目的:观察针刺5-HT7R跨膜信号通路的影响,探讨其与改善PCPA失眠大鼠中枢5-HT障碍机制的关系。方法:64只大鼠经随机、PCPA失眠造模及筛选后,共分4组各16只;正常组、模型组无特殊处理,针刺组给予针刺失眠方合脐内环穴干预,非穴组予针刺非穴处理,治疗6天后分离大鼠海马匀浆,ELISA法检测5-HT7R和cAMP含量,免疫印迹检测Gαs表达;统计学分析5-HT7R与cAMP相关性。结果:PCPA模型大鼠海马5-HT7R和cAMP明显降低,针刺治疗组5-HT7R和cAMP含量均显著升高;各组二指标间呈显著正相关性;四组间Gαs相对表达无统计学差异。结论:针刺调控5-HT7R/Gαs/cAMP信号通路起到改善PCPA失眠作用,这种改善与中枢5-HT递质障碍机制有一定的相关性。 Objective: To observe the effects of the acupuncture of combined method of the acupoint-prescription of umbilical inner acupoints and the insomnia acupoint-prescription on 5-HT7R transmembrane signal pathway and explore its relationship with improving the mechanism of central 5-HT disorder in PCPA insomnia model rats. Methods: After being randomly divided, modeled and screened for PCPA insomnia model, 64 rats were divided into four groups, and each group was 16. The normal group and the model group were given no special treatment, the acupuncture observation group was acupunctured the combined acupoints of the acupoint-prescription of umbilical inner acupoints and the insomnia acupoint-prescription. The non-acupoint group was dealed with acupuncture non-acupoint. After six days of treatment, in the hippocampus homogenates of the rats, the content levels of 5-HT7R and cAMP were detected by enzyme-linked immunoassay, and their correlation was statistically analyzed. The expression of Gαs was detected by western blot. Results: The contents of 5-HT7R and cAMP in hippocampal of PCPA insomnia model rats in control group were significantly reduced, while that of the acupuncture treatment group was significantly increased, and there was a significantly positive correlation between them in each group. The relative expression of Gαs in hippocampus was no statistical difference among the four groups. Conclusion: The cupuncture method could improve PCPA insomnia by regulating the 5-HT7R/Gαs/cAMP signaling pathway. This regulating mechanism is related to the acupuncture method improving the pathogenesis of central 5-HT transmitter disorder of insomnia.
目的:观察针刺5-HT7R跨膜信号通路的影响,探讨其与改善PCPA失眠大鼠中枢5-HT障碍机制的关系。方法:64只大鼠经随机、PCPA失眠造模及筛选后,共分4组各16只;正常组、模型组无特殊处理,针刺组给予针刺失眠方合脐内环穴干预,非穴组予针刺非穴处理,治疗6天后分离大鼠海马匀浆,ELISA法检测5-HT7R和cAMP含量,免疫印迹检测Gαs表达;统计学分析5-HT7R与cAMP相关性。结果:PCPA模型大鼠海马5-HT7R和cAMP明显降低,针刺治疗组5-HT7R和cAMP含量均显著升高;各组二指标间呈显著正相关性;四组间Gαs相对表达无统计学差异。结论:针刺调控5-HT7R/Gαs/cAMP信号通路起到改善PCPA失眠作用,这种改善与中枢5-HT递质障碍机制有一定的相关性。
