【作者】 王俊英；

【导师】 刘俊岭；

【作者基本信息】 中国中医科学院 ， 中西医结合基础， 2008， 硕士

【摘要】 研究背景针刺治疗疾病具有累积效应现在已是众多研究者所公认的事实。但是目前关于针刺累积效应的具体机理尚不明确,未见到系统的研究报道。已有的动物实验结果表明:反复针刺所产生的累积效应与神经系统的特别是中枢神经系统对针刺信息的储存、编码等活动密切相关;下丘脑、海马等边缘系统、中枢胆碱能系统参与针刺镇痛的过程。我们实验室前期的工作证明大鼠下丘脑、海马囊泡乙酰转移酶（VAChT）、胆碱乙酰转移酶（ChAT）等参与针刺镇痛的累积效应;去卵巢（OVX）引起的记忆功损伤减弱了针刺的效应。为了从不同的环节进一步确认下丘脑、海马胆碱能神经元在针刺累积性镇痛效应中的作用,本研究在结扎坐骨神经造成压迫性损伤（chronic constrictive injury,CCI）/及OVX大鼠记忆损害的动物模型上,从乙酰胆碱降解酶胆碱酯酶（AChE）和细胞内蛋白激酶PKA的活性的改变、以及毒蕈碱M₁受体m RNA表达的变化,探讨针刺累积效应的机制。研究方法Wistar雌性大鼠103只,随机分为正常对照组（n=15）,慢性压迫损伤（CCI）组（n=15）,CCI+EA2天（D）组（n=15）,CCI+EA2周（W）组（n=15）,去卵巢（OVX）+CCI（n=13）,OVX+CCI+EA2D（n=15）,OVX+CCI+EA2W（n=15）组。Morris水迷宫测试检查OVX+D-半乳糖皮下注射动物学习记忆能力的变化。结扎坐骨神经造成CCI慢性痛模型,电针双侧“足三里”-“阳陵泉”穴（2/15Hz,1-2mA,1次/D）。用辐射热照射大鼠双侧足底引起的缩腿反应潜伏期（PWL）的差值作为痛敏分数（HS）判断动物的痛反应。在麻醉状态下,一部分（每组8-10例）动物常规灌流取脑,冰冻切片,用酶组织化学方法来检测下丘脑弓状核（ARC）区、视上核（SON）区和室旁核（PVN）区,及海马CA1区AChE的表达;用免疫组化法检测其PKA活性。另一部分动物（每组5例）取脑组织匀浆后,用RT-PCR的方法来检测M₁受体m RNA的表达的变化。结果1)电针对CCI大鼠痛阈变化的影响CCI术18天后,与正常组（0.22±0.68）比较,各组HS的绝对值都明显升高（P＜0.05）。在单纯CCI动物上,与CCI对照组（-4.22±0.33）比,CCI术后第18天CCI+EA2D（-2.76±0.32）、CCI+EA2W组（-0.42±0.68）HS的绝对值显著减小（P＜0.05）;CCI+EA2W组（-0.42±0.68）HS绝对值明显低于CCI+EA2D组（-2.76±0.32,P＜0.05）。在OVX动物上,OVX+CCI+EA2W组（-2.37±0.29）和CCI+EA2D组（-3.11±0.32）HS绝对值明显低于OVX+CCI组（-4.66±0.45,P＜0.05）;OVX+CCI+EA2W组HS绝对值（-2.37±0.29）明显低于CCI+EA2D组（-3.11±0.32,P＜0.05）。提示电针可以改善CCI动物的痛敏状态,并具有累加性。CCI+EA2W组HS的绝对值（-0.42±0.68）显著低于OVX+CCI+EA2W组（-2.37±0.29,P＜0.05）,说明记忆损伤动物的电针累积效应明显降低。2)电针对CCI大鼠下丘脑三核团、海马组织胆碱能神经纤维AChE的活性变化的影响:坐骨神经结扎后,CCI组ARC（144.43±4.68）、SON（135.14±6.95）AChE的表达显著少于正常对照组ARC（126.27±4.35）、SON（122.2±7.5）（P＜0.05,灰度值越小,表达量越大）。电针2天后,CCI+EA2D组AChE的表达在ARC（133.32±8.03）、SON（111.24±6.01）显著增多（P＜0.05）。CCI+EA2W组下丘脑AChE的表达PVN（100.11±12.71）、ARC（117.25±4.12）和SON区（95.27±7.23）显著多于CCI对照组和CCI+EA2D组（P＜0.05）。复合模型组中的表达趋势与单纯CCI模型组相同,电针后AChE的表达累积性上调。OVX+CCI+EA2W组AChE的表达ARC（133.23±11.48）、和SON区（113.92±7.11）均显著少于CCI+EA2W组同一脑区（P＜0.05）。海马CA1区各组AChE的表达与下丘脑区各组的表达趋势一致。结果说明:CCI后,下丘脑和海马组织AChE的活性有所抑制,电针可使AChE的活性增强,并有累积性上调;去除卵巢,动物记忆能力受到损害后,仍有累积效应存在,但明显减弱。3)电针对CCI大鼠下丘脑、海马组织毒蕈碱M₁受体mRNA表达的影响CCI后,下丘脑组织M₁受体mRNA的表达量（0.39±0.25）与正常对照组（0.83±0.40）比较显著性降低（P＜0.05）;CCI+EA2W组M₁受体mRNA表达量（0.88±0.16）显著高于CCI对照组（0.39±0.25）和CCI+EA2D组（0.59±0.21,P＜0.05）。记忆损伤动物中,OVX+CCI组下丘脑M₁受体mRNA的表达量（0.29±0.11）明显少于正常对照组（0.83±0.40,P＜0.05）;OVX+CCI+EA2W组M₁受体mRNA的表达量（0.76±0.35）显著高于OVX+CCI（0.29±0.11）和OVX+CCI+EA2D组（0.41±0.22,P＜0.05）。这说明电针可以累积性上调M₁受体mRNA的表达。海马区M₁受体mRNA的表达趋势与下丘脑区的表达变化趋势类似:OVX+CCI+EA2W组（1.00±0.20）M₁受体mRNA的表达显著少于CCI+EA2W（1.37±0.37）,提示电针上调M₁受体mRNA表达的累积作用记忆损伤后减弱。4)电针对CCI大鼠下丘脑三核团、海马CA1区神经元PKA的表达变化的影响CCI+EA2W组下丘脑PVN（99.28±19.33）、ARC（100.18±14.52）和SON（103.26±17.12）区PKA的表达均明显高于正常对照组PVN（133.54±6.31）,ARC（127.84±18.91）,SON（133.53±21.34）（P＜0.05;灰度值越小,表达量越大）,也显著高于CCI+EA2D组PVN（124.35±6.3）,ARC（121.77±14.66）,SON（118.61±9.64,P＜0.05）。OVX+CCI+EA2W组PVN（107.48±12.39）、ARC（117.11±19.87）和SON（129.12±16.96）区PKA表达亦均明显高于OVX+CCI组PVN（145.96±8.02）,ARC（134.24±18.43）,SON（148.42±10.7（P＜0.05）,说明EA2W可使PKA的表达显著上调,且有累积性作用。OVX+CCI+EA2W组ARC和SON区PKA的表达均显著低于CCI+EA2W组（P＜0.05）,提示EA上调PKA表达的累积效应在记忆功能减退的动物上明显减弱。海马PKA的表达与SON等下丘脑核团PKA的表达趋势类似。结论1）电针“足三里”-“阳陵泉”可明显减轻CCI后出现的痛反应,且有累积效应;记忆损伤后减弱电针的效应;2)电针可显著减轻CCI引起的下丘脑、海马AChE、毒蕈碱M₁受体mRNA表达的抑制作用,累积性上调AChE、PKA及M₁受体mRNA的表达;记忆损伤后电针的上述效应降低。因此,电针“足三里”-“阳陵泉”减轻慢性痛的累积效应与其上调下丘脑和海马组织中AChE、M₁受体mRNA、PKA的表达作用、与动物神经记忆功能有密切联系。本研究结果为反复针刺治疗产生的累积效应提供了系统而详实的实验依据。更多还原

