“肾脑相济”电针疗法对“天癸竭”围绝经期抑郁症作用的机制研究

发布时间：2018-01-05 08:22

本文关键词：“肾脑相济”电针疗法对“天癸竭”围绝经期抑郁症作用的机制研究　出处：《辽宁中医药大学》2016年博士论文　论文类型：学位论文

【摘要】：目的:在“肾脑相济”理论的指导下,观察“补肾益精填髓”的电针疗法对“天癸竭”围绝经期抑郁症的作用,并探讨其作用机制,为临床治疗围绝经期抑郁症提供指导和实验依据。材料与方法:选取17周龄健康雌性SD大鼠120只,随机分为正常组、模型组、药物组、电针组和假手术组,每组各24只。2只一笼正常饲养。1.行为学指标检测:各组大鼠在造模开始前先进行行为学指标的观测,行为学指标有旷场实验中总移动距离和旷场中心区域停留时间;糖水偏好实验中糖水消耗量、总液体消耗量和糖水消耗率;强迫游泳实验中大鼠在水中静止时间。2.制备围绝经期抑郁症模型:正常组大鼠不接受造模,正常饲养。模型组、药物组和电针组大鼠摘除双侧卵巢去势,假手术组不切除卵巢,只在卵巢附近切除与卵巢等大的脂肪。手术后大鼠恢复两周,之后进行连续5天的阴道涂片,确定手术组大鼠无动情周期。认为切除卵巢去势成功。之后正常组大鼠正常饲养不接受刺激,模型组、药物组、电针组和假手术组的大鼠均接受为期21天的慢性不可预见温和刺激(CUMS)。刺激包括:冰水游泳、热水游泳、禁水禁食、夹尾、束缚限制行动、倾斜笼子和昼夜颠倒。每种刺激随机出现,且每种刺激在21天中出现次数不少于3次。21天刺激结束后,通过行为学指标的变化确定大鼠围绝经期抑郁症模型造模是否成功。3.电针治疗:正常组大鼠正常喂养,不接受治疗。电针组和假手术组的大鼠接受28天的电针治疗,根据“肾脑相济”理论,选取百会、肾俞、三阴交穴,每日电针治疗1次,持续20分钟。药物组大鼠选用三环类抗抑郁药物盐酸氯米帕明作为对照,用盐酸氯米帕明灌胃,每日灌胃1次。治疗结束后通过行为学指标的变化评价电针对于围绝经期抑郁症大鼠的治疗效果。4.行为学指标观察结束后,麻醉处死所有大鼠取材,大鼠大脑石蜡切片用于观察大鼠海马结构和神经细胞形态变化;分离大鼠海马组织,用酶联免疫吸附法检测海马组织中脑源性神经营养因子(BDNF)、酪氨酸激酶受体B(Trk B)、c AMP反应元件结合蛋白(CREB)、B细胞淋巴瘤2(Bcl-2)和Bcl-2相关X蛋白(Bax)的含量,并用实时荧光定量PCR的方法检测海马组织内BDNF、Trk B、CREB、Bcl-2和Bax的m RNA表达量。结果:1.造模开始前各组大鼠在旷场实验中总移动距离和中心区域停留时间无差异(p0.05)。糖水实验中,糖水消耗量、总液体消耗量和糖水消耗率无差异(p0.05);强迫游泳实验中静止不动时间无差异(p0.05)。2.双侧卵巢摘除术后,除正常组和假手术组外,各组大鼠阴道涂片均无动情周期。3.经过21天慢性不可预见温和刺激后,旷场实验结果:与正常组相比,其余各组大鼠的总移动距离各组均小于正常组,差异有统计学意义(p0.05)。其余各组大鼠旷场中心区域停留时间各组均小于正常组,差异有统计学意义(p0.05)。4.经过21天慢性不可预见温和刺激后,糖水实验结果:与正常组比较,其余各组大鼠糖水消耗率均小于正常组,差异有统计学意义(p0.05)。与模型组比较,假手术组大鼠糖水消耗率高于模型组,差异有统计学意义(p0.05)。5.