【作者】 李卫东；

【导师】 图娅；

【作者基本信息】 北京中医药大学 ， 针灸推拿学， 2009， 博士

【摘要】 抑郁症是一种高发病率和高致死率的精神疾患,产生了严重的社会经济负担。目前抑郁症的病理生理学机制不是很清楚。目前抗抑郁治疗,主要以单胺类递质的调控作为治疗靶点。而这种方式的治疗仅能缓解30%患者的症状。而且,临床常用的各种抗抑郁药物都存在着各种副作用和疗效延迟效应。针灸疗法属于传统中医疗法,具有整体调节的特点,动物实验研究和临床报道均表明针灸治疗抑郁症的有效性,与传统药物治疗相比,它可以避免药物长期应用的毒副作用,而且是整体多靶点治疗。电针虽然抗抑郁有效,但具体作用机制不明。近年来随着分子生物学技术的深入研究,发现抑郁症的发病与信号通路有一定相关性。基于上述考虑,我们选择慢性应激抑郁动物模型对电针抗抑郁的分子机制进行一定探究。选择与抑郁症密切相关的两条信号通路,运用多种方法对信号通路的上下游指标进行检测,并选择作用较为明显的信号通路进行阻断剂实验,以期发现电针抗抑郁的可能作用途径,并比较电针在生理状态下与病理状态下的作用不同。研究方法和结果:1.建立慢性应激抑郁模型,并采用Open-field test、糖水实验、体质量测量来综合评价模型复制是否成功。造模结束后,模型组大鼠旷场试验的水平穿越格数格、竖立次数次、糖水实验消耗量、体质量增加量均显著低于空白组(P<0.05);与模型组相比,旷场试验的水平穿越格数、竖立次数、糖水实验消耗量、体质量增加变化,电针组均显著高于模型组(P<0.05);而电针组与百优解组上述指标之间没有差异(P>0.05)。2.在模型复制成功的基础上,进行了流式细胞技术Annexin V-FITC/PI双染法、PI单染法检测大鼠海马神经元凋亡率。Annexin V-FITC/PI双染法结果显示:模型组应激第21天海马神经元凋亡率高于空白组(P<0.05);电针组、百优解组均低于模型组(P<0.05);电针组低于百优解组(P<0.05)。PI单染法检测结果显示:模型组与空白组之间没有显著差异(P>0.05);而电针组与模型组之间有显著性差异(P<0.05);百优解组与电针组之间差异不显著(P>0.05)。3.在模型复制的基础上,并结合流式细胞结果,我们进行了电针抗细胞凋亡的机制探究;运用荧光定量PCR方法对大鼠海马组织Bcl-2mRNA、BadmRNA的表达检测。结果显示:Bcl-2mRNA表达电针组较模型组为高(P<0.05),模型组与百优解组之间没有差异(P>0.05)。BadmRNA的表达各组之间没有差异,但是模型组的Bad表达有增高趋势,电针组和百优解组均有下降趋势。4.cAMP-PKA信号通路与抑郁症的神经再生和细胞凋亡密切相关。因此,我们选择了该通路的关键指标进行检测;放射免疫法对海马组织中cAMP含量的检测;采用免疫组化法对PICA的表达进行检测;采用Western blotting方法对CREB的含量进行检测。结果显示:cAMP含量模型组大鼠海马组织中最低,空白组最高,二者之间有显著性差异(P<0.05);电针组、百优解组大鼠海马组织cAMP含量较模型组高,并与模型组有显著性差异(P<0.05);电针组与百优解组大鼠海马组织中的cAMP含量之间没有差异(P>0.05)。大脑皮质中各组之间的PKA表达之间没有差异;海马组织中,百优解组与模型组之间PICA的表达有显著性差异(P<0.05)。皮质CREB的含量电针组较模型组有一定升高,但是差异不显著(P>0.05)。海马CREB的含量:以百优解组最高,但是各组之间没有显著性差异。5.ERK信号通路与抑郁症的发病关系密切,参与了抑郁症的神经再生和细胞凋亡机制。因此我们对该通路的关键指标进行了探究。采用Western blotting方法对该通路的上下游指标进行了检测。结果显示:各组大鼠海马组织和皮质组织中Ras、c-raf、p-c-raf、ERK蛋白含量之间差异不显著。皮质组织中电针组p-ERK的水平和百优解组的p-ERK水平明显增高,均与模型组相比较有显著性差异(P<0.05)。同时模型组较空白组p-ERK水平为低(P<0.05)。而在海马组织中检测的结果显示:模型组的p-ERK水平较空白组低(P<0.05);电针组较模型组为高(P<0.05)。但是药物组虽然较模型组有增高趋势,但是两组之间无显著性差异(P>0.05)。皮质中电针组RSK的水平较模型组为高,与模型组相比较有显著性差异(P<0.05);而且模型组与空白组之间有显著性差异(P<0.05)。百优解组与模型组相比较没有差异(P>0.05)。海马中电针组与百优解组均有升高趋势,但是统计学上与模型组之间无显著性差异(P>0.05)。6.通过对与抑郁症发病密切相关的两条信号通路的检测,我们发现电针对ERK信号通路效果较好,因此,我们选择对ERK通路的关键点ERK进行了阻断剂研究,选用了慢性强迫游泳应激模型,结合动物行为学测试评价阻断剂的效果和电针抗抑郁的作用机制。结果显示:应激处理第7天,模型组与空组之间相比较体质量有极显著性差异(P<0.01)。应激处理第13天,模型组的体质量与空白组相比较有显著性差异(P<0.05);模型组的体质量变化与电针组之间有显著性差异(P<0.05);PD98059组大鼠体质量与电针组之间也有显著性差异(P<0.05)。第13天的测试结果中可见模型组较空白组的水平穿越格数、竖立次数明显下降,二组之间有极显著性差异(P<0.01);同时电针组较PD98059组、模型组均有显著性差异(P<0.05)。竖立次数电针+PD98059组与电针组有显著性差异(P<0.05)。大鼠皮质和海马中ERK水平各组之间没有显著性差异。皮质中的p-ERK电针组较模型组为高,二组之间有显著性差异(P<0.05);电针+PD98059组的p-ERK较电针组为低,二组之间有显著性差异(P<0.05);海马中,各组之间没有显著性差异。皮质中BDNF各组之间没有显著性差异。海马中电针+PD98059组的BDNF较电针组为低,二组之间有显著性差异(P<0.05);电针组的BDNF较模型组为高,二组有显著性差异(P<0.05)。结论:1.行为学测试结果提示慢性应激可以引起大鼠的活动量降低和探究行为减少,体质量增加量减少;而电针干预可以改善慢性应激抑郁大鼠行为学表现;证明模型复制成功且电针干预慢性应激抑郁模型有效。2.流式细胞的检测结果提示模型组大鼠海马细胞凋亡较电针组、百优解组严重;电针干预可以减低海马细胞凋亡;同时也表明慢性应激可以引起海马组织细胞凋亡发生。3.大鼠海马组织Bcl-2mRNA、BadmRNA表达的检测结果提示慢性应激可引起细胞凋亡相关基因的改变,而电针抗慢性应激抑郁与增加抗凋亡基因Bcl-2mRNA的表达和抑制促凋亡基因BadmRNA的表达有一定关系。4.慢性应激模型大鼠海马组织中cAMP含量低于电针组、百优解组,提示电针、药物干预可以对cAMP产生积极作用;百优解组PKA海马表达明显高于模型组,而电针组与模型组没有差异;同时,CREB的含量各组之间有没有差异。由此,我们认为慢性应激可减低海马组织中cAMP的含量,减少PKA的表达,而百优解可起到相反的作用;电针对该通路的影响与药物不同,产生较少作用。5.慢性应激可以一定程度上引起大鼠脑内ERK通路表达抑制,而电针可以起到相反作用。电针主要活化ERK通路上的关键指标ERK,从而对下游产生影响而发挥抗调亡和抗抑郁作用。电针干预、药物干预均能对该通路产生一定影响,尤其以电针的作用较为明显。6.阻断剂实验中,动物所表现出来的探究行为和体质量变化表明慢性强迫游泳实验模型复制成功;磷酸化ERK的变化以及下游与神经再生密切相关的蛋白BDNF的检测结果提示:电针对ERK通路的确有影响,而且这种作用可以被PD98059一定程度上所阻断。更多还原