【Abstract】 BackgroundIt is a generally acknowledged that repeated acupuncture stimulation of acupoints has an accumulative effect in the treatment of diseases.But its underlying mechanism remains unknown,and no any systematic studies have been reported up to now.Experimental studies have indicated that the cumulative effect of repeated acupuncture has a close relationship with the nervous system,especially with the processing,storage,and coding of the central nervous system.The limbic system including hypothalamus and hippocampus,and the central cholinergic system have been shown to participate in the process of the accumulative analgesic effect of electroacupuncture（EA）.Our research results have revealed that hypothalamic and hippocampal neuronal vesicles acetyltransferase （VAChT）,choline acetyltransferase（CHAT） play a certain role in EA-induced accumulative analgesic effect in chronic constrictive injury （CCI） rats;and the memory impairment caused by ovariectomized（OVX） may weaken the effects of acupuncture.In order to confirm the relationship between the accumulative analgesic effect of EA and the cholinergic neurons in the hypothalamus and hippocampus from different links,the present study was designed to further explore the mechanism underlying EA-induced accumulative analgesic effect from the activity of acetylcholinesterase（ACHE,a catabolic enzyme of acetylcholine） and intracellular protein kinase A（PKA）,and changes of muscarinic M₁ receptor mRNA expression and neuron-memory in rats with chronic neuropathy pain and or OVX-induced memory impairment.Materials and MethodsA hundred and three female Wistar rats（230-250g） were randomized into normal control,CCI,CCI+EA2D,CCI+EA2W,OVX+CCI,OVX+CCI+EA2D and OVX+CCI+EA2W groups,with 15 cases in each except OVX+CCI group（n=13）. Neuro-memory impairment was induced by OVX plus subcutaneous injection of D-galactose.CCI pain model was established by ligature of the right sciatic nerve with surgical suture.EA（2/15Hz,1-2 mA,30 min） was daily applied to bilateral "Zusanli"（ST36） and "Yanglingquan"（GB34） for 2 days（2D） and 2 weeks（2W） respectively.Hyperalgesic scores（HS） were detected with radiation-heat irradiation.After deeply anesthetized（25% urethane and 1.5%cholorase,0.5ml/100g）,the rats（8-10/group） were perfused transcardially and the brain tissue was dissected out to be sectioned at 60μm on a freezing microtome.AChE activity and PKA activity of hypothalamic arcuate nucleus（ARC）,supraoptic nucleus（SON） and paraventricular nucleus（PVN） and Hippocampal CA1 of different groups were measured respectively with the enzyme histochemistry and immunohistochemical methods.Other 35 anesthetized rats（5/group） were executed on the ice-plate and their brain tissue was taken out.Muscarinic M₁ receptor