心理应激对大鼠咬肌肌紧张度及脑内神经递质变化的影响及药物干预研究

发布时间：2018-02-11 01:40

本文关键词： 心理应激束缚应激咬肌能量代谢自由基代谢三叉神经运动核神经递质　出处：《第四军医大学》2014年博士论文　论文类型：学位论文

【摘要】：随着我国经济的快速发展、社会的剧烈变革、人们生活方式的转变，来自于学业、工作、家庭等各方面的精神压力不断困扰着人们，由此造成的心理疾病和心身疾病的发病率逐年攀升。负面性的生活事件产生的心理应激对人体造成的影响不仅体现在精神疾病方面，大量的证据也表明心理应激刺激在颞下颌关节紊乱病、夜磨牙症等口颌系统疾病的发生和发展过程中都扮演了重要的角色；研究也证实长时间的应激刺激不仅会引起焦虑、抑郁等心理改变，还会导致口颌肌肉的病理变化，以及咀嚼肌的紧张度升高。心理应激环境下出现的咀嚼肌肌紧张度显著上升，可能不仅仅是应激触发的一种机体反应，同时也可能是心理应激导致颞下颌关节紊乱病产生或加重的内在病理过程。然而到目前为止，学界对这一现象内在机制的认识仍为空白，了解这一现象的内在机制，并据此建立有效的预防措施和合理的治疗方法对心理应激相关口颌系统疾病的治疗将产生深远的影响。基于以上原因，我们建立了大鼠慢性束缚应激模型，并采用肌肉电生理技术、高效液相色谱技术、Elisa、Western Blot、qRT-PCR等分子生物学技术，对长期束缚应激对大鼠咬肌及脑内相关核团的影响，及相应变化的内在机制进行了研究，同时对拉莫三嗪在上述病态变化中的治疗效果进行了评估。本研究共分为2大部分，具体内容如下：第一部分心理应激动物模型的建立及评价采用慢性束缚应激（chronic restraint stress，CRS）方法建立心理应激模型，将大鼠随机分入对照组、心理应激组、20mg/kg低剂量药物组、30mg/kg中剂量药物组和40mg/kg高剂量药物组，在束缚应激3周后，对大鼠体重进行检测，同时采用糖水偏好实验、旷场实验等行为学评估方法以及促肾上腺皮质激素（adrenocorticotropichormone, ACTH）、皮质酮（corticosterone, CORT）血清学激素测定对慢性束缚应激模型进行评估。实验结果显示，心理应激后大鼠体重较应激前显著下降（p＜0.05）；糖水偏好程度显著降低（p＜0.005），应激大鼠在旷场中移动总距离及移动速率显著降低（p＜0.005），中央停留时间显著延长（p＜0.005）；应激大鼠血清CORT（p＜0.005）和ACTH的含量显著升高（p＜0.005）。以上结果证实，本实验所建立的慢性束缚应激大鼠模型存在明显的应激相关的行为学和血清学特征，可以满足后续实验的要求。第二部分心理应激对大鼠咬肌肌紧张度及脑内神经递质变化的影响及药物干预研究 1.心理应激对大鼠咬肌能量代谢的影响及药物干预研究应激后大鼠咬肌内肌酸激酶（creatine kinase, CK）、乳酸脱氢酶（lactatedehydrogenase, LDH）和乳酸（lactate, LD）的含量显著升高（p0.005），磷酸肌酸（creatine phosphate, CP）含量显著降低（p0.005）；30和40mg/kg剂量的拉莫三嗪可以有效逆转应激引起的上述变化（p0.005）；20mg/kg剂量的拉莫三嗪则无明显效果(p＞0.005)。 2.心理应激对大鼠咬肌肌电图(electromyography, EMG)的影响及药物干预研究不论在清醒安静状态还是清醒活动状态下，应激后大鼠咬肌肌紧张度较应激前均显著升高(p＜0.05)；30和40mg/kg剂量的拉莫三嗪可以有效逆转应激后出现的肌紧张水平上升（p0.005），20mg/kg剂量的拉莫三嗪则无明显效果(p＞0.005)。 3.心理应激对大鼠脑内自由基代谢的影响及药物干预研究应激后大鼠脑中中央杏仁核(central amygdaloid nucleus, CeA)、小细胞网状结构(parvocellular reticular nucleus, PCRt)及三叉神经运动核（motor nucleus of trigeminalnerve, Vm)内谷胱甘肽过氧化物酶（glutathione peroxidase, GSH-Px）、过氧化氢酶（catalase, CAT）、超氧化物歧化酶（superoxide dismutase, SOD）的活性较对照组均出现显著下降（p0.005）；丙二醛（malondialdehyde, MDA）的含量则显著升高（p0.005）；30和40mg/kg剂量的拉莫三嗪可以有效逆转心理应激引起的上述变化（p0.005）；20mg/kg剂量的拉莫三嗪则无明显心理应激对抗效果(p＞0.005)。 4.心理应激对大鼠脑内神经递质变化的影响及药物干预研究应激后大鼠脑中上述核团内谷氨酸（glutamate，，Glu）含量、谷氨酰胺酶（glutaminase，GLS）活性、囊泡谷氨酸转运体蛋白1(vesicular glutamate transporter1，VGluT1)及囊泡谷氨酸转运体蛋白2(vesicular glutamate transporter2，VGluT2)的含量等均显著升高（p0.005），谷氨酰胺合成酶（glutamine synthetase，GS）的活性显著下降（p0.005）；杏仁核内NMDAR2a（N-methyl-D-aspartate receptor2a）表达水平显著升高（p0.005）；三叉神经运动核内NMDAR1（N-methyl-D-aspartate receptor1）表达显著升高（p0.005）；20、30和40mg/kg剂量的拉莫三嗪对上述变化无明显的治疗效果（p0.005）。通过本研究我们发现：慢性束缚应激会造成大鼠咬肌肌紧张度增高、咬肌能量代谢异常；而咬肌肌紧张度增高是由三叉神经运动核内谷氨酸的水平增高所引起；GLS和GS的活性改变可能是导致应激后三叉神经运动核内谷氨酸浓度升高的主要原因，VGluT1和VGluT2的表达上调可能对细胞外谷氨酸水平产生了影响，而NMDAR1的表达增加可能导致了NMDA受体的表达上调，继而导致神经元细胞对谷氨酸浓度的增加更为敏感，这些因素共同作用引发了三叉神经运动核内运动神经元的异常神经冲动，最终导致了咬肌肌紧张度的升高；此外应激还导致了三叉神经运动核组织的氧化应激损伤；应激后三叉神经运动核内谷氨酸浓度增加的部分原因可能源于来自于其上级神经元—小细胞网状结构及杏仁核的兴奋性神经递质的增加；30、40mg/kg剂量的拉莫三嗪针对谷氨酸浓度浓度的干预效应并不依赖于其对谷氨酸代谢酶、囊泡谷氨酸转运体蛋白及谷氨酸受体亚基的调节作用，而可能与拉莫三嗪抑制谷氨酸向突触间隙内的释放有关。本研究初步阐明了心理应激导致咬肌肌紧张度增高现象的内在原因，并且证实了拉莫三嗪对抗心理应激负效应的潜在治疗效果，丰富了心理应激导致口颌系统疾病的致病机理，为未来制定应激相关疾病的治疗方法提供了实验参考依据。
