【作者】 王星杰； 崔陶陶； 梁晓宇； 朱富群； 杨志新；

【Author】 Wang Xingjie;Cui Taoao;Liang Xiaoyu;Zhu Fuqun;Yang Zhixin;ChengDe Medical College,Hebei Key Laboratory of Nerve Injury and Repair;HeiLongjiang University of Chinese Medicine;

【机构】 承德医学院,河北省神经损伤与修复重点实验室； 黑龙江中医药大学；

【摘要】 目的观察针刺内关、外关对失眠大鼠中枢海马及穴区组织内5-HT、5-HIAA、5-HT_1AR及5-HT₄R含量的影响,探究基于相对穴理论针刺内关、外关治疗失眠症的作用机制。方法使用随机数字表从80只Wistar大鼠中分出12只作为空白组,其余大鼠进行失眠模型复制,造模2d后于造模成功的大鼠中选取60只,重新随机分为6组（每组12只）:模型组、单穴内关组、单穴外关组、透刺组、对刺组,空白组,采用腹腔注射对氯苯丙氨酸（PCPA）混悬液制作大鼠失眠模型,各针刺组针刺相应腧穴,针刺得气后针柄接华佗牌IV型电针治疗仪,刺激参数为频率2Hz/100Hz,电流强度1mA,以针柄轻微颤动且大鼠能保持安静为宜,每次电针20min。每日1次,连续6d。使用ELISA法检测中枢海马及穴区组织上清液5-HT及5-HIAA含量,荧光原位杂交检测中枢海马及穴区组织5-HT₄m RNA的表达,免疫组化检测中枢海马与穴区组织内5-HT_1AR的表达。结果与空白组相比较,模型组中枢海马及穴区内5-HT及5-HIAA含量明显减少,针刺"相对穴"内关穴与外关穴可使中枢海马内5-HT及5-HIAA表达水平升高,并且针刺"相对穴"对刺组与透刺组较单穴内关组和单穴外关组改善程度显著,针刺后,外周穴区组织5-HT及5-HIAA含量升高,并且针刺"相对穴"对刺组与透刺组较单穴内关组与单穴外关组改善程度明显（P<0.05）,且对刺组与透刺组无明显差异,单穴内关组与单穴外关组亦无明显差异（P>0.05）。免疫组化结果显示失眠大鼠模型中枢海马及穴区组织中5-HT_1AR表达减少,通过针刺干预后,可以提高失眠大鼠模型中枢海马内5-HT_1AR的表达,且针刺"相对穴"对刺组与透刺组提高失眠大鼠海马内5-HT_1AR的表达较单穴内关组与单穴外关组更加显著（P<0.05）。通过荧光原位杂交结果显示,失眠大鼠中枢海马及穴区组织内5-HT₄m RNA表达减少,针刺干预后失眠大鼠中枢海马及穴区组织内5-HT₄m RNA表达水平均有提高,且针刺"相对穴"对刺组与透刺组较单穴内关组和单穴外关组表达更为显著（P<0.05）,对刺组与透刺组两组间无明显差异;单穴内关组与单穴外关组两组间无明显差异（P>0.05）。结论针刺相对穴"内关""外关"可增加失眠大鼠中枢海马及穴区组织内5-HT及代谢产物5-HIAA的含量,使失眠大鼠中枢海马及穴区组织内5-HT_1AR、5-HT₄mRNA的表达水平提高,从而治疗失眠。更多还原

【Abstract】 Objective To observe the effect of acupuncture at Neiguan and Waiguan on the content of 5-HT,5-HIAA,5-HT_1AR and 5-HT₄R in the central hippocampus and acupoint area of insomnia rats,and to explore the mechanism of acupuncture at Neiguan and Waiguan based on relative acupoint theory in the treatment of insomnia.Methods Twelve of 80 Wistar rats were selected as the blank group by random number table,and the remaining rats were used to replicate the insomnia model.After 2 days of modeling,60 rats were selected and randomly divided into 6 groups（12 rats in each group）:model group,single acupoint Neiguan group,single acupoint Waiguan group,penetrating acupuncture group,opposing acupuncture group and blank group.The insomnia model of rats was made by intraperitoneal injection of p-chlorophenylalanine（PCPA） suspension.Each acupuncture group was acupunctured at the corresponding acupoints for 20 min each time for 6 days.ELISA was used to detect the content of 5-HT and 5-HIAA in the supernatant of hippocampus and acupoint tissue.Fluorescence in situ hybridization was used to detect the expression of 5-HT₄R in hippocampus and acupoint tissue.Immunohistochemistry was used to detect the expression of 5-HT_1AR in hippocampus and acupoint tissue.Results Compared with the blank group,the contents of 5-HT and 5-HIAA in the central hippocampus and acupoint area of the model group were significantly reduced.Acupuncture at Neiguan and Waiguan could increase the expression levels of 5-HT and 5-HIAA in the central hippocampus,and the acupuncture group and the penetration acupuncture group were significantly improved compared with the single acupoint Neiguan group and the single acupoint Waiguan group.After acupuncture,the contents of 5-HT and 5-HIAA in the peripheral acupoint area increased,and the acupuncture group and the penetration acupuncture group were significantly improved compared with the single acupoint Neiguan group and the single acupoint Waiguan group（P<0.05）.The results of immunohistochemistry showed that the expression of 5-HT_1AR in the hippocampus and acupoint area of the insomnia rat model decreased.After acupuncture intervention,the expression of 5-HT_1AR in the hippocampus of the insomnia rat model could be improved,and the acupuncture ’ relative acupoint ’ was improved.The expression of 5-HT_1AR in the hippocampus of insomnia rats in the acupuncture group and the penetration acupuncture group was more significant than that in the single-point Neiguan group and the single-point Waiguan group（P<0.05）.The results of fluorescence in situ hybridization showed that the expression of 5-HT₄R in the central hippocampus and acupoint area of insomnia rats decreased,and the expression level of 5-HT₄R in the central hippocampus and acupoint area of insomnia rats increased after acupuncture intervention.The expression of 5-HT₄R in the acupuncture group and the penetration group was more significant than that in the single-point Neiguan group and the single-point Waiguan group（P<0.05）,and there was no significant difference between the two groups.There was no significant difference between the single acupoint Neiguan group and the single acupoint Waiguan group（P>0.05）.Conclusion Acupuncture relative acupoints ’ Neiguan ’ and ’ Waiguan ’ can increase the content of 5-HT and metabolite 5-HIAA in the central hippocampus and acupoint area of insomnia rats,and increase the expression level of 5-HT_1AR and 5-HT₄R in the central hippocampus and acupoint area of insomnia rats,so as to treat insomnia.更多还原