Liu Jie, Wang Meng-Meng, Yang Dian-Hui, Cui Xin-Xin
Department of Traditional Chinese Medicine, The Second Hospital of Shandong University, Jinan 250033, China.
College of Acupuncture-moxibustion and Tuina, Shandong University of Traditional Chinese Medicine, Jinan 250355.
Zhen Ci Yan Jiu. 2022 Jun 25;47(6):510-6. doi: 10.13702/j.1000-0607.20210691.
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.
In the first part of this study, forty male Wistar rats were randomized into control, model, routine acupuncture and "Shugan Tiaoshen" acupuncture groups (=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 (=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.
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 (<0.05), and the beha-vioral score had no significant difference among the model and two acupuncture groups before intervention (>0.05). Whereas the expression levels of OPRM, OPRD and OPRK mRNAs and proteins had a slight increase in the model group (>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 (<0.01, <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 (<0.01,<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 (<0.05, <0.01).
"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.
观察“疏肝调神”针刺法对偏头痛大鼠行为反应、前扣带回皮质组织中阿片受体表达及血清中炎症因子的影响，以探讨其改善偏头痛的作用机制。
本研究第一部分将40只雄性Wistar大鼠随机分为对照组、模型组、常规针刺组和“疏肝调神”针刺组（每组10只），第二部分将另外40只雄性Wistar大鼠随机分为低、中、高剂量μ阿片受体（OPRM）阻断剂CTOP组和PBS组（每组10只，用于验证阿片受体在“疏肝调神”效应中的作用）。采用皮下注射硝酸甘油建立偏头痛模型。常规针刺取“百会”（GV20）及双侧“风池”（GB20），“疏肝调神”针刺取GV20、双侧GB20、“内关”（PC6）和“太冲”（LR3），留针30分钟。对行为反应（抓头、咬尾、爬笼及往返次数）进行评分。采用ELISA法检测血清IL-1β、IL-6和TNF-α，分别采用荧光定量PCR和Western blot法检测前扣带回皮质中阿片受体μ、δ和κ（OPRM、OPRD、OPRK）mRNA和蛋白的表达水平。在本研究的第二部分，在每次“疏肝调神”针刺干预前30分钟，将CTOP溶液（浓度为20μg/μL、10μg/μL和5μg/μL，5μL）或PBS注入双侧前扣带回皮质喙部，随后观察行为变化并检测血清IL-1β、IL-6和TNF-α的含量。
造模后，模型组的行为评分、血清IL-1β、IL-6和TNF-α含量与对照组相比显著升高（<0.05），干预前模型组与两个针刺组的行为评分无显著差异（>0.05）。而模型组OPRM、OPRD和OPRK mRNA和蛋白的表达水平略有升高（>0.05）。干预后，“疏肝调神”针刺组和常规针刺组的行为评分、血清IL-1β、IL-6和TNF-α含量均显著降低，OPRM（“疏肝调神”组为2.150倍）以及OPRD和OPRK mRNA（“疏肝调神”组为1.066倍和0.805倍）和蛋白（“疏肝调神”组为2.273倍、0.901倍和0.893倍）的表达水平与模型组相比显著上调（<0.01，<0.05），表明mRNA表达上调幅度最大的是OPRM。两个针刺组比较，“疏肝调神”组的行为评分、血清IL-1β、IL-6和TNF-α含量显著更低，OPRM、OPRD和OPRK mRNA和蛋白的表达水平明显更高（<0.01，<0.05）。本研究第二部分结果显示，注射OPRM拮抗剂CTOP后，“疏肝调神”针刺在降低行为评分及血清IL-1β、IL-6和TNF-α含量方面的治疗效果减弱，与PBS相比，高、中、低剂量拮抗剂组的减弱程度依次为最小、中等和最大（<0.05，<0.01）。
“疏肝调神”针刺可减轻偏头痛大鼠的疼痛，这可能与其上调阿片受体（尤其是OPRM）表达及抑制前扣带回皮质炎症反应的作用有关。