[Abstract]:The psychological stress caused by psychological stress is not only caused by psychological changes such as anxiety , depression and so on , but also can be the intrinsic pathological process of psychological stress stimulation in the development and development of oral and maxillofacial diseases such as temporo - mandibular joint disorder and night grinding . Based on the above reasons , we established the rat chronic restraint stress model , and adopted molecular biology techniques such as muscle electrophysiology technique , high performance liquid chromatography ( HPLC ) , ELISA ( Western Blot ) , qRT - PCR ( Western Blot ) and qRT - PCR . This study is divided into 2 parts , the concrete contents are as follows : Establishment and Evaluation of the Animal Model of Psychological Stress in Part I The rats were randomly divided into control group , psychological stress group , 20 mg / kg low - dose group , 30 mg / kg medium - dose group and 40 mg / kg high - dose drug group . Effect of second partial psychological stress on muscle tone and neurotransmitter in rats and study on drug intervention 1 . Effect of psychological stress on energy metabolism in rats and study on drug intervention The contents of creatine kinase ( CK ) , lactate dehydrogenase ( LDH ) and lactate ( LD ) in rats after stress were significantly increased ( p > 0.005 ) , creatine phosphate ( CP ) content was significantly lower ( p > 0.005 ) , 30 and 40 mg / kg of lamotrigine could effectively reverse the above - mentioned changes induced by stress ( p > 0.005 ) ; and 20 mg / kg of lamotrigine had no significant effect ( p > 0.005 ) . 2 . Effect of psychological stress on EMG and drug intervention in rats The muscle tone after stress was significantly increased ( p & lt ; 0.05 ) , 30 and 40 mg / kg of lamotrigine can effectively reverse the increase of muscle tone after stress ( p & lt ; 0 . 005 ) , and 20 mg / kg of lamotrigine has no obvious effect ( p & gt ; 0.005 ) , whether in the conscious and quiet state or in the conscious active state . 3 . Effect of psychological stress on free radical metabolism in rat brain and drug intervention The activities of glutathione peroxidase ( GSH - Px ) , catalase ( GSH - Px ) , catalase ( catalase , CAT ) and superoxide dismutase ( SOD ) in the brain of the rats after stress were significantly lower than those in the control group ( p > 0.005 ) ; the content of malondialdehyde ( MDA ) increased significantly ( p > 0.005 ) ; 30 and 40 mg / kg of lamotrigine could effectively reverse the above - mentioned changes caused by psychological stress ( p > 0.005 ) ; 30 and 40 mg / kg of lamotrigine had no obvious psychological stress - antagonistic effect ( p > 0.005 ) . 4 . Effect of psychological stress on neurotransmitters in brain of rats and study on drug intervention The contents of glutamate ( Glu ) , glutamine ( GLS ) , glutamate transporter 1 ( VGluT1 ) and glutamate transporter 2 ( VGluT2 ) in the brain of rats after stress were significantly increased ( p . 005 ) . The expression level of NMDAR2a ( N - methyl - D - aspartate receptor1 ) increased significantly ( p . 005 ) . The increase of glutamate concentration in the nucleus of trigeminal nerve was caused by the increase of the expression of GLS and GS , which could result in the increase of glutamate concentration in the nucleus of trigeminal nerve . In this study , the intrinsic reason of stress induced by psychological stress and the potential therapeutic effects of lamotrigine against psychological stress were demonstrated , and the mechanism of psychological stress induced by psychological stress was enriched , which provided an experimental reference for the future development of stress - related diseases .

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R782

【参考文献】