【作者】 陈立兵；

【导师】 葛卫红；

【作者基本信息】 浙江中医药大学 ， 中药学， 2019， 博士

【摘要】 目的1.探索建立基于慢性不可预知应激法(Chronic Unforeseeable Stress,CUS)首发造模和盐酸丙咪嗪治疗和康复后复发抑郁模型并固化造模方法;2.探索首发抑郁模型大鼠和复发抑郁模型大鼠的神经生物学变化,并基于此探寻抑郁复发易感性的预测因子;3.探讨星形胶质细胞的降解剂硫酸软骨素酶(Chondroitinase-ABC,ChABC)对抑郁模型大鼠复发易感性的作用,同时,通过反应性星形胶质细胞(Reactive astrocytes,RA)的活化和抑制的研究和相关蛋白因子表达变化研究,进一步探讨RA增加抑郁复发易感性的机制;4.研究芍药苷联合ChABC对复发抑郁模型大鼠的治疗作用,初步探索芍药苷联合ChABC的作用机制,并探求建立芍药苷联合ChABC用药的最佳方案。方法1.研究复发抑郁模型大鼠的神经生物学变化并探寻抑郁复发易感性的预测因子。首先,取160～180 g大鼠,预养1 w后,给予持续3 w慢性不可预知应激,制备首发抑郁模型;然后,在首发抑郁模型的基础上,模型动物每天腹腔注射盐酸丙咪嗪进行为期4 w的治疗,治疗期间,于每次给药1 h后给予CUS应激;治疗期结束后,给予2 w的康复期,期间动物不接受任何应激或处置;最后,在康复期结束后,再次给予CUS应激,持续2 w制备大鼠复发抑郁模型。期间,分别于第3 w后、第7 w后和第11 w后,进行动物行为学检查,采用体重变化、糖水消耗实验、游泳实验、旷场实验等方法考察比较抑郁模型大鼠首发期和复发期的行为学改变,利用Y迷宫新奇事物探索实验考察抑郁模型大鼠首发期和复发期抑郁模型大鼠的认知能力区别。采用透射电镜法观察抑郁模型大鼠首发期和复发期海马区神经元超微结构改变;利用冰冻切片、免疫荧光法和激光共聚焦技术检测两个时期各组大鼠海马区胶质原纤维酸性蛋白(Glial Fibrillaryacidic Protein,GFAP)和神经元核(Neuronal Nuclear,Neu N)表达,以观察复发抑郁模型大鼠的海马区星形胶质细胞结构改变及神经元表达情况。运用酶联免疫吸附试验(Enzyme-Linked Iimmunosorbent Assay,ELISA)检测大鼠血清促肾上腺皮质激素(Adrenocorticotropic Hormone,ACTH)、皮质酮(Corticosterone,CORT)含量,考察复发抑郁模型大鼠下丘脑-垂体-肾上腺轴的改变。运用ELISA、Western blot技术检测大鼠血清或海马齿状回(Dentate Gyrus,DG)区环磷酸腺苷(Cyclic Adenosine Monophosphate,c AMP)、脑源性神经营养因子(Brain-Derived Neurotrophic Factor,BDNF)、反应元件结合蛋白(c AMP-Response Element Dinding Protein,CREB)和纤维母细胞生长因子2(Fibroblast Growth Factor 2,FGF2)蛋白表达,考察复发抑郁模型大鼠脑神经内分泌改变,明确复发抑郁模型大鼠脑内神经细胞结构和功能改变。比较首发和复发抑郁模型大鼠行为学改变及神经细胞结构和功能改变,试图寻找两者神经内分泌改变,找到有效的防止抑郁复发的病理靶标。2.RA降解剂ChABC降低抑郁模型大鼠复发易感性研究。制备复发抑郁模型,并在康复期(第7 w造模结束时),ChABC组大鼠脑微量注射给予ChABC,复发模型组和假手术组大鼠给予同体积的生理盐水,其余同上。比较复发模型组和ChABC组大鼠行为学改变,考察ChABC对抑郁复发易感性的影响。利用冰冻切片及免疫荧光技术测定大鼠海马区硫酸软骨素蛋白多糖(Chondroitin Sulfate Proteoglycans-1,CSPG1)、突触素-Ⅰ(synapsin-Ⅰ)、及溴脱氧尿苷(Bromo-2-deoxyuridine,Brd U)及GFAP表达,观察神经元再生、突触形成及反应性星形胶质细胞的相关性,初步明确RA引起复发易感性作用机制。3.芍药苷联合ChABC对复发抑郁模型大鼠的治疗作用研究。采用复发抑郁模型,考察不同时间点给予芍药苷与ChABC联合用药对复发抑郁模型大鼠行为学影响,选择最优治疗时间窗,并检测最优联合治疗组的大鼠海马区Brd U的表达,模拟促神经再生和去RA的联合治疗过程,运用Western blot技术检测磷酸化信号转导与转录激活因子(Phosphorylated Signal Transducer and Activator of Transcription 3,p-STAT3)神经神经营养因子BDNF、脑源性神经营养因子前体(Precursor of Brain-Derived Neurotrophic Factor,pro BDNF)及其受体p75神经营养因子受体(p75 Neurotrophin Receptor,p75NTR)蛋白表达,考察其在联合用药治疗抑郁复发的作用机制。结果1.和同期正常组比较,(1)首发抑郁模型和复发抑郁模型大鼠体重、糖水偏好指数、Y迷宫新臂停留时间和水平穿越格子数均明显下降,游泳不动时间和大便粒数明显上升,说明复发和首发抑郁模型具有明显的抑郁样行为和存在认知能力下降;且从多个指标的P值上看复发模型大鼠比首发抑郁模型大鼠具有更严重的抑郁样行为和认知障碍。(2)首发和复发抑郁模型大鼠海马神经元超微结构呈现线粒体、突触等损伤现象,两者损伤程度相似。(3)首发模型组大鼠海马区DG区GFAP阳性细胞荧光强度明显降低;复发抑郁模型大鼠海马区星形胶质细胞呈现胞体膨胀、肥大,突触增厚、浓染或重叠等活化状态,表现为荧光强度增加。(4)首发模型组大鼠海马区Neu N阳性细胞数明显下降,而复发模型组未见明显差异。(5)复发模型组大鼠血清CORT和ACTH表达明显升高,下丘脑-垂体-肾上腺轴(Hypothalamic-Pituitary-Adrenal Axis,PHA)明显上调呈激活状态。(6)复发模型组大鼠c AMP、CREB和BDNF蛋白表达降低,神经营养因子及其相关信号通路c AMP-CREB-BDNF信号通路明显受到抑制。(7)首发抑郁模型大鼠多个指标表达和复发模型大鼠的变化趋势一致,但星形胶质细胞表达上呈现差异,前者负表达,后者正表达。2.(1)与假手术组比较,复发模型组大鼠经过2 w再应激后,体重、糖水偏好指数明显下降、游泳不动时间明显上升,出现抑郁样行为;而ChABC组大鼠在同样持续应激条件下相应指标没有显著差异,未出现抑郁样行为;ChABC组在大鼠糖水偏好指数和游泳不动时间与复发模型组比较有显著差异。(2)与假手术组比较,复发模型组大鼠海马DG区CSPG1和GFAP蛋白表达均明显升高,而Synapsin-Ⅰ和Brd U的蛋白均明显降低;ChABC可以逆转复发抑郁模型组大鼠海马DG区CSPG1、GFAP、Synapsin-Ⅰ蛋白表达的变化,但对Brd U表达的影响未见显著性差异。3.(1)与复发模型组比较,不同给药时间点芍药苷与ChABC联合用药,各组大鼠糖水偏好指数和游泳不动时间具有显著差异;与ChABC单用组比较,芍药苷1组(即再应激前1 w开始给予芍药苷治疗)对复发抑郁模型大鼠糖水偏好指数有明显提升作用,但游泳不动时间没有明显改变,说明两者联合用药的抗抑郁复发效果具有一定增强作用。