电针“内关”对炎性痛小鼠脑内食欲素1受体的影响

Effect of electroacupuncture at “Neiguan”(PC6) on pain and brain orexin 1 receptor in mice with inflammatory pain

目的:观察电针“内关”对足掌注射完全弗氏佐剂(CFA)小鼠疼痛反应的影响,探讨其脑内食欲素1受体(OX1R)-内源性大麻素1受体(CB1R)通路机制。方法:SPF级成年雄性C57BL/6小鼠按两部分实验随机分组,第一部分实验分为对照组、模型组、电针组,第二部分实验分为对照组、模型组、电针组、电针+纳洛酮(Naloxone)组和电针+OX1R拮抗剂(SB33486)组。于小鼠左后肢足掌皮下注射20μL CFA建立炎性痛模型。电针组、电针+Naloxone组和电针+SB33486组给予电针双侧“内关”,疏波,频率2 Hz,强度2 mA,20 min/次,1次/d,连续5 d;电针+Naloxone组和电针+SB33486组在电针干预的基础上于第5天分别给予腹腔注射Naloxone和SB33486。采用Tail-flick法和Von Frey法检测小鼠热痛阈值和机械痛阈值,实时荧光定量PCR法检测小鼠中脑导水管周围灰质(PAG)中β-内啡肽mRNA表达水平;免疫荧光法检测小鼠下丘脑外侧区(LH)中OX1R及PAG腹外侧区(vlPAG)中CB1R阳性细胞表达数量。结果:与对照组比较,模型组小鼠热痛阈值、机械痛阈值均降低(P<0.001),PAG中β-内啡肽mRNA表达水平降低(P<0.001),LH内的OX1R及vlPAG内的CB1R阳性细胞数量减少(P<0.05,P<0.001)。与模型组比较,电针组热痛阈、机械痛阈值明显升高(P<0.001),LH内的OX1R和vlPAG内的CB1R阳性细胞数量增多(P<0.01,P<0.001)。与电针组比较,电针+Naloxone组机械痛阈值差异无统计学意义,电针+SB33486组机械痛阈值则降低(P<0.01),且电针+SB33486组小鼠LH内的OX1R和vlPAG内的CB1R阳性细胞数量减少(P<0.001)。结论:电针“内关”可通过激活脑内OX1R-CB1R通路实现对CFA小鼠的镇痛效应,且该效应不依赖于阿片类镇痛物质。

Objective To observe the effect of electroacupuncture(EA) at “Neiguan”(PC6) on pain response in mice injected with complete Freund's adjuvant(CFA) in the hind paw,so as to investigate the mechanism of orexin 1 receptor(OX1R)-endogenous cannabinoid 1 receptor(CB1R) pathway in acupuncture analgesia.Methods A total of 48 male C57BL/6 mice were used in the present study.In the first part of this study,18 mice were randomized into control,model and EA groups,with 6 mice in each group.In the second part of this study,30 mice were randomized into control,model,EA,EA+Naloxone,EA+OX1R antagonist(SB33486) groups,with 6 mice in each group.Inflammatory pain model was established by subcutaneous injection of 20 μL CFA solution in the left hind paw.EA(2 Hz,2 mA) was applied to bilateral PC6 for 20 min,once a day for 5 consecutive days.The mice in the EA+Naloxone and EA+SB33486 groups were intraperitoneally injected with naloxone(10 mg/kg) or SB33486(15 mg/kg) 15 min before EA intervention on day 5,respectively.Tail-flick method and Von Frey method were used to detect the thermal pain threshold and mechanical pain threshold of mice.Quantitative real-time PCR was used to detect the expression level of β-endorphin mRNA in periaqueductal gray(PAG) of mice.The expression of OX1R positive cells in the lateral hypothalamic area(LH) and CB1R positive cells in the ventrolateral periaqueductal gray(vlPAG) were detected by immunofluorescence.Results Compared with the control group,the thermal pain threshold and mechanical pain threshold of the model group were decreased(P<0.001),the expression level of β-endorphin mRNA in PAG was decreased(P<0.001),and the numbers of OX1R positive cells in LH and CB1R positive cells in vlPAG were decreased(P<0.05,P<0.001).Compared with the model group,the thermal pain threshold and mechanical pain threshold of the EA group were significantly increased(P<0.001),and the numbers of OX1R positive cells in LH and CB1R positive cells in vlPAG were increased(P<0.01,P<0.001).Compared with the EA group,the mechanical pain threshold in the EA+SB33486 group was significantly decreased(P<0.01),but there was no significant difference in the mechanical pain threshold between the EA+Naloxone group and EA group,and the numbers of OX1R positive neurons in LH and CB1R positive neurons in vlPAG were decreased in the EA+SB33486 group(P<0.001).Conclusion EA at PC6 can achieve analgesic effect on CFA mice by activating the OX1R-CB1R pathway in the brain,and this effect is opioid-independent.