【作者】 富文俊；

【导师】 徐志伟；

【作者基本信息】 广州中医药大学 ， 中医基础理论， 2009， 博士

【摘要】 1、研究目的慢性应激发生时,HPA轴兴奋性提高,促肾上腺皮质激素释放激素(Cortico-trophin releasing hormone,CRH)分泌增多,引起糖皮质激素水平升高,从而动员储能,适应应激反应。若机体长期处于应激状态HPA轴功能持续亢进,高皮质酮/醇血症将对大脑海马神经元细胞造成严重损伤,继发引起海马神经元内N-甲基-D-天门冬氨酸受体(NMDAR)过度激活及海马神经元内糖皮质激素受体(iGR)表达减少或其活性降低,引起HPA轴负反馈功能减弱,而这将进一步加重高皮质酮/醇血症,形成一恶性循环。因此慢性应激损伤的一个重要环节即为高皮质酮/醇血症,本论文即以此为切入点,通过地塞米松抑制试验(DEXT)检验慢性应激模型是否成功,用逍遥散和GR-受体阻滞剂干预慢性应激损伤大鼠,观察其大脑室旁核区促肾上腺皮质激素释放激素(CRH)mRNA、海马神经元细胞内内糖皮质激素受体(iGR)及NR各亚型的表达状况,试图从NMDAR、GC/GR(iGR)、HPAA这一传导通路解释逍遥散对慢性应激的调控机理。2、研究方法复制慢性不可预知应激大鼠模型,运用地塞米松抑制试验加以验证,以糖皮质激素受体阻滞剂RU-38486及中药复方逍遥散干预,探讨逍遥散对慢性应激损伤大鼠的调控作用及机理:1)在前期试验基础上,复制慢性不可预计应激模型;2)取地塞米松抑制试验的大鼠血清,应用酶联免疫吸附剂测定(Enzyme LinkedImmunosorbnent Assay ELISA)检测其血清皮质酮水平;3)取慢性应激大鼠模型的各组大鼠脑部石蜡切片,以原位杂交法检测其下丘脑室旁核促肾上腺皮质激素释放激素(Corticotrophin releasing hormone,CRH)mRNA的表达水平;4)取慢性应激大鼠模型的各组大鼠脑部石蜡切片,以荧光免疫组化的方法检测其海马区糖皮质激素受体(intracellular GLucocorticoid receptor,iGR)的表达水平;5)取慢性应激大鼠模型的各组大鼠脑部石蜡切片,以荧光免疫组化的方法检测其海马区N-甲基-D-天门冬氨酸(NMDA)受体部分亚型(NR1、NR2A、NR2B)的表达水平。3、研究结果1)在地塞米松抑制试验中,单纯慢性应激损伤组大鼠血清皮质酮水平未见明显下降,其地塞米松抑制试验呈阴性,GR受体阻滞剂RU-38486组与中药复方逍遥散组大鼠的血清皮质酮水平明显下降,其地塞米松抑制试验呈阳性;2)慢性应激损伤模型组大鼠的下丘脑室旁核CRH mRNA表达明显增多,海马神经元细胞内糖皮质激素受体(iGR)阳性表达明显降低,海马区NR1、NR2A、NR2B阳性表达明显升高;3) GR受体阻滞剂RU-38486组与中药复方逍遥散组大鼠的CRH mRNA阳性表达水平明显下降;4) GR受体阻滞剂RU-38486组与中药复方逍遥散组大鼠的海马神经元细胞内糖皮质激素受体(iGR)阳性表达水平显著上升;5) GR受体阻滞剂RU-38486组与中药复方逍遥散组大鼠的海马区NR1、NR2A、NR2B阳性表达水平显著下降。4、研究结论根据本文的研究我们可以推出以下结论:经地塞米松抑制试验检验,证实本实验所采用的慢性应激大鼠模型是成功的;逍遥散可以显著下调慢性应激大鼠海马神经元细胞中的NR1、NR2A、NR2B的表达,促进其海马神经元细胞内糖皮质激素受体(iGR)的阳性表达增加,降低其高皮质酮血症,促使其下丘脑室旁核区CRH mRNA表达下调,从而达到部分恢复慢性应激大鼠HPA轴负反馈功能的作用,起到缓减诸应激症状的疗效。更多还原