通过糖水偏好指数指标发现几组芍药苷联合ChABC的抗抑郁效果,以芍药苷1组最好,而芍药苷2组(ChABC组大鼠再应激开始同时给予芍药苷治疗)和3组(ChABC组大鼠再应激1 w后开始给予芍药苷治疗)的治疗效果没有明显增强作用。(2)与复发模型组比较,芍药苷1组的大鼠海马区Brd U阳性细胞数明显升高,逆转复发模型大鼠脑区神经再生低表达,而ChABC对神经再生没有明显作用。(3)与假手术组比较,复发模型组大鼠p-STAT3及p75NTR呈现高表达,而pro BDNF蛋白表达没有显著性差异。结论1.复发模型大鼠具有明显的抑郁样行为,存在认知能力和海马神经元超微结构损伤现象,HPA轴内分泌明显上调,BDNF及其关联信号通路相关蛋白表达因子明显受到抑制。复发较首发造模更容易成模,造模时间缩短了1 w,呈现抑郁复发易感性。2.首发和复发抑郁模型大鼠存在神经内分泌及结构差异,多数指标表达呈正向相关;但首发抑郁模型大鼠星形胶质细胞低表达,复发抑郁模型星形胶质细胞高表达,呈现活化状态;活化的星形胶质细胞(RA)可以作为抑郁复发预测因子。3.RA降解剂ChABC具有降低抑郁复发易感性作用;RA可能通过增加复发模型大鼠海马DG区CSPG1表达,影响神经元突触可塑性及抑制神经再生介导抑郁复发易感性。4.芍药苷与ChABC两者联合用药的抗抑郁复发效果优于ChABC单用,而且于再应激前1 w开始给予芍药苷联合ChABC进行治疗的抗抑郁复发效果最好,两者增效作用可能是ChABC通过减轻星形胶质细胞的过度活化与芍药苷促神经再生达到联合治疗复发抑郁作用;且两者联合用药可以抑制复发模型大鼠p75NTR蛋白表达。5.研究将有助于发现抑郁症易复发的新神经生物学指标和阐明其神经细胞分子病理学机制,为有效防治抑郁症复发提供对策,为进一步药物结合RA治疗复发抑郁症提供联合治疗方法。更多还原

【Abstract】 Objectives1.To explore the modeling method for rats with recurrent depression basing on the treatment with chronic unpredictable stress（CUS）and imipramine hydrochloride for the first-episode depression rats;2.To explore the neurobiological changes of the first-episode depression and recurrent depression model in rats,and then to explore predictors of depression relapse susceptibility based on founds of the neurobiological changes;3.To investigate the effects of chondroitin sulfate（ChABC）,a depressant of astrocytes,on relapse susceptibility in depression model rats.Meanwhile,the mechanism of RA increased depression relapse susceptibility was further explored by studying the activation and inhibition of reactive astrocytes（RA）and the changes in the expression of related protein factors.4.To explore the therapeutic effects of paeoniflorin combined with ChABC on Rats with relapsed depression,preliminarily explore the mechanism of paeoniflorin combined with ChABC,and try to establish a good regimen for paeoniflorin combined with ChABC,furthermore,try to find the optimal medication time window.MethodsPart Ⅰ:This part of the page is to study the neurobiological changes of recurrent depression model rats and to search for predictors of recurrent depression susceptibility.The first-episode depression model Rats were prepared by chronic unpredictable stress（CUS）,and the relapse depression model Rats were prepared by CUS again after treatment and recovery with imipramine hydrochloride.And then,the behavioral changes of depression model Rats between first-episode depression model and relapse depression model Rats were investigated by sugar water consumption experiment,swimming experiment,open field experiment and body weight measurement,et al.Meanwhile,the difference of cognitive ability was investigated by Y maze novelty exploration experiment,the ultrastructural changes of neurons in the hippocampus were observed by transmission electron microscopy（TEM）.The expression of chondroitin sulfate proteoglycans 1（CSPG1）,glial fibrillary acidic protein（GFAP）and Neuronal nuclear（Neu N）in hippocampal area of Rats in different periods were detected by frozen section,immunofluorescence and laser confocal technique,so as to observe the structure and function changes of astrocytes and the expression of neurons in relapse depression model Rats.The