PCPA失眠,脐内环合失眠穴方针刺;5-HT7R,环磷酸腺苷,Gαs
Benhua Luo1*, Yu Huang1, Xiaolin Wu2, Yuqiu Li2
Guangxi University of Traditional Chinese Medicine, Nanning Guangxi
Received: Nov. 30th, 2021; accepted: Mar. 22nd, 2021; published: Mar. 29th, 2021
Objective: To observe the effects of the acupuncture of combined method of the acupoint-prescription of umbilical inner acupoints and the insomnia acupoint-prescription on 5-HT7R transmembrane signal pathway and explore its relationship with improving the mechanism of central 5-HT disorder in PCPA insomnia model rats. Methods: After being randomly divided, modeled and screened for PCPA insomnia model, 64 rats were divided into four groups, and each group was 16. The normal group and the model group were given no special treatment, the acupuncture observation group was acupunctured the combined acupoints of the acupoint-prescription of umbilical inner acupoints and the insomnia acupoint-prescription. The non-acupoint group was dealed with acupuncture non-acupoint. After six days of treatment, in the hippocampus homogenates of the rats, the content levels of 5-HT7R and cAMP were detected by enzyme-linked immunoassay, and their correlation was statistically analyzed. The expression of Gαs was detected by western blot. Results: The contents of 5-HT7R and cAMP in hippocampal of PCPA insomnia model rats in control group were significantly reduced, while that of the acupuncture treatment group was significantly increased, and there was a significantly positive correlation between them in each group. The relative expression of Gαs in hippocampus was no statistical difference among the four groups. Conclusion: The cupuncture method could improve PCPA insomnia by regulating the 5-HT7R/Gαs/cAMP signaling pathway. This regulating mechanism is related to the acupuncture method improving the pathogenesis of central 5-HT transmitter disorder of insomnia.
Keywords:PCPA Insomnia, The Combined Acupuncture of the Acupoint-Prescription of Umbilical Inner Acupoints and the Insomnia Acupoint-Prescription, Serotonin Receptor 7 Subtypen (5-HT7R), Cyclic Adenosine Monophosphate (cAMP), Gαs