mRNA expression was detected by reversed transcription-polymerase chain reaction（RT-PCR） after homogenate.Results:1) Effect of EA on pain threshold in CCI ratsAfter CCI,the absolute values of HS of all groups increased significantly in comparison with normal control group（0.22±0.68, P＜0.05）.In simple CCI rats,compared with CCI group（-4.22±0.33）,the absolute values of HS of both CCI+EA2D（-2.76±0.32） and CCI+EA2W （-0.42±0.68） groups decreased markedly（P＜0.05）,and that of CCI+EA2W group（-0.42±0.68） was evidently lower than that of CCI+EA2D group （-2.76±0.32,P＜0.05） on the 18th day after CCI（P＞0.05）.In memory impairment rats,the absolute values of the HS of both OVX+CCI+EA2W （-2.37±0.29）and CCI+EA2D（-3.11±0.32）groups were significantly lower than that of OVX+CCI group（-4.66±0.45,P＜0.05）.Compared with OVX+CCI+EA2D（-3.11±0.32） group,the absolute values of HS of OVX+CCI+EA2W（-2.37±0.29） group decreased markedly（P＜0.05）,suggesting that electroacupuncture treatment can improve the animal’s pain sensitivity after CCI,and has an accumulative effect.The absolute value of HS of CCI+EA2W（-0.42±0.68） was apparently lower than that of OVX+CCI+EA2W group（-2.37±0.29,P＜0.05）,indicating that the memory impairment weakens the above mentioned effects of EA.2) Effect of EA on AChE activity of cholinergic nerve fibers in three nuclei of hypothalamus and hippocampus in CCI model ratsAfter CCI,AChE expression of ARC（144.43±4.68） and SON（135.14±6.95） in CCI group were significantly lower than ARC（126.27±4.35）,SON （122.2±7.5） in normal control group（P＜0.05,the bigger the integral grey value,the less the expression of ACHE）.In comparison with normal control group,AChE expression in the ARC（133.32±8.03） and SON（111.24±6.01） in CCI+EA2D group increased significantly（P＜0.05）.Compared with CCI control group and CCI+EA2D group,AChE expression in PVN（100.11±12.71）,ARC（117.25±4.12） and SON（95.27±7.23） in CCI+EA2W group increased significantly（P＜0.05）.AChE expression in ARC,SON and PVN in OVX+CCI,OVX+CCI+EA2D and OVX+CCI+EA2W groups presented the similar tendency in comparison with CCI,CCI+EA2D and CCI+EA2W groups after the EA treatment,and EA-induced upregulation of AChE expression has a cumulative efficacy.AChE expression of OVX+CEI+EA2W group in ARC（133.23±11.48） and SON（113.92±7.11） was significantly lower than that of CCI + EA2W group in the same brain regions（P＜0.05）.AChE expression in hippocampal CA1 area showed the similar tendency.These results showed that after CCI,the activity of AChE was down-regulated in hypothalamus and hippocampus,EA of ST36-GB34 can increase the activity of ACHE,and induce a cumulative increase;OVX-induced memory impairment（estrin level-decrease） attenuates EA-induced effects,in spite of still existing in OVX rats.3) Effect of EA on muscarinic M₁ receptor mRNA expression in hypothalamus and hippocampus in CCI ratsAfter CCI,M₁ receptor mRNA expression（0.39±0.25） in hypothalamus was considerably lower than that of normal control group（0.83±0.40, P＜0.05）.Compared with CCI