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不同电针对慢性应激抑郁大鼠海马神经营养因子及谷氨酸代谢相关蛋白的影响
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摘要
抑郁症是精神科最常见的自杀率最高的疾病之一,其发病率逐年升高、就诊率治疗率低,抑郁症导致的自杀及自残给家庭带来痛苦,给社会带来沉重负担。
     抑郁症发病的病理生理机制尚不完全清楚,主要治疗药物以单胺类神经递质为调控靶点,但疗效不甚理想,无法缓解所有症状,且存在各种副作用。
     针灸治疗抑郁症疗效确切,无毒副作用,而且是整体调节,多靶点治疗。近年来针灸治疗抑郁症是研究热点之一,大量研究表明针灸能从多个角度,多靶点、多通道起到抗抑郁作用。
     关于星形胶质细胞、谷氨酸代谢、谷氨酸转运体及受体,与抑郁症的关系属于研究初步阶段,神经营养因子前体蛋白的研究也不多见,本实验选取脉冲电针和音乐电针干预慢性应激抑郁大鼠,对上述研究领域相关指标进行初筛,以期发现电针抗抑郁的新靶点,并比较两种电针疗效的异同点。
     研究方法与结果
     1.应用慢性应激刺激结合孤养造成抑郁大鼠模型,采用开野实验、体质量测量、糖水偏好实验来评价抑郁模型复制是否成功。造模完成后,模型对照组大鼠开野试验的水平运动次数、垂直运动次数、体质量增加量、糖水偏好比例均显著低于空白对照组(P<0.01)说明造模成功;与模型对照组相比,氟西汀组、脉冲电针组、音乐电针组均明显升高(P<0.05);在糖水偏好实验及体质量测定中,音乐电针组大鼠优于脉冲电针组。
     2.采用尼氏海马神经元染色来评价抑郁模型复制是否成功,并且评价三种干预方法的疗效。模型对照组海马CA1区尼氏神经元数量明显低于空白对照组,说明造模成功。氟西汀、脉冲电针、音乐电针均可改善慢性应激刺激及孤养对大鼠海马神经元的损害作用,对海马神经元细胞有一定的保护作用。其中音乐电针对慢性应激抑郁大鼠海马CA1区神经元的保护作用优于脉冲电针,且有优干氟西汀的趋势,但无显著性差异。
     3.用免疫组化法检测海马BDNF阳性神经元的平均光密度,Elisa法检测血清中BDNF、t-PA及PAI-1的含量。慢性应激刺激结合孤养能造成大鼠海马CA1区及血清中BDNF含量降低,氟西汀、脉冲电针及音乐电针均能逆转这种变化,并且在改善脑内BDNF含量方面,音乐电针优于氟西汀及脉冲电针,在改善血清中BDNF含量方面,音乐电针优于氟西汀,与脉冲电针疗效相当。与空白对照组比较,模型对照组大鼠血清t-PA/PAI-1明显降低(P<0.01),表示mBDNF比例降低。与模型对照组比较,氟西汀组、脉冲电针组和音乐电针组大鼠血清t-PA/PAI-1明显上升(P<0.05),且音乐电针组明显高于脉)中电针组(P<0.05),表明mBDNF比例较高,有较好的神经元保护作用。
     4.用免疫组化法检测GFAP阳性细胞平均光密度,RT-PCR法检测海马GFAP、EAAT1及EAAT2mRNA含量。与空白对照组比较,模型对照组GFAP阳性细胞平均光密度明显降低(P<0.01);与模型对照组比较,氟西汀组、脉冲电针组、音乐电针组GFAP阳性细胞平均光密度明显增加(P<0.05)。音乐电针组GFAP阳性细胞平均光密度高于脉冲电针组但无统计学意义(P>0.05),高于氟西汀组有显著性差异(P<0.05)。慢性应激抑郁模型组海马EAAT1mRNA的表达明显降低,与空白对照组比较有显著性差异(P<0.01);与模型对照组比较,氟西汀组有升高趋势但无显著性差异(P=0.059);与模型对照组比较,脉冲电针组、音乐电针组EAAT1mRNA表达明显上升,结果有显著性差异(P<0.05)。慢性应激抑郁模型组海马EAAT2mRNA的表达明显降低,与空白对照组比较有显著性差异(P<0.01);与模型对照组比较,氟西汀组有升高趋势但无显著性差异(P>0.05);与模型对照组比较,脉冲电针组、音乐电针组EAAT2mRNA表达明显上升,结果有显著性差异(P<0.05)。
     5.高效液相色谱法检测海马Glu含量,RT-PCR法检测海马G1uR1、G1uR2、NR1及NR2B mRNA含量。慢性应激抑郁模型对照组海马Glu含量明显上升,G1uR1、G1uR2mRNA表达明显降低,NR1、NR2B mRNA表达明显升高,与空白对照组比较有显著性差异(P<0.05)。氟西汀可升高G1uR2mRNA的表达并降低NR1、NR2B mRNA的表达(P<0.05),对G1uR1mRNA表达的影响没有统计学意义(P>0.05)。脉冲电针和音乐电针可显著升高G1uR1、G1uR2mRNA的表达并降低NR1、NR2B mRNA的表达(P<0.05)。音乐电针在改善G1uR1、G1uR2、NR1mRNA表达方面优于氟西汀及脉冲电针(P<0.05)。
     结论
     1行为学实验结果提示本实验慢性应激刺激结合孤养造模成功。氟西汀及两种电针干预均可改善慢性应激抑郁大鼠行为学表现。其中音乐电针在改善慢性应激抑郁大鼠自主活动、探究性方面有优于氟西汀及脉冲电针的趋势,但无统计学意义。在改善大鼠体质量增加减缓、快感缺失和奖励机制敏感性方面音乐电针明显优于脉冲电针。
     2氟西汀、脉冲电针、音乐电针均可改善慢性应激刺激及孤养对大鼠海马神经元的损害作用,对海马神经元细胞有一定的保护作用。其中音乐电针对慢性应激抑郁大鼠海马CA1区神经元的保护作用优于脉冲电针,且有优于氟西汀的趋势,但无显著性差异。
     3在改善慢性应激抑郁模型大鼠海马内BDNF含量方面,音乐电针优于氟西汀及脉冲电针,在改善血清中BDNF含量方面,音乐电针优于氟西汀,与脉冲电针疗效相当。在提高t-PA/PAI-1比值,进而提高plasmin活性、促进pro-BDNF水解为mBDNF方面音乐电针优于脉冲电针
     4三种干预方法均可显著改善慢性应激抑郁模型大鼠海马内GFAP蛋白及mRNA的表达,从而起到改善慢性应激抑郁大鼠行为学的作用,其中脉冲电针及音乐电针在提高GFAP蛋白及mRNA方面均优于氟西汀,两种电针之间疗效相当。在提高EAAT1、EAAT2mRNA表达方面,脉冲电针、音乐电针有显著作用且效果相当,氟西汀组有上升趋势但无统计学差异。因此,在改善GFAP及EAAT1、EAAT2mRNA表达方面,脉冲电针、音乐电针优于氟西汀。
     5慢性应激刺激结合孤养能使大鼠海马内Glu含量倍增,GluRl、G1uR2mRNA表达降低,NR1、NR2B mRNA表达升高,氟西汀、脉冲电针、音乐电针能逆转这种改变,降低谷氨酸浓度,从而保护神经元,其中音乐电针在改善Glu及GluR1、GluR2、NR1mRNA表达方面优于氟西汀及脉冲电针。
Objective
     Depression is one of the most common diseases in psychiatric apartment, which has the highest suicide rate. The incidence increased year by year. But the attendance rate is quite low. Suicide and self-inflicted caused by Depression give lots of pain to the families. And it is a large burden for the society. The pathophysiology of depression is not fully understood. The medicines are mainly focused on monoamine neurotransmitter, but it not goes well. Acupuncture is the most popular treatment for the depression for the past few years. A lot of researches proved it can'treat depression in several ways. In our study, the treatment methods are the pulse electro-acupuncture and music electro-acupuncture. The rat is chronic unpredictable mild stress model. In order to find a new target for antidepressant, compare the two kinds of electro acupuncture efficacy.