Copyright © 2021 by author(s) and Hans Publishers Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY 4.0).
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5-羟色胺受体7亚型(5-HT7R)是与睡眠和觉醒密切相关的经典5-HT神经递质一个亚型,它对睡眠的昼夜节律有一定调节作用,但与中枢5-HT促睡眠机制有着一定关系,但具体怎样不甚清楚。针刺失眠方合脐内环穴针刺是临床失眠症的有效 [
健康SPF级Wistar雄性大鼠80只,体重(250 ± 15) g,由广西医科大学动物实验中心提供;室温20℃~25℃饲养,室内采光按照白昼循环模式,定时喂食,自由饮水,适应性喂养15 d。
对氯苯丙氨酸(PCPA)试剂盒:美国Alfa Aesar,批号:H30Y014;I抗Gαs (Santa cruz,货号:Sc-135914),II抗:Goat anti-Mouse IgG (Bioswamp,货号:SAB43706);大鼠5-HT、5-HT7R、cAMP ELISA试剂盒(均Bioswamp):武汉基因美生物科技有限公司,批号:GR-201912、201907。
低温高速离心机(SIGMA3-18K,德国Sigma公司);Bio-Tek 800酶标仪、mini protean 3 cel电泳仪(美国BIO-RAD公司);洗板机(芬兰Thermo Labsystems公司);培养箱(lHH.CP-01W);匀浆仪(FLUKO-PRO200)。
采用随机数字表法将80只Wistar大鼠随机分为正常组16只、预用模型复制大鼠64只。预用模型复制大鼠采用腹腔注射对氯苯丙氨酸(PCPA)法进行模型复制,筛选后将造模成功的48只大鼠按随机数字表法分为失眠方加脐内环针组、西药组、非穴组3组,每组各16只。四组各16只动物按分组随机号顺序再顺编为1~16号;在各自干预完成后,每组取1~6号动物进行免疫印迹检测,每组其余7~16号动物再进行酶联免疫等相关检测。
对氯苯丙氨酸(PCPA),临用前按大鼠45 mg/(l ml∙100 g)用量,用弱碱性生理盐水(pH 7~8)配置成混悬液备用。
于上午8:00左右将大鼠按l ml/100 g剂量腹腔注射PCPA混悬液,每日1次,连续注射2日,在第2次腹腔注射PCPA 28 h~30 h后,动物出现入睡率显著降低,或昼夜节律消失,呈现兴奋、烦躁、易激惹,白天活动不停,整体观察与正常组有明显不同者,表明模型复制成功。
正常组腹腔注射相同体积的弱碱性生理盐水。
4组于造模成功后次日开始实验干预治疗。
1) 正常组与模型组:不予任何治疗,均只给予同失眠方加脐内环针组相同时间及相同强度的抓摸刺激(7 min)。
2) 失眠方加脐内环针组:
① 先给予针刺脐内环穴治疗。
取穴:脐内环穴,取心肝脾肺肾六穴点。脐内环穴在大鼠神阙穴 [
针刺方法:以25 × 13 mm粗细毫针,由内向外以10˚~15˚刺入2~4 mm,针后不行捻转提插手法,每穴点留针30 s,共计3 min。
②再给予针刺失眠方穴治疗。
取穴 [
以上失眠方针刺加脐内环针刺每次干预治疗,共7 min。
3) 非穴组:针刺双侧肋下各两个固定非穴点 [
以上4组按相应干预方法,每日治疗1次,共6次。
针灸干预方案完成后第2天,即处死各组动物,开颅迅速取出整脑,冰上分离各组大鼠海马,冷生理盐水中洗去血液,电子天平上称重后置于−80℃冰冻保存备用待测。
取海马组织,按1:9比例加入0.9%氯化钠溶液,组织匀浆器低温下匀浆,4℃,3000 r/min离心20 min,取上清,置于−20℃保存待测。采用双抗体夹心法检测5-HT7R、cAMP含量。按试剂盒说明书设立标准品孔、样品孔和空白对照孔,标准品孔加入不同浓度的标准品50 µl。样品孔先加待测样本10 µl,再加样本稀释液40 µl。标准品孔和样本孔每孔加入辣根酶标记抗体10 µl,37℃水浴锅温育60 min,后洗板5次,吸水纸拍干。每孔加入底物A、B各50 µl,37℃避光孵育15 min。加入终止液50 µl。450 nm波长测定各孔的吸光度(OD)值。计算机根据各标准品吸光度以及标准品浓度,计算检测样品5-HT7R、cAMP的含量。
主要步骤:① 总蛋白提取:大鼠迅速断头后,分离新鲜海马组织,加适量蛋白裂解液和蛋白酶抑制剂,冰上研磨、匀浆,12,000转,4℃离心,收集上清液,分装,−80℃保存;② BCA法蛋白定量;③ 蛋白电泳;④ 转膜;⑤ 封闭;⑥ 一抗孵育;⑦ 二抗孵育;⑧ 显定影。应用Quantity One软件计算各组目标蛋白条带的灰度值,并与同一膜上相应同组β-actin条带的灰度值进行比较,将该蛋白相对表达量的比值用于各组统计分析。
采用SPSS16.0软件进行统计分析,实验数据以均数±标准差( x ¯ ± s )表示,组间比较用单因素方差分析,P < 0.05为差异有统计学意义。
1) 对PCPA大鼠海马5-HT、5-HT7R、cAMP含量( x ¯ ± s )的影响,如表1。
指标 分组 | 5-HT (ng/ml) | 5-HT7R (pg/ml) | cAMP (nmol/L) |
---|---|---|---|
