Lan Yuye, Jing Xianghong, Zhou Ziyu, Rao Yiqing, Wang Kaichen, Qin Renjie, Wu Yisong, Sun Jingjing, Zhang Ke, Liu Xinyue, Wang Zixiao, Xu Jiahao, Zhao Minzhen, Yuan Xiao Cui, Liu Yongmin, Zhang Hong, Hu Xuefei, Pan Huilin, Hou Tengfei, Li Man
Department of Neurobiology, School of Basic Medicine, Tongji Medical College, Key Laboratory of Neurological Diseases of Hubei Province and National Education Ministry, Huazhong University of Science and Technology, Wuhan, 430030, China.
Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
Chin Med. 2024 Dec 24;19(1):176. doi: 10.1186/s13020-024-01048-z.
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.
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.
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.
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.
慢性炎性疼痛是一种普遍存在的病症，电针（EA）是一种有效的治疗方法，但其机制尚未完全明确。AMP激活的蛋白激酶（AMPK）作为关键的能量传感器，参与疼痛缓解及电针效应。电针可能通过增加内源性大麻素、上调CB2受体（CB2R）以及刺激β-内啡肽（β-END）发挥作用。本研究旨在验证电针是否通过CB2R激活AMPK来调节β-END并减轻疼痛。
采用完全弗氏佐剂（CFA）建立炎性疼痛模型，连续6天每日进行电针治疗，针刺穴位为“足三里”（ST36）和“上巨虚”（ST37）。使用von Frey细丝评估机械阈值、用热板评估热阈值来评价疼痛敏感性。采用超高效液相色谱串联质谱法（UPLC-MS/MS）定量测定内源性大麻素2-花生四烯酸甘油酯（2-AG）和花生四烯乙醇胺（AEA）的水平。通过蛋白质免疫印迹法检测CB2R和β-END的表达水平以及AMPK的激活情况。应用免疫荧光双标记法观察CD68阳性巨噬细胞内AMPK激活及β-END表达情况。本研究纳入野生型和CB2R基因敲除小鼠，以阐明CB2R在电针诱导AMPK激活中的作用。
CFA诱导的炎性疼痛模型小鼠表现出机械性异常疼痛和热痛觉过敏。电针在对炎性疼痛发挥镇痛作用时，激活了炎症皮肤组织中的AMPK。预先给予AMPK抑制剂化合物C可显著抑制电针对疼痛缓解的作用。电针提高了炎症皮肤组织中β-END的表达，而化合物C可使其逆转，表明AMPK在电针诱导β-END表达中具有调节作用。此外，与CFA组相比，电针显著上调了炎症皮肤组织中2-AG、AEA的水平以及CB2Rs的表达。在野生型小鼠中，电针激活巨噬细胞中的AMPK，而CB2基因敲除则降低了电针激活这些细胞中AMPK的能力。
电针通过CB2R激活AMPK，增强炎症皮肤中β-END的表达以减轻炎性疼痛。本研究揭示了内源性大麻素、内啡肽和AMPK在电针镇痛作用中的新联系，突出了CB2R-AMPK-β-END途径。