control group（0.39±0.25） and CCI+EA2D group （0.59±0.21）,M₁ receptor mRNA expression of CCI+EA2W group（0.88±0.16） was increased significantly（P＜0.05）.In OVX+CCI model,M₁ receptor mRNA expression of OVX+CCI control group（0.29±0.11） was markedly lower than that of normal control group（0.83±0.40,P＜0.05）.Compared with OVX+CCI （0.29±0.11） and OVX+CCI+EA2D group（0.41±0.22）,the expression of M₁ receptor mRNA expression（0.76±0.35） in OVX+CCI+EA2W group was markedly increased（P＜0.05）,suggesting a cumulative increase of M₁ receptor mRNA expression after EA treatment.The expression of M₁ receptor mRNA in hippocampus tissue had a similar trend in comparison with hypothalamus tissue.The expression of M₁ receptor mRNA in OVX+CCI+EA2W group （1.00±0.20） was lower than that of CCI+EA2W group （1.37±0.37）,suggesting an attenuation of EA-induced cumulative effect in up-regulating M₁ receptor mRNA expression after memory impairment in OVX rats.4) Effect of EA on PKA activity in hypothalamus and hippocampus in CCI ratsResults of immunohistochemistry showed that the expressions of PKA of PVN（99.28±19.33,integral grey value）,ARC（100.18±14.52） and SON （103.26±17.12） of hypothalamus in CCI+EA2W group were significantly higher than those in PVN（133.54±6.31）,ARC（127.84±18.91） and SON （133.53±21.34） in normal control group;and PVN（124.35±6.3）,ARC （121.77±14.66）,SON（118.61±9.64） in CCI+2D group（P＜0.05）.PKA expressions of PVN（107.48±12.39）,ARC（117.11±19.87） and SON （129.12±16.96） in OVX+CCI+EA2W group were also markedly higher than PVN （145.96±8.02）,ARC（134.24±18.43） and SON（148.42±10.7,P＜0.05） in OVX+CCI group,displaying upregulation and cumulative increase of PKA expression after EA2W.PKA expression of PKA of ARC and SON in OVX+CCI+EA2W group was markedly lower than that in CCI+EA2W group（P＜0.05）,suggesting an apparent attenuation of EA-induced cumulative upregulation effect of PKA expression in OVX rats.The changing tendency of PKA expression in hippocampal CA1 region was similar to that of hypothalamic SON after repeated EA of ST36-GB34 in CCI and OVX+CCI rats.Conclusion1) EA of "Zusanli"（ST36）-"Yanglingquan"（GB34） is able to significantly alleviate pain sensitivity caused by CCI,and has a cumulative effect,which is weakened obviously after neuron-memory injury.2) EA can significantly reduce CCI-induced inhibition of the expression of ACHE,M₁ receptor mRNA in hypothalamus and hippocampus,and has an accumulative upregulation effect on ACHE,PKA and M₁ receptor mRNA expression,which are closely associated with the animals’ neuron-memory ability.Therefore,there is a close relationship between the accumulative analgesic effect of EA and its resultant increase of the expression of ACHE,M₁ receptor mRNA and PKA in hypothalamus and hippocampus,and the function of neuron-memory is involved in the cumulative effect of repeated EA treatment.Results of the present study provide a set of detailed experimental evidence for repeated acupuncture-induced cumulative effect in the treatment of chronic pain.更多还原