【Abstract】 Depression is a mental disorder with high prevalence and mortality,resulting in massive socioeconomic burden.The pathophysiology mechanism of depression has been poorly understood.Nowdays it is most porpular therapeutic target to modulate monoaminergic neurotransmission.But this therapeutic method only just alleviate the symptoms of 30%patients.Furthermore,all the antidepressant drugs exist side effects and lag period for their clinical effects.Acupuncture belong to traditional Chinese medicine and can regulate the balance for human body.The datas,from the animal experiments and clinical reports,show that acupuncture is effective for depression.It has little side effect and can play roles on multiple targets.Although acupuncture is effective for depression,the mechanism for the efficiency is still not clear.The pathogenesis of depression may be relevant to molecular signaling pathway.So chronic stress animal model was chosen for the study.Two signaling pathways were chosen for the mechanism study on acupuncture antidepression.The upper stream and down stream indexes were detected.At the same time,the special inhibitor was chosen for the signaling pathway.We hope to make it clear for the mechanism of acupuncture antidepression.Methods and Results1.Chronic stress depression animal model was established,and Open field test, sucrose intake,and body weight balance were used to evaluate the model.After stress,compared with control group rats,the model group rats’crossing numbers,rearing times,the level of sacchar-consumption,body weight changes,were apparently less than those of control group rats’(P<0.05).In comparison with model group,the crossing numbers,rearing times,body weight changes,the level of sacchar-consumption,being apparently increasing in electroacupuncture group and Prozac group(P<0.05),those indexes were not apparently differences between electroacupuncture group and Prozac group(P>0.05).2.Annexin V-FITC/PI double staining and PI single staining flow cytometric method were used to detect the hippocampal apoptotic rates.Annexin V-FITC/PI double staining result indicated that hippocampal apoptotic rates in model group was higher than control group(P<0.05),apoptotic rates of EA group and Pozac group was lower than model group(P<0.05),EA group was more lower than Pozac group(P<0.05).PI single staining result indicated that there was no difference between model group and control group(P>0.05),apoptotic rates of EA group was signifant different from model group(P<0.05),there was no difference between Pozac group and EA group(P>0.05).3.Real-time PCR method was used to detect Bcl-2mRNA and BadmRNA expression.The results showed that hippocampal Bcl-2mRNA expression for EA group was more higher than model group(P<0.05),there was no difference between EA group and Pozac group(P>0.05).BadmRNA expression in model group was higher than EA group and Pozac group,but the difference was not significant.4.cAMP-PKA signaling pathway is more relative to depression.So the signaling was chosen to detect.cAMP in hippocampus was detected by radioimmunoassay method.Immunohistochemistry method was used to detect PKA expression,and Western blotting method was used to detect CREB protein.The results showed that the level of cAMP in hippocampus for model group is lower than control group(P<0.05), cAMP level for EA group and Pozac group were more higher than model group(P <0.05),there was