contents of cortisol（CORT）and adrenocorticotropic hormone（ACTH）in serum were measured by enzyme-linked immunosorbent assay method（ELISA）to investigate the changes of hypothalamic-pituitary-adrenal axis（HPA）in relapse depression model Rats.The expressions of cyclic adenosine monophosphate（c AMP）,c AMP-response element binding protein（CREB）,Brain derived neurotrophic factor（BDNF）and Fibroblast growth factor 2（FGF2）in both the serum and the hippocampus DG area of Rats were detected by ELISA and Western blot,in order to investigate the neuroendocrine changes and the structure and function changes of neurons in the brain of relapse depression model Rats.We attempt to find out the neuroendocrine changes between first-episode depression model and relapse depression model Rats by comparing the behavioral changes and the structure,function changes in both of the depression model rats,so as to find effective drug targets which prevent recurrence of depression.Part Ⅱ:Study on the mechanism of ChABC in reducing the susceptibility to the recurrence of depression.The relapse depression model Rats were prepared by the methods described in PartⅠ.And then,Rats in the ChABC group were given ChABC by microinjection in the brain,while Rats in the relapse model group were given normal saline of the same volume,during the rehabilitation period.The rest treatments were same as PartⅠ.Behavioral changes of rats in the relapse model group and ChABC group were compared,to investigate the influence of ChABC on the susceptibility to depression recurrence.The expressions of CSPG,synaptophysin,Bromo-2-deoxyuridine（Brd U）and GFAP in the hippocampus of rats were determined by frozen section and Immunofluorescence（IF）technique,so as to observe the correlation of neuron regeneration and synaptic formation with reactive astrocytes,and to preliminarily identify the mechanism of the recurrent susceptibility of depression.Part Ⅲ:The relapse depression model Rats were prepared and adopted to investigate the effect of paeoniflorin combined with ChABC on the behavior of Rats.The optimal treatment time window was selected by behavioral examination,and then,to simulate the combined treatment process of nerve regeneration and dereactive astrocytes by detecting the expression of Brd U and CSPG in the hippocampal area of Rats in the optimal combined treatment group.The expression of precursor of brain-derived neurotrophic factor（pro BDNF）and its receptor p75 Neurotrophin receptor（p75NTR）were detected by Western Blot,so as to investigate the mechanism of NOTCH signaling pathway,which is closely related to axon or synaptic regeneration and astrocytes,in the combined treatment of relapse depression.ResultsPart Ⅰ:Compared with the control group,firstly,the weight,sugar preference index,new arm dwell time,non-swimming time,horizontal crossing lattices of both the first episode depression model and relapse depression model Rats were all significantly decreased.Meanwhile,the number of fecal grains was significantly increased.The results showed that the models of both first-episode and relapse depression had significant depression-like behavior and