Electroacupuncture alleviates inflammatory pain through AMPK-autophagy signaling and suppression of iNOS and IL-1β in inflamed tissues.

Abstract

Electroacupuncture (EA) effectively alleviates inflammatory pain, yet its underlying mechanisms remain incompletely understood. Emerging evidence implicates AMP-activated protein kinase (AMPK) and autophagy in modulating inflammation, but whether they mediate EA's analgesic action is unknown. This study tests the hypothesis that EA relieves inflammatory pain by activating AMPK in macrophages, thereby triggering autophagy and suppressing pro-inflammatory mediators (iNOS and IL-1β) in inflamed tissues. In a mouse model of complete Freund's adjuvant (CFA)-induced inflammatory pain, we assessed pain behavior and inflammatory markers following EA. Pharmacological modulators- AMPK antagonist Compound C, AMPK activator AICAR, and autophagy inhibitor 3-methyladenine (3-MA) - were applied to dissect the AMPK- autophagy-cytokine cascade. Complementary in vitro experiments using NR8383 macrophages with a autophagy reporter (stubRFP-sensGFP-LC3 Lentivirus) further validated the pathway. EA not only reduced CFA-induced hyperalgesia but also concurrently augmented AMPK phosphorylation, enhanced autophagic flux, and diminished iNOS and IL-1β levels in injured skin. Mirroring these effects, AICAR alone produced analgesia, increased autophagy, and lowered cytokine expression. Conversely, Compound C blocked EA's actions on autophagy and inflammatory markers, while 3-MA abrogated EA's analgesic and anti-inflammatory effects. Macrophage experiments corroborated the in vivo results, confirming that AMPK activation drives autophagy and subsequent suppression of iNOS/IL-1β. Our findings establish a mechanistic axis whereby EA engages AMPK in macrophages to upregulate autophagy, which in turn curtails iNOS and IL-1β production, ultimately mitigating inflammatory pain. This work unveils a novel cellular pathway for EA's anti-nociceptive effect and highlights AMPK-autophagy as a potential therapeutic target in pain management.