基于CRFR-BDNF通路对前额叶及海马突触可塑性的调控探讨逍遥散改善焦虑的作用机制

【作者】 李琳；

【导师】 姜幼明；

【作者基本信息】 北京中医药大学 ， 中医养生康复学， 2024， 硕士

【摘要】 研究背景慢性束缚应激(chronic restraint stress,CRS)可诱导前额叶皮质及海马CA1区突触前释放谷氨酸神经递质,增强突触效能,产生长时程增强,进而诱发焦虑。BDNF在中枢神经系统突触功能和可塑性中起关键作用,与TrkB结合调控突触前谷氨酸释放和突触后谷氨酸受体表达。CRS后边缘系统CRFR1过量表达是焦虑的重要机制,前期研究发现,逍遥散通过抑制边缘系统CRFR1过度激活改善焦虑,而CRFR1与BDNF-TrkB信号通路密切相关,应激状态下,边缘系统CRFR1过度表达,激活BDNF启动子,导致BDNF转录、翻译和表达增加,进而过度激活BDNF-TrkB信号通路,引起突触效能增强,诱导焦虑发生。因此,研究逍遥散是否通过CRFR-BDNF通路调控突触可塑性改善焦虑具有重要意义。因此,本研究复制慢性束缚应激焦虑症小鼠模型,运用行为学、透射电镜、分子生物学实验等方法,探究逍遥散通过影响CRFR-BDNF通路对前额叶皮质和海马CA1区突触可塑性的调控,从而改善焦虑的作用机制。研究目的本实验通过观察CRFR-BDNF通路在前额叶皮质及海马突触可塑性调控中的作用,探究逍遥散改善焦虑的作用机制。研究方法1.构建焦虑症模型:将40只小鼠随机分为空白对照组(con)、模型组(crs)、逍遥散组(xys)、阳性对照药组(llxp),每组10只,除con组外,其余3组连续14天给予慢性束缚应激,并于束缚后0.5h进行灌胃,xys组以0.65g/kg,0.2mL/10g灌胃逍遥散悬液,llxp组以0.46mg/kg,0.2mL/10g灌胃劳拉西泮悬液,con组和crs组灌胃同积体的蒸馏水,连续观察小鼠一般状态及体质量变化,并在第0,7,14天运用糖水实验评价小鼠快感缺失状态,第15-17天,运用高架十字迷宫实验、旷场实验、新环境进食抑制实验评价小鼠焦虑水平。2.形态学实验:运用透射电镜拍摄前额叶皮质和海马CA1区神经元及其内部线粒体结构、突触前膜内谷氨酸神经递质囊泡含量和突触数量。3.分子生物学实验:运用免疫组化、qRT-PCR检测小鼠前额叶皮质和海马CA1区CRFR1的蛋白及mRNA表达水平,运用Western blot检测小鼠前额叶皮质及海马CA1区BDNF、TrkB、p-TrkB表达水平,探究逍遥散对CRFR-BDNF通路表达水平的影响。研究结果1.模型评价及行为学实验:14天CRS成功复制焦虑症小鼠模型,符合模型评价标准。通过观察小鼠一般状态和体重变化,发现crs组小鼠毛发晦暗,束缚和灌胃时反抗程度激烈;xys组和llxp组小鼠毛发光亮,束缚和灌胃时嘶叫挣扎反抗程度较轻,各组小鼠体重未出现明显差异。行为学实验中,与con组相比,crs组小鼠糖水消耗量减少,开放臂进入时间和次数比、旷场中央区进入时间和次数均降低,进食潜伏期提高;与crs组相比,xys组和llxp组小鼠糖水消耗量升高,开放臂进入时间和次数比、旷场中央区进入时间和次数增多,进食潜伏期降低。2.形态学实验:透射电镜观察前额叶皮质和海马CA1区神经元和突触结构发现,(1)con组小鼠神经元体积较大,细胞膜光滑完整,细胞质清晰,神经核圆形,核膜光滑、完整,核仁可见;小鼠神经元内线粒体双层膜结构完整,线粒体嵴结构清晰可见;突触前膜内可见一定数量的谷氨酸神经递质囊泡及少量的GABA神经递质囊泡;可见一定数量的突触,突触结构清晰。(2)crs组小鼠神经元固缩,细胞膜卷曲,细胞结构清晰度较差,神经核固缩,形状不规则,核深染;小鼠神经元内线粒体双层膜结构破损,线粒体肿胀,线粒体嵴断裂消融;突触前膜内谷氨酸能神经递质囊泡增多;突触数量增多,突触颜色加深。(3)xys组小鼠神经元未见固缩,细胞膜光滑完整,细胞质较为清晰,神经核圆形,核膜光滑、完整,核仁可见。小鼠线粒体双层膜结构完整,线粒体嵴结构较为完整清晰;突触前膜谷氨酸神经递质囊泡含量减少;突触数量相对减少,突触颜色较crs组相比略有减淡。3.分子生物学实验:(1)crs组小鼠前额叶皮质和海马CA1区CRFR1蛋白及mRNA的相对表达量较con组升高;与crs组小鼠相比,xys和llxp组小鼠CRFR1蛋白及mRNA的相对表达量降低。(2)crs组小鼠BDNF、TrkB、p-TrkB的相对表达量较con组升高,与crs组小鼠相比,xys和llxp组小鼠BDNF、TrkB、p-TrkB相对表达量降低。研究结论逍遥散通过抑制焦虑小鼠前额叶皮质和海马CA1区CRFR1、BDNF、TrkB、p-TrkB的过度表达,减少前额叶皮质及海马CA1区中突触前膜谷氨酸含量,对慢性束缚应激诱导的焦虑症发挥改善作用。更多还原