cognitive decline.Moreover,depression-like behavior and tendence of cognitive decline were more obvious in the relapse model Rats than in first-episode depression model Rats from the p-value of multiple indicators.The ultrastructure of hippocampal neurons in both first episode depression model and relapse depression model Rats showed mitochondrial and synaptic damage,and the degrees of damage were similar to each.The fluorescence intensity of GFAP positive cells in the DG area of the hippocampus was significantly decreased in the first-episode depression model Rats.It showed the hippocampal astrocytes were reactived in relapse depression model Rats,the cytons are dilating and swelling out,its axons were thickened,hyperchromatism or overlapped,manifest as the fluorescence intensity and cell area of RA increased.There was no significant change in the number of Neu N positive cells in the hippocampus of the rats neither in the first model group or the relapse model group.The expression of corticosterone（CORT）and adrenocorticotropic hormone（ACTH）in serum CORT and ACTH were significantly increased in the relapse model group,and the hypothalamic-pituitary-adrenal axis（PHA）was obviously rised.The expressions of c AMP,CREB and BDNF proteins were decreased in the relapse model group,the neurotrophic factors and its related signaling pathway camp-creb-bdnf pathway were significantly inhibited.Seventhly,the interesting thing is that there was a positive correlation between the expression of various indicators in the first-episode depression model and the relapse depression model Rats while there was a negative correlation in the expression and function of astrocytes.Part Ⅱ:Compared with the sham-operated Group,firstly,the Rats in the relapse model Group showed depression-like behaviors after 2 weeks of CUS,such as significant difference in loss of weight,decline of sugar water preference index,rise of non-swimming time,while the Rats in ChABC Group showed no obvious depression-like behaviors or significant difference in such index.However,there were significant differences between the ChABC Group and the relapse model Group in the sugar water preference index and non-swimming time.Secondly,the protein expressions of CSPG1 and GFAP in the Rats’hippocampus of the relapse depression model Group were significantly increased,while Synapsin-Ⅰand Brd U were significantly decreased.ChABC could reverse the changes in CSPG1,GFAP and Synapsin-Ⅰof protein expressions,but not Brd U.Part Ⅲ:Compared with relapse depression model Group,Rats in the Paeoniflorin combined with ChABC Group 1 showed significant differences in sugar water preference index and non-swimming time.While compared with the ChABC group,the Groups of paeoniflorin combined with ChABC showed significant rise of sugar water preference index,but no significant differences in non-swimmingt time,indicating that the combined use of the two drugs had a certain enhancing effect on the anti-depression recurrence.It showed that the optimal combined treatment time window was at the 8^th week,the therapeutic effect of paeoniflorin combined with ChABC at 9^th and 