针刺对肝郁型失眠大鼠行为学及海马5-HT_(1A)R和5-HT_(2A)R表达的影响

Effects of acupuncture on behaviors and expression levels of 5-HT_(1A)R and 5-HT_(2A)R in the hippocampus of insomnia rats due to liver Qi stagnation

目的:观察“疏肝调神”针法对肝郁型失眠大鼠行为学及海马5-羟色胺1A受体(5-HT1AR)和5-羟色胺2A受体(5-HT2AR)表达的影响。方法:从70只Wistar大鼠中随机选取10只作为空白组,其余采用慢性夹尾刺激结合腹腔注射对氯苯丙氨酸复制肝郁型失眠大鼠模型。选取造模成功大鼠50只随机分为模型组、抓取组、西药组、针刺组和假针刺组,每组10只。抓取组采用和针刺组相同的方式抓取;西药组予以艾司唑仑溶液灌胃;针刺组采用“疏肝调神”针法针刺百会、内关、神门和太冲;假针刺组针刺四个非经非穴点。所有组别每日干预1次,连续7d。采用戊巴比妥睡眠实验评价治疗7 d后各组大鼠睡眠潜伏期(SL)、睡眠持续时间(ST);高架十字迷宫实验(EPMT)监测各组大鼠开臂进入次数比例(OE%)和开臂停留时间比例(OT%);旷场实验(OFT)监测大鼠直立次数、水平跨格次数、粪便粒数;蛋白免疫印迹法检测大鼠海马区5-HT1AR和5-HT2AR的表达。结果:与空白组相比,模型组大鼠体质量增长幅度、ST、OE%、OT%、直立次数、水平跨格次数及海马5-HT1AR表达明显降低(P<0.01),SL及海马5-HT2AR表达明显升高(P<0.01)。治疗后,与抓取组相比,针刺组和西药组大鼠体质量、ST、OT%、OE%、直立次数、水平跨格次数及海马5-HT1AR表达明显升高(P<0.01或P<0.05),SL及海马5-HT2AR表达明显降低(P<0.01或P<0.05)。与西药组相比,针刺组OT%、SL及海马5-HT1AR升高更显著(P<0.05)。与假针刺组相比,针刺组大鼠体质量、OT%、OE%、ST、直立次数、水平跨格次数及海马5-HT1AR表达明显升高(P<0.01),SL及海马5-HT2AR表达明显降低(P<0.01)。结论:“疏肝调神”针法不仅可以改善睡眠,还能改善肝郁型失眠大鼠异常行为和情绪,且对异常情绪的改善作用优于西药组。其作用机制可能与针刺调节海马5-HT1AR和5-HT2AR的表达有关。

Objective:To observe the effects of liver-soothing spirit-regulating acupuncture on the behaviors and expression levels of 5-hydroxytryptamine receptor 1A(5-HT1AR)and 5-hydroxytryptamine receptor 2A(5-HT2AR)in the hippocampus of insomnia rats due to liver Qi stagnation.Methods:Ten of the 70 Wistar rats were randomly selected as the blank group,and the remaining rats were subjected to preparing the insomnia model due to liver Qi stagnation by the chronic tail clamping stimulation combined with intraperitoneal injection of para-chlorophenyl alanine.Fifty successful model rats were randomly divided into a model group,a grasp group,a Western medicine group,an acupuncture group,and a sham acupuncture group,with 10 rats in each group.Rats in the grasp group were grabbed in the same way as those in the acupuncture group.Rats in the Western medicine group were given estazolam solution by intragastric administration.Rats in the acupuncture group received liver-soothing spirit-regulating acupuncture at Baihui(GV20),Neiguan(PC6),Shenmen(HT7),and Taichong(LR3);rats in the sham acupuncture group received acupuncture at 4 non-meridian non-point spots.All groups were treated once a day for 7 d.Pentobarbital sleep test was used to evaluate the sleep latency(SL)and sleep time(ST)of rats in each group after 7 d of intervention;the proportion of the number of entry into the open arms(OE%)and the proportion of the residence time in the open arms(OT%)were monitored by elevated plus maze test(EPMT);the open field test(OFT)was used to monitor the numbers of standing upright,horizontal cross-grid,and fecal grains in rats.The expression levels of 5-HT1AR and 5-HT2AR in rat hippocampus were detected by Western blotting.Results:The body mass gain,ST,OE%,OT%,upright standing times,horizontal cross-grid times,and the 5-HT1AR expression level in the hippocampus were significantly decreased(P<0.01),while the SL and 5-HT2AR expression level in the hippocampus were significantly increased(P<0.01)in the model group compared with the blank group.After intervention,the body mass,ST,OT%,OE%,upright standing times,horizontal cross-grid times,and hippocampal 5-HT1AR expression level were significantly increased(P<0.01 or P<0.05),while the SL and hippocampal 5-HT2AR expression level were significantly decreased(P<0.01 or P<0.05)in the acupuncture group and the Western medicine group compared with the grasp group.Compared with the Western medicine group,OT%,SL,and hippocampal 5-HT1AR expression level were significantly increased in the acupuncture group(P<0.05).The body mass,OT%,OE%,ST,upright standing times,horizontal cross-grid times,and the 5-HT1AR expression level in the hippocampus were significantly increased(P<0.01),while the SL and 5-HT2AR expression level in the hippocampus were significantly decreased(P<0.01)in the acupuncture group compared with the sham acupuncture group.Conclusion:Liver-soothing spirit-regulating acupuncture can not only improve sleep but also improve the abnormal behaviors and mood of insomnia rats due to liver Qi stagnation,and the improvement effect on abnormal mood is better than that of Western medicine.The mechanism may be related to the regulation of 5-HT1AR and 5-HT2AR expression levels in the hippocampus by acupuncture.