【Abstract】 Objective:In this study,we investigated the mechanism by which Xiaoyao San improves anxiety through its modulation of the CRFR-BDNF pathway in regulating synaptic plasticity in the prefrontal cortex and hippocampus.Chronic restraint stress(CRS)can induce glutamate neurotransmitter release in the presynaptic cortex and hippocampal CA1 region,enhancing synaptic efficacy,promoting long-term potentiation,and inducing anxiety.BDNF plays a crucial role in synaptic function and plasticity within the central nervous system by modulating presynaptic glutamate release and postsynaptic glutamate receptor expression via TrkB binding.The upregulation of CRFR1 within the limbic system following CRS is an important mechanism underlying anxiety.Previous studies have demonstrated that Xiaoyao San alleviates anxiety by inhibiting overactivation of CRFR1 within the limbic system,which is closely associated with BDNF-TrkB signaling pathway.Under stressful conditions,overexpression of CRFR1 within the limbic system activates BDNF promoter leading to increased transcription,translation,and expression of BDNF.This subsequently triggers an overactivated BDNF-TrkB signaling pathway resulting in enhanced synaptic efficacy and induction of anxiety.Therefore,it is highly significant to investigate whether Xiaoyao San improves anxiety by modulating synaptic plasticity through regulation of the CRFR-BDNF pathway.In this study,we replicated the mouse model of chronic restraint stress anxiety disorder and employed behavioral,transmission electron microscopy,and molecular biology experiments to investigate Xiaoyao San’s modulation of synaptic plasticity in the prefrontal cortex and hippocampal CA1 region through its impact on the CRFR-BDNF pathway,thereby ameliorating anxiety.Methods:1.Construction of anxiety model:Forty mice were randomly divided into four groups,including a blank control group(con),a model group(crs),a Xiaoyao San group(xys),and a positive control drug group(llxp),with ten mice in each group.Except for the Con group,the other three groups were subjected to chronic restraint stress for 14 consecutive days and then orally administered with either Xiaoyao San suspension at 0.65g/kg and 0.2mL/10g,lorazepam suspension at 0.46mg/kg and 0.2mL/10g for llxp or distilled water as conglomerate for both con and crs groups after being restrained for half an hour daily during this period of time.The general state,body weight changes,as well as anhedonia states of mice were continuously observed on day 0,day 7,and day 14 by sucrose preference test(SPT)while elevated plus-maze test(EPM),open-field test(OFT),novelty-suppressed feeding test(NSFT)were conducted from day15-17 to evaluate their anxiety levels accordingly.2.Morphological experiments:Transmission electron microscopy was used to photograph neurons along with their internal mitochondrial structure in prefrontal cortexes and hippocampal CA1 regions;glutamate neurotransmitter vesicles content;number of synapses in presynaptic membranes.3.Molecular biology experiments:Immunohistochemistry and qRT-PCR detected protein/mRNA expression levels of CRFR1 in prefrontal cortexes and hippocampal CA1 regions while Western blot detected BDNF/TrkB/p-TrkB expression levels therein so that we could explore how Xiaoyao San affects CRFR-BDNF pathway’s expression level.Results:1.Model evaluation and behavioral experiments:The 14-day CRS successfully replicated the anxiety mouse model,meeting the criteria for model evaluation.By observing the overall condition and weight changes of mice,it was observed that mice in the crs group exhibited dark hair color and displayed fierce resistance during restraint and gavage procedures.In contrast,mice in the xys