Arthritis is an inflammatory disease that is primarily caused by trauma, infection, and inflammation. To date, arthritis cannot be completely cured, and patients with arthritis experience long-term pain and often require long-term medications [1], [2]. To the best of our knowledge, pain and inflammation are the primary symptoms of arthritis; however, their specific pathogenesis remains unclear [3], [4], [5].

Manual acupuncture (MA) is a type of therapy in traditional Chinese medicine. In MA, the skin or tissues is stimulated via twisting and lifting motions. This method exerts therapeutic effects in several pain-associated diseases, including rheumatoid arthritis, acute migraines, acute renal colic, osteoarthritis, and inflammatory pain [6], [7], [8], [9], [10], [11]. Recently, acupuncture was discovered to effectively reduce pain by regulating opioid, cytokine, and adenosine secretion in patients with arthritis [12]. Moreover, various inflammatory and pain diseases can be treated at the ST36 acupoint based on its unique qualities, particularly its analgesic and anti-inflammatory effects [13]. Therefore, ST36 is frequently used as an analgesic acupoint in acupuncture [14]. As expected, acupuncture at ST36 alleviated inflammation and pain in rats with colitis [15]. In addition, acupuncture exerts analgesic effects by increasing Cx43 expression at the ST36 acupoint in the skin [16]. Meanwhile, another study has revealed that acupuncture decreased joint pain in complete Freund’s adjuvant (CFA)-induced arthritis rats by inhibiting the expression of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 [17]. These studies indicate that MA exerts analgesic effects in CFA-induced arthritis rats.

The transcription factor nuclear factor B (NF-κB) plays a vital role in mediating inflammatory processes, both in innate and adaptive immune responses. Furthermore, it can regulate CFA-induced arthritis under MA [18], [19], [20]. MA inhibited the NF-κB signaling pathway in rats with vascular dementia and ameliorated inflammation-associated cognitive impairment [21]. Electroacupuncture could downregulate the expression of IL-1β, TNF-α, TRPV1, and NF-κB receptors in colonic tissues by acting on the ST36 acupoint site of rats [22]. Collectively, these data suggest that acupuncture confers analgesic and anti-inflammatory benefits in CFA-induced arthritis rats by inhibiting the NF-κB signaling pathway.

Adenosine is an endogenous mediator that primarily exerts its physiological functions by activating four adenosine receptors: A1, A2A, A2B, and A3 adenosine receptors (A3R) [23]. Interestingly, studies have indicated that MA can increase local adenosine levels at the ST36 acupoint [24]. Recently, a study revealed the potential of A3R as a target in cancer, inflammatory diseases, and pain [25]. However, the mechanism by which A3R participates in acupuncture analgesia remains unclear. As mentioned above, MA at the ST36 acupoint increases adenosine levels in the acupoint area and significantly decreases pain and inflammation in rats with CFA-induced arthritis [24], [26]. Meanwhile, another study has revealed that 2-Cl-IB-MECA (an A3R agonist) or MRS-3777 (an A3R antagonist) increases or decreases the paw withdrawal threshold (PWT) of rats [27]. Therefore, whether the analgesic effects of MA are associated with A3R expression in the acupoint area is worth discussing.

In this study, an arthritis rat model was developed by injecting CFA into the left ankle joints. Thereafter, the rats were subjected to MA (ST36 acupoint) for 3 days. Furthermore, the role of MA in regulating pain, inflammation, and A3R was explored. In addition, A3R agonist (IB-MECA) and A3R antagonist (Reversine) were injected into ST36 before MA to explore the effect of A3R in inflammation under MA treatment in rats with arthritis.