no difference between EA group and Pozac group(P>0.05).PKA expressin in cortex was almost similar in each groups,Hippocampal PKA expression in Pozac group was stronger than model group(P<0.05).CREB protein level in cortex for EA group was higher than model group,CREB protein level hippocampus for Prozac group was higher than model group.5.ERK is another important signaling pathway for depression.Western blotting method was used to detect ERK signaling pathway.The data showed that Ras,c-raf,p-c-raf,ERK protein level was almost similar in each groups.p-ERK level in cortex for EA group and Pozac group were higher than model group(P<0.05).p-ERK level in cortex for model group was lower than control group(P<0.05).p-ERK level in hippocampus for model group was lower than control group(P<0.05),p-ERK level for EA group was higher than model group(P<0.05).RSK protein level in cortex for EA group was higher than model group(P<0.05),and RSK level in cortex for model group was lower than control group(P<0.05),there was no difference for cortex RSK level between Pozac group and model group(P>0.05).Although RSK level in hippocampus increased for EA group and Pozac group,there was no different from model group (P>0.05).6.Inhibitor was used to detect the ERK signaling pathway function.Chronic forced swimming stress model was established.Open field test and body weight balance were used to evaluate the model.The results showed that,compared with control group, the crossing,rearing times,body weight changes for model group was significant different on day 7(P<0.01) and day13(P<0.05).The open field test and body weight changes for EA group was more apparent.EA group and EA+PD98059group was significant different for crossing times(P<0.05).ERK level in cortex was almost similar for each group.p-ERK level in cortex of EA group was higher than model group (P<0.05) and EA+PD98059 group(P<0.05).p-ERK level in hippocampus for each group was almost similar.There were no differences for BDNF level in cortex of each group.BDNF level in hippocampus of EA group was higher than model group (P<0.05) and EA+PD98059 group(P<0.05).Conclusions1.The data from open field test,sucrose intake,body weight changes showed that chronic stress could cause the rats to move less,suppress body weight gain of rats, decreasing of sucrose intake,whereas electroacupuncture could improve them.2.Flow cytometric results indicated that chronic stress could cause hippoampal apoptotic rates increasing,but electroacupuncture could decrease it.3.Real-time PCR results indicated that chronic stress could cause Bcl-2mRNA decreasing and BadmRNA increasing in hippocampus,but electroacupuncture could reverse it.4.Chronic stress could cause cAMP level decreasing in hippocampus,but electroacupuncutre and Pozac could reverse it.Pozac could cause PKA expression increasing under chronic stress.We thought that Pozac was more effective than electroacupuncture on cAMP-PKA signaling pathway.5.Chronic stress could inhibit the ERK signaling pathway,but electroacupuncture could active ERK signaling pathway under stress by phosphorate ERK and play some roles on antiapoptosis and antidepression.6.Chronic forced swimming model could be used for inhibitor study.The results indicated that electroacupuncture could effect ERK pathway expression,and PD98059 could inhibit its function.更多还原