Electroacupuncture ameliorates inflammatory pain through CB2 receptor-dependent activation of the AMPK signaling pathway

Abstract

Background Chronic inflammatory pain is a pervasive condition, and electroacupuncture (EA) is an effective treatment, but its mechanisms are not fully understood. AMP-activated protein kinase (AMPK), a key energy sensor, is involved in pain relief and EA’s effects. EA may work by increasing endocannabinoids, upregulating CB2 receptors (CB2R), and stimulating β-endorphin (β-END). This study tests if EA activates AMPK via CB2R to modulate β-END and reduce pain. Methods The inflammatory pain model was established with Complete Freund’s adjuvant (CFA), and EA was administered daily for six consecutive days, targeting the acupoints “Zusanli” (ST36) and “Shangjuxu” (ST37). Pain sensitivity was evaluated using Von Frey filaments for mechanical thresholds and a hot plate for thermal thresholds. Ultra-high Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS) was used to quantitatively determine the levels of endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA). The expression levels of the CB2R and β-END were measured by Western blotting, along with the activation of AMPK. Immunofluorescence double-labeling was applied to visualize AMPK activation and β-END expression within CD68-positive macrophages. The study encompassed both wild-type and CB2R gene knockout mice, elucidating the role of CB2R in EA-induced AMPK activation. Results CFA-induced inflammatory pain model mice exhibited mechanical allodynia and thermal hyperalgesia. EA activated AMPK in the inflamed skin tissue when it exerted analgesic effect on the inflammatory pain. Pre-administration of the AMPK inhibitor Compound C significantly inhibited the effect of EA on pain relief. EA elevated β-END expression in inflamed skin tissue, which was reversed by Compound C, indicating that AMPK has a regulatory role in EA inducing β-END expression. In addition, EA significantly upregulated the levels of 2-AG, AEA and the expression of CB2Rs in the inflamed skin tissue compared with the CFA group. In wild-type mice, EA activates AMPK in macrophages, while CB2 knockout reduced EA's ability to activate AMPK in these cells. Conclusion EA activates AMPK through CB2R, enhancing β-END expression in inflamed skin to alleviate inflammatory pain. This study reveals a new link between endocannabinoids, endorphins, and AMPK in analgesic effects of EA, highlighting the CB2R-AMPK-β-END pathway.