【Abstract】 Studies found that the acute stress has no distinctive impact to the construction of hippocampus.But under the station of chronic stress,the neurons of hippocampus would be atrophy even to death.The concrete mechanism is still unknown,but maybe the neural pathway of HPA- N-methyl -D-aspartic acid receptors(NMDAR) - GLucocorticoid receptor(GR)has played a significant role in the station.Under the physiological condi -tion,the pathway can exert a sere of biological effect on maintaining the excitable transmit between neurons and the excitability of neurons and brain.1、ObjectiveIf Chronic stress happen,HPA axis excitability would rise,excretion of Corticotrophin releasing hormone(CRH) would rise,corticosteroid level in blood would rise,body potential would be activated,then body can adapt the chronic stress.But if body under stress too long time,HPA axis ’function would keep exciting,high corticosteroid level would damnify brain hippocampus nerve cell badly,result in NMDAR of hippocampus nerve cell excess activation,GR especially iGR expression would reduce; HPA axis’ s feedback would be weaken,all of this would aggravate high corticosteroid level in blood.So,the high corticosteroid level in blood is a important sign of chronic stress.This paper based on this theory, verify the chronic stress model by the DEXT.Then we treat the model by Xiaoyaosan and RU-38486,observing the expression of CRH mRNA、GR(iGR)、NMDAR subfamily,studying the mechanism of Xiaoyaosan’ s regulation chronic stress modle.2、MethodsWe studying the mechanism of Xiaoyaosan’ s regulation on chronic stress model in the way that replicating the chronic stress model, verifying the chronic stress model through the DEXT,treating the model by Xiaoyaosan and RU-38486.1) detect the corticosteroid level in the serum of the rat with DEXT by Enzyme Linked Immunosorbnent Assay(ELISA);2) detect the expressions of Corticotrophin releasing hormone(CRH) mRNA in the brain paraffin sections of the chronic stress rat by insituhybridization;3) detect the expressions of intracellular GLucocorticoid receptor(iGR) in the brain paraffin sections of the chronic stress rat by fluorescence Immunohistochemsitry;4) detect the expressions of intracellular N-methyl-D-aspartic acid receptors subspecies(NR1、NR2A、NR2B) in the brain paraffin sections of the chronic stress rat by fluorescence Immunohistochemsitry.3、Result1) In our research,the disorder of the HPAA’ s function in the chronic stress rat include that the high corticosteroidlevel in blood,the high expressions of Corticotrophin releasing hormone(CRH) mRNA in hypothalamus paraventricular nucleus,the low expressions intracellular GLucocorticoid receptor(iGR) in brain hippocampus, the high expressions of intracellular N-methyl-D-aspartic acid receptors subspecies(NR1、NR2A、NR2B) in brain hippocampus;2) The level of corticosteroid in blood in group of the chronic stress rat doesn’t decilne in DEXT,by contraries,the level of corticosteroid in blood of the chronic stress rat in DEXT of Xiaoyaosan group and RU-38486 group decline obviously:3) The expression of the Corticotrophin releasing hormone(CRH) mRNA at hypothalamus paraventricular nucleus of the rat in single chronic stress group raise obviously;the expression of the Corticotrophin releasing hormone(CRH) mRNA at hypothalamus paraventricular nucleus of the rat in Xiaoyaosan group and RU-38486 group doesn’t ascend;4) The expression of intracellular GLucocorticoid receptor(iGR) at brain hippocampus of the rat in single chronic stress group decline obviously;the expression of intracellular GLucocorticoid receptor(iGR) at brain hippocampus of the rat in Xiaoyaosan group and RU-38486 group doesn’t decline;5) The expression of intracellular N-methyl-D-aspartic acid receptors subspecies(NR1、NR2A、NR2B) in brain hippocampus of the rat in single chronic stress group raise obviously;The expression of intracellular N-methyl-D-aspartic acid receptors subspecies(NR1、NR2A、NR2B) in brain hippocampus of the rat in Xiaoyaosan group and RU-38486 group doesn’t ascend.4、ConclusionBased on our research,we can make the flowing conclusion:chronic stress modle in our research is successful,Xiaoyaosan can depress the expressions of intracellular N-methyl-D-aspartic acid receptors subspecies(NR1、NR2A、NR2B) in brain hippocampus of the chronic stress rat,raise the expressions of intracellular GLucocorticoid receptor(iGR) in brain hippocampus,depress the level of corticosteroid in blood, depress the expression of the Corticotrophin releasing hormone(CRH) mRNA in hypothalamus paraventricular nucleus,partly resume the function of the HPAA on feedback of the chronic stress rat.Xiaoyaosan can effectively depress the excitability of HPA axis caused by the chronic stress,regulate function of HPA axis through which above to protect the neurons from damage,resume the feedback function of the HPA axis partly.更多还原