电针激活CB2受体增强细胞自噬功能缓解炎性痛的机制

Mechanism of Electroacupuncture Inhibiting Inflammatory Pain by Activating CB2 Receptor to Regulate Autophagy

目的观察电针对野生型小鼠和内源性大麻素2型受体(Canabinoid receptor 2,CB2受体)基因敲除小鼠完全弗氏佐剂(Complete Freund’s adjuvant,CFA)诱导的炎性痛的缓解作用和炎症皮肤组织中细胞自噬功能的影响;激活CB2受体模拟电针作用,观察其对NR8383巨噬细胞自噬功能以及线粒体受损情况的影响,探讨电针能否通过激活CB2受体增强炎症皮肤组织自噬功能并清除受损线粒体从而缓解炎性痛。方法采用热板测痛法测定小鼠的热痛阈;采用von Frey丝,以“up and down”方法测定小鼠机械痛阈;采用Western blot检测野生型和CB2受体基因敲除小鼠足背皮肤组织中自噬相关蛋白p62和微管相关蛋白轻链3(light chain 3,LC3)的表达及NR8383巨噬细胞中p62和LC3的表达;采用流式细胞术检测受损线粒体的数量。结果①CFA诱导炎性痛模型的野生型小鼠和CB2受体基因敲除小鼠的热痛阈值和机械痛觉阈值均较溶媒对照组显著下降(P<0.01);电针可显著提高野生型炎性痛模型小鼠的热痛阈值和机械痛阈值(P<0.05),而对CB2受体基因敲除炎性痛模型小鼠无显著影响(P>0.05)。②电针可翻转炎性痛模型的野生型小鼠炎症皮肤组织中自噬相关蛋白p62表达增加和LC3-Ⅱ/Ⅰ比值降低,从而促进炎症皮肤组织中细胞自噬功能,而对CB2受体基因敲除炎性痛模型小鼠无影响;③采用CB2受体激动剂AM1241激活NR8383巨噬细胞中CB2受体可减少p62蛋白表达,升高LC3-Ⅱ/Ⅰ比值,从而改善CFA引起的自噬功能减弱,该作用可被CB2受体拮抗剂AM630所翻转;④采用CB2受体激动剂AM1241激活NR8383巨噬细胞中CB2受体可减少受损线粒体的数量,该作用可被CB2受体拮抗剂AM630所翻转,提示CB2受体的激活能促进巨噬细胞的自噬作用,从而清除受损的线粒体。结论电针通过激活CB2受体,增强炎症皮肤组织细胞自噬功能,清除受损的线粒体,从而缓解炎性痛。

Objective In this study,we attempted to investigate whether Electroacupuncture(EA)could promote the autophagy function in macrophages of inflammatory skin tissues by activating CB2 receptor,thus relieving inflammatory pain induced by CFA in mice,and whether activation of CB2 receptor in NR8383 macrophages cell line can simulate the effect of EA on the autophagy function and mitochondrial damage.Methods Inflammatory pain model was induced by CFA injection into the planta the hind paw of wildtype and CB2 knockout mice.EA or sham EA was applied on the left Huantiao(GB30)and Yanglingquan(GB34)sites.Thermal hyperalgesia was determined with the Hargreaves test.Mechanical sensitivity was assessed with von Frey filaments.NR8383 microphage cell line was used to study the effect of CB2 activation on macrophage function induced by CFA.The expression level of autophagy protein LC3 and p62 in wildtype and CB2 knockout mice skin tissue and NR8383 cell line were determined by Western blot.And flow cytometry analysis was applied to detect damaged mitochondria and mitochondrial superoxide.Results CFA significantly reduced the thermal and mechanical pain threshold in both wildtype and CB2 knockout mice,comparing with the vehicle control groups(P<0.01).EA significantly inhibited thermal and mechanical hyperpathia induced by CFA in wildtype mice(P<0.05),but had no effect on CB2 knockout mice with CFA(P>0.05).CFA significantly increased the expression of p62 protein and decreased LC3-II/I ratio,which was inversed by EA in wildtype mice but wasn't affected by EA in CB2 knockout mice.CFA increased the expression of p62 protein and decreased LC3-II/I ratio in NR8383 cell line,which were inversed by CB2 agonist AM1241.CFA increased mitochondria damage,which were then attenuated by CB2 agonist AM1241.Conclusion The analgesic effect of EA on inflammatory pain induced by CFA was mediated by activation of CB2 receptor,which promoted the autophagy function and the clearance of damaged mitochondria in macrophage.