正常组 | 510.06 ± 50.01 | 2352.08 ± 126.58 | 102.78 ± 11.00 |
模型组 | 198.96 ± 28.54## | 1485.42 ± 103.43## | 40.32 ± 6.73## |
针刺组 | 406.82 ± 33.26##** | 1949.45 ± 159.93##**△△ | 80.93 ± 6.40##** |
非穴组 | 219.71 ± 39.93## | 1420.17 ± 99.36## | 39.34 ± 7.37## |
F值 | 149.83 | 122.316 | 150.11 |
P值 | 0.000 | 0.000 | 0.000 |
表1. 各组大鼠海马5-HT、5-HT7R、cAMP含量的影响
注:与正常组比较:#P < 0.05,##P < 0.01;与模型组比较:*P < 0.05,**P < 0.01;与非穴组比较,△P < 0.05,△△P < 0.01。
从表1可以看出:PCPA失眠模型组5-HT、5-HT7R、cAMP含量均较正常组非常显著性降低,通过针刺后5-HT、5-HT7R、cAMP含量均得到升高,均具有统计学意义(均P < 0.01),均显示有经穴作用的特异性。
2) 各组5-HT、5-HT7R、cAMP检测指标相互间的相关性,如表2。
分组 相关性分析 | 正常组 | 模型组 | 针刺组 | 非穴组 | |
---|---|---|---|---|---|
5-HT与5-HT7R | 相关系数 | 0.810 | 0.914 | 0.844 | 0.768 |
P值 | 0.005 | 0.000 | 0.002 | 0.010 | |
回归方程 | Y = 1306.61 + 2.05x | Y = 826.56 + 3.311x | Y = 299.17 + 4.057x | Y = 1000.43 + 1.91x | |
5-HT7R与cAMP | 相关系数 | 0.861 | 0.813 | 0.803 | 0.891 |
P值 | 0.001 | 0.004 | 0.005 | 0.001 | |
回归方程 | Y = −73.239 + 0.075x | Y = −38.305 + 0.053x | Y = 18.321 + 0.032x | Y = −54.56 + 0.066x | |
5-HT与cAMP | 相关系数 | 0.845 | 0.832 | 0.814 | 0.736 |
P值 | 0.002 | 0.003 | 0.004 | 0.015 | |
回归方程 | Y = 7.897 + 0.186x | Y = 1.289 + 0.0.196x | Y = 17.26 + 0.157x | Y = 9.495 + 1.36x |
表2. 各组大鼠海马5-HT、5-HT7R、cAMP指标间相关性分析表
表2可见,各组5-HT与5-HT7R之间、5-HT7R与cAMP、5-HT与cAMP之间均呈正相关性关系,各组均有统计学意义(均P < 0.05)。
3) 对PCPA大鼠海马Gαs//β-actin含量与相对表达的影响,如表3、图1。
分组(n = 6) | 正常组 | 模型组 | 非穴组 | F值 | P值 |
---|---|---|---|---|---|
Gαs/β-actin表达 | 0.738±0.106 | 0.531±0.149 | 0.537±0.132 | 2.587 | 0.111 |
表3. 各组大鼠海马Gαs//β-actin含量的影响
注:与正常组比较:#P < 0.05,##P < 0.01;与模型组比较:*P < 0.05,**P < 0.01;与非穴组比较,△P < 0.05,△△P < 0.01。
图1. 各组PCPA大鼠海马Gαs/β-actin蛋白相对表达
图1中,A5、B5、C5、D5代表动物分组及动物编号,图示各组PCPA大鼠海马Gαs /β-actin相对表达量,模型组和非穴组较正常组表达要弱,灰度浅,但从与β-actin相对表达情况看(表2),各组间比较差异未达到统计学意义(P = 0.111)。
失眠症的病机关键为水火失调、阴阳失衡。失眠穴方是治疗失眠症的针刺验方 [
睡眠发生与睡眠–觉醒机制相关的中枢神经递质含量发生改变密切相关 [
广西自然科学基金项目任务书,2018GXNSFAA138076,桂科计字[
罗本华,黄 羽,吴小玲,李玉秋. 针刺对5-HT7R跨膜信号通路的影响与其改善PCPA失眠中枢5-HT障碍机制的关联性探讨Study the Acupuncture Effect on 5-HT7R Transmembrane Signaling Pathway and the Exploration of the Relationship between This Effect and the Acupuncture Improving the Pathogenesis of Central 5-HT Transmitter Disorder in PCPA Insomnia[J]. 中医学, 2021, 10(02): 261-267. https://doi.org/10.12677/TCM.2021.102036