Objective: To observe the effect of "Shugan Tiaoshen"(liver-soothing and mind-regulating) acupuncture on behavior reactions, opioid receptor expressions in the anterior cingulate cortex tissue and inflammatory factors in the serum in migraine rats, in order to explore its mechanism underlying improvement of migraine.
Methods: In the first part of this study, forty male Wistar rats were randomized into control, model, routine acupuncture and "Shugan Tiaoshen" acupuncture groups (n=10/group), and in the second part, other 40 more male Wistar rats were randomized into low, medium and high dosage of blocker of μopioid receptor (OPRM）CTOP5 and PBS groups (n=10/group, for validating the involvement of opioid receptor in the effect of "Shugan Tiaoshen"). The migraine model was established by subcutaneous injection of glyceryl trinitrate. Routine acupuncture was applied to "Baihui" (GV20) and bilateral"Fengchi" (GB20), and "Shugan Tiaoshen" acupuncture applied to GV20, and bila-teral GB20, "Neiguan" (PC6) and "Taichong" (LR3), with the needles retained for 30 min. Behavior responses (head scratching, tail biting, cage climbing and number of going there and back) were scaled. Serum IL-1β, IL-6 and TNF-α were detected by ELISA, and the expression levels of opioid receptor μ, δ and κ (OPRM, OPRD, OPRK) mRNAs and proteins in the anterior cingulate cortex were detected by fluorescence quantitative PCR and Western blot separately. In the second part of this study, CTOP solution (5μL at concentrations of 20μg/μL,10μg/μL and 5μg/μL) or PBS was injected into the bilateral rostral portions of anterior cingulate cortex 30 min before every "Shugan Tiaoshen" acupuncture intervention, followed by observing the behavioral changes and assaying the contents of serum IL-1β, IL-6 and TNF-α.
Results: After modeling, the behavioral score, serum IL-1β, IL-6 and TNF-α contents were significantly increased in the model group relevant to the control group (P<0.05), and the beha-vioral score had no significant difference among the model and two acupuncture groups before intervention (P>0.05). Whereas the expression levels of OPRM, OPRD and OPRK mRNAs and proteins had a slight increase in the model group (P>0.05). After the intervention, the behavioral score, serum IL-1β, IL-6 and TNF-α contents were significantly decreased and the expression levels of OPRM, OPRD and OPRK mRNAs (2.150, 1.066 and 0.805 folds in the "Shugan Tiaoshen" group) and proteins (2.273, 0.901 and 0.893 folds in the "Shugan Tiaoshen" group) notably up-regulated in both "Shugan Tiaoshen" and routine acupuncture groups relevant to the model group (P<0.01, P<0.05), showing that the biggest up-regulation of mRNA expression was OPRM. Comparison between two acupuncture groups showed that the behavioral score, and serum IL-1β, IL-6 and TNF-α contents were significantly lower, and the expression levels of OPRM, OPRD and OPRK mRNAs and proteins obviously higher in the "Shugan Tiaoshen" group than those in the routine acupuncture group (P<0.01,P<0.05). Results of the second part of this study showed that after injection of antagonist CTOP of OPRM, the therapeutic effect of "Shugan Tiaoshen" acupuncture was weakened in the reduction of behavioral score and serum IL-1β, IL-6 and TNF-α contents, being minimal, moderate and maximum in the high, medium and low dose of antagonist relevant to PBS in sequence (P<0.05, P<0.01).
Conclusion: "Shugan Tiaoshen" acupuncture can mitigate pain in migraine rats, which may be associated with its function in up-regulating the expressions of opioid receptors (especially OPRM), and in inhibiting inflammatory reaction in the anterior cingulate cortex.
目的：观察疏肝调神针法对偏头痛大鼠行为学、血清炎性因子及前扣带皮层阿片受体表达水平的影响，探讨其介导阿片受体参与偏头痛治疗的机制。方法：Wistar大鼠按随机数字表法分为对照组10只、造模组30只。于大鼠颈后平坦区域皮下注射硝酸甘油制备偏头痛模型，造模成功的30只大鼠随机分为模型组、常规针刺组、疏肝调神组各10只。常规针刺组取“百会”、双侧“风池”，疏肝调神组取“百会”、双侧“风池”“内关”“太冲”，两组均留针30 min。干预前后评估大鼠行为学评分，干预后采用实时荧光定量PCR法、Western blot法检测大鼠脑组织前扣带皮层μ、δ和κ阿片受体(OPRM、OPRD、OPRK)mRNA及蛋白表达，ELISA法检测血清炎性因子白细胞介素(IL)-1β、IL-6、肿瘤坏死因子-α（TNF-α）含量。取另外40只造模成功的大鼠按随机数字表法分为抑制剂高、中、低剂量组及PBS组，分别于干预前颅内注射5 μg高、中、低浓度的疏肝调神组治疗后表达变化最明显的OPRM抑制剂，PBS组注射等量PBS溶液，之后各组均行疏肝调神针法干预；干预前后评估大鼠行为学评分，干预后ELISA法检测血清IL-1β、IL-6、TNF-α含量。结果：干预前其余3组大鼠行为学评分均高于对照组(P<0.01)，干预后常规针刺组及疏肝调神组行为学评分均低于模型组(P<0.01)，疏肝调神组低于常规针刺组(P<0.01)。模型组大鼠血清IL-1β、IL-6、TNF-α含量高于对照组(P<0.01)，常规针刺组、疏肝调神组均低于模型组(P<0.05)，疏肝调神组低于常规针刺组(P<0.05)；模型组大鼠前扣带皮层中OPRM、OPRD、OPRK mRNA和蛋白表达水平均略高于对照组，但差异无统计学意义(P＞0.05)，常规针刺组及疏肝调神组均高于模型组(P<0.01，P<0.05)，疏肝调神组高于常规针刺组(P<0.01，P<0.05)。与模型组比较，干预后疏肝调神组阿片受体中以OPRM上调倍数最大，由此回复实验选择OPRM抑制剂进行注射。干预后，高、中、低剂量组及PBS组行为学评分均低于同组干预前(P<0.01，P<0.05)，且3个抑制剂组均高于PBS组（P<0.01，P<0.05）；高、中、低剂量组血清IL-1β、IL-6、TNF-α含量均高于PBS组(P<0.01，P<0.05)。结论：疏肝调神针法通过上调阿片受体，尤其OPRM的表达，抑制炎性反应，从而发挥对偏头痛的治疗作用。.
Keywords: Interleukin-6; Migraine; Opioid receptors;Interleukin-1β; Tumor necrosis factor-α; “Shugan Tiaoshen” (liver-soothing and mind-regulating) acupuncture.