10^th week showed no significant differences with ChABC Group from the changes of sugar water preference index.Compared with Rats in the relapse depression model group,the number of Brd U positive cells in the hippocampal area of rats in Paeoniflorin+ChABC Group 1 was significantly increased,which reversed the nerve regeneration,while treat with ChABC only had no significant effect on nerve regeneration.Compared with the sham-operated Group,the expression of p-STAT3 and p75NTR in the relapse depression model Group was enhanced,but the expression of pro BDNF had no significant difference.Compared with Rats in the relapse depression model group,the ChABC reduced the expression of p-STAT3 and promoted the expression of BDNF m RNA in the hippocampus.The paeoniflorin combined with ChABC could inhibit the expression of p75NTR protein in Rats of relapse depression model Group.ConclusionsFirstly,the Rats in relapse depression model showed obvious depression-like behavior,such as decreasing of cognitive ability,demagement of the ultrastructure of hippocampal neurons,significantly up-regulated neuroendocrine in the HPA,and significantly inhibition in expression of BDNF and protein expression factors in related signaling pathways.Compared with the first episode,it was easier to get the relapse model Rats,the relapse time was shortened by one week,which showed an obvious susceptibility to relapse depression.Secondly,there were significant differences in neuroendocrine and ultrastructure of neurons between the first-episode and the relapse depression model Rats,and the expressions of multiple objects were positively correlated.However,the expression of astrocytes in rats of first episode model was low while relapse model was high.It showed that the astrocytes were activated in relapse model Rats,the activated astrocytes might be used as a predictor of depression recurrence.Thirdly,the RA depressant ChABC might reduce the susceptibility to relapse of depression,and RA might mediate and enhance the susceptibility to relapse of depression by increasing the expression of CSPG1 in the hippocampal DG region,affectting the synaptic plasticity of neurons and inhibitting nerve regeneration,et al.Fourthly,treated with paeoniflorin that combined with ChABC one week before Re-CUS,might have the best effect of anti-relapse of depression.The possible mechanism of combined treatment of paeoniflorin and ChABC for relapse of depression mingt be that ChABC alleviated the overactivation of astrocytes,while paeoniflorin could pomote nerve regeneration and protect nerve.Moreover,it might be related to the inhibition of p75NTR protein expression in relapse depression Rats.Fifthly,we hypothesized that RA might be a predictor of relapse susceptibility,based on this study.Furthermore,this study might be helpful for searching new neurobiological indicators of relapse of depression and elucidating its molecular pathological mechanism of relapse,and providing countermeasures for the effective prevention and treatment of relapse depression.However it also provided new therapeutic schemes for relapse depression based on function of RA.更多还原