase-1. This process is triggered by various inflammasome complexes, including NLRP3, NLRC4, NLRP1, and AIM2, which are essential components of the innate immune system. Upon activation, these inflammasomes stimulate the release of pro-inflammatory cytokines such as IL-1β and IL-18 [17], [58], [59]. Several studies have provided evidence of NLRP3 inflammasome activation in rheumatoid arthritis (RA) and other autoimmune diseases [60], [61]. The specific interplay between NLRP3/caspase-1 and other cytokines leads to inflammatory cell death [62], triggering the release of significant quantities of IL-1β and IL-18. This process drives both systemic and localized osteolysis in arthritis [63], [64].
Our findings indicate a significant notable increase in the expression of NLRP3 and caspase-1 in the synovium among CIA rats. However, EA treatment was able to mitigate these changes. These results suggest that the inhibitory effect of EA on the inflammatory response is multifaceted, targeting multiple components involved in the inflammatory cascade.
A variety of transcription factors and signaling mediators play a crucial role in regulating the polarization state of macrophages, determining whether inflammation persists or terminated [63]. Among these, the GSDME-dependent pyroptosis signaling pathways is considered classical and pivotal factors [65]. The degradation of GSDMD promotes M1 polarization and pyroptosis [66], leading to the release of a significant number of mature inflammatory cytokines. In this study, it was observed that EA treatment effectively blocked GSDME-dependent pyroptosis signaling pathways, thereby suppressing M1 polarization and inflammation associated with JIA.