group and llxp group had bright hair color and showed mild resistance during these procedures.There were no significant differences in weight among the groups.In behavioral experiments,compared to the con group,mice in the crs group demonstrated reduced sucrose consumption,decreased time spent entering open arms as well as a lower frequency ratio of open arm entries,decreased time spent entering central area of an open field test along with a lower frequency of such entries,and increased feeding latency.Furthermore,when compared to the crs group,mice in both xys and llxp groups exhibited increased sucrose consumption,increased time spent entering open arms as well as a higher frequency ratio of open arm entries,increased time spent entering central area of an open field test along with a higher frequency of such entries,meanwhile they showed decreased feeding latency.2.Morphological experiment:Transmission electron microscopy observation revealed that neurons in the prefrontal cortex and hippocampal CA1 area exhibited distinct morphological characteristics under different experimental conditions.In the con group,neurons displayed larger volumes with smooth and intact cell membranes,clear cytoplasm,round nuclei with smooth nuclear membranes,and visible nucleoli.The mitochondrial bilayer structure was complete,along with clearly visible mitochondrial cristae.Additionally,presynaptic membranes contained a certain number of glutamate neurotransmitter vesicles and a small amount of GABA neurotransmitter vesicles.Synapses were also observed with clear synaptic structures.In contrast,mice in the crs group showed neuronal pyknosis characterized by condensed nuclei,curling cell membranes leading to poor structural clarity,deep staining of irregularly shaped nuclei,damaged mitochondrial bilayer structure accompanied by swelling and broken ablation of mitochondria cristae.Moreover,an increase in glutamate neurotransmitter vesicles within the presynaptic membrane as well as an increased number of synapses with deeper synaptic coloration were observed.Interestingly,mice in the xys group did not exhibit neuronal pyknosis but rather displayed similar morphological features to those seen in the control group including smooth and intact cell membranes,clear cytoplasmic content surrounding round nuclei with smooth nuclear membranes and visible nucleoli.The mitochondrial bilayer structure remained intact while the mitochondrial cristae structure appeared relatively complete and clear compared to that observed in the crs group.Notably reduced levels of glutamate neurotransmitter vesicles within presynaptic membranes were detected alongside a relative reduction in synapse numbers which exhibited slightly lighter colors compared to those found in the crs group.3.Molecular biological experiments:The relative expression levels of CRFR1 protein and mRNA in the prefrontal cortex and hippocampal CA1 region of mice in the crs group were higher compared to those in the con group.However,a decrease was observed in the relative expression levels of CRFR1 protein and mRNA in both the xys and llxp groups when compared to the crs group.In the crs group,there was an increase in the relative expression levels of BDNF,TrkB,and p-TrkB compared to the con group.Conversely,a decrease was observed in these relative expression levels for both xys and llxp groups when compared to the crs group.Conclusions:Xiaoyao San can inhibit the over-expression of CRFR1,BDNF,TrkB and p-TrkB in the prefrontal cortex and hippocampal CA1 region of anxiety mice,reduce the content of glutamate in the presynaptic membrane in the prefrontal cortex and hippocampal CA1 region,and improve the anxiety induced by chronic restraint stress.更多还原