     Method and Result
     1Using open field test, weight test, sugar preference evaluate the model of depression. The open cross, open rear, weight gain, sugar preferences in model group, which are lower than blank control group (P<0.01). This also shows the model of depression is made successfully. The treatment groups are higher than model group (P<0.05). In sugar preferences, the Music electro acupuncture is better than pules acupuncture.
     2Using nissl body test evaluate the effect of three treatments. Model control group is lower than blank control group, Three treatments all can protect the neure in the hippocampus. Music electro acupuncture is better than pules acupuncture.
     3Using immunohistochemical methods show the BDNF, and Elisa method show the BDNF, t-PA and PAI-1. Three treatments can stop the reducing of BDNF. Music electro acupuncture is better than Fluoxetine in hippocampus. Three treatments can raise the t-PA/PAI-1, and music elector acupuncture is better than pules acupuncture.
     4Using immunohistochemical methods show the GFAP, and RT-PCR show the CFAP, EAAT1and EAAT2mRNA. Three treatments all can raise the GFAP, and music electro acupuncture is better than Fluoxetine (P<0.05). Three treatments raise the EAAT1mRNA and EAAT2mRNA (P<0.05).
     5Using HPLC methods show the Glu, and RT-PCR show the G1uR1, G1uR2, NR1and NR2B mRNA. Fluoxetine raise G1uR2mRNA and reduce NR1, NR2B mRNA (P<0.05). Music electro acupuncture and pules acupuncture can raise GluR1,GluR2mRNA, reduce NR1, NR2B mRNA. Music electro acupuncture is better than pules acupuncture in GluR1, GluR2and NR1mRNA.
     Conclusion
     1Behavior results suggest the successful modeling. Fluoxetine and two kinds of electro-acupuncture can improve behavioral performance. Music electro acupuncture is better than the other two treatments in autonomous and exploratory movements. Music electro acupuncture is better than pules acupuncture in weight gain, pleasant sensation and reward mechanism.
     2The music electro acupuncture for the protective effect of chronic stress depression hippocampal CA1neurons, compared with pules electro-acupuncture, but no significant difference to fluoxetine.
     3Chronic stress stimulation combined the singly housed cause hippocampal CA1region and serum BDNF levels decreased, fluoxetine, pulse electro-acupuncture and music EA able to reverse the changes. In improving the brain BDNF content, Music electro-acupuncture is superior to fluoxetine and pulse electro-acupuncture. In improving the BDNF levels, music electro-acupuncture is superior to fluoxetine, and pulse electro-acupuncture treatment.
     4Three treatments can change the GFAP and mRNA in hippocampus. In Improving GFAP and EAAT1, EAAT2mRNA, pulse electro acupuncture and music EA are superior to fluoxetine.
     5The three intervention can reduce the contents of Glu increased GluR1GluR mRNA reduce NR1and NR2B mRNA. Music electro acupuncture is better than pules acupuncture and fluoxetine in Glu, GluR1, GluR2, NR1mRNA.
引文
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