经过21天慢性不可预见温和刺激后,强迫游泳实验结果:与正常组比较,其余各组大鼠水中静止时间均大于正常组,差异有统计学意义(p0.05)。6.经过28天的电针和药物治疗后,旷场实验结果:与正常组比较,模型组大鼠的总移动距离小于正常组,中心区域停留时间小于正常组,差异有统计学意义(p0.05)。与模型组比较,药物组、电针组和假手术组的总移动距离大于模型组,旷场中心区域停留时间大于模型组,差异有统计学意义(p0.05)。7.经过28天的电针和药物治疗后,糖水实验结果:与正常组比较,其余各组大鼠糖水消耗率均少于正常组,差异有统计学意义(p0.05)。与模型组比较,假手术组、电针组和药物组的糖水消耗率高于模型组,差异有统计学意义(p0.05)。与电针组比较,假手术组糖水消耗率高于电针组,差异有统计学意义(p0.05)。8.经过28天的电针和药物治疗后,强迫游泳实验结果:与正常组比较,模型组大鼠静止时间大于正常组,差异有统计学意义(p0.05)。与模型组比较,药物组、电针组和假手术组的静止时间小于模型组,差异有统计学意义(p0.05)。9.经过28天的电针和药物治疗后,在光镜下观察各组大鼠海马石蜡切片,正常组大鼠海马CA3区锥体细胞层和齿状回区颗粒细胞层细胞数量多、排列整齐,神经元结构完整,胞核及胞质染色均匀、清晰。尼氏染色可见神经元胞质内含有丰富的尼氏体,呈紫蓝色,神经元细胞轴突树突形态结构完整。模型组大鼠海马CA3区细胞数量少,细胞间隙增大,排列疏松不齐,甚至出现细胞层中断,尼氏染色可见神经元细胞核消失,细胞质萎缩、出现空洞,轴突树突结构不完整,甚至出现细胞结构消失。药物组、电针组和假手术组的情况介于两者之间。10.经过28天的电针和药物治疗后,海马组织酶联免疫吸附法结果:与正常组相比,模型组大鼠海马BDNF蛋白含量明显降低,差异有统计学意义(p0.05);与模型组比较,药物组、电针组和假手术组均高于模型组,差异有统计学意义(p0.05);与电针组比较,假手术组的数值高于正常组,差异有统计学意义(p0.05)。与正常组相比,模型组大鼠海马Trk B蛋白含量明显降低,差异有统计学意义(p0.05);与模型组比较,药物组、电针组和假手术组均高于模型组,差异有统计学意义(p0.05);与电针组比较,假手术组的数值低于正常组,差异有统计学意义(p0.05)。与正常组相比,模型组大鼠海马CREB蛋白含量降低,差异有统计学意义(p0.05);与模型组比较,药物组、电针组和假手术组含量高于模型组,差异有统计学意义(p0.05);与电针组比较,假手术组含量高于电针组,差异有统计学意义(p0.05)。与正常组相比,模型组大鼠海马中Bcl-2蛋白含量低于正常组,Bax含量高于正常组,差异有统计学意义(p0.05);与模型组比较,药物组、电针组和假手术组Bcl-2含量高于模型组,Bax含量低于模型组,差异有统计学意义(p0.05)。与正常组比价,模型组大鼠Bcl-2/Bax比例关系大于正常组,差异有统计学意义(p0.05);与模型组比较,药物组、电针组和假手术组Bcl-2/Bax比例关系大于模型组,差异有统计学意义(p0.05);与电针组比较,假手术组Bcl-2/Bax比例关系大于电针组,差异有统计学意义(p0.05)。11.经过28天的电针和药物治疗后,海马组织实时荧光定量PCR结果:与正常组相比,模型组大鼠海马BDNF m RNA相对表达量低于正常组,差异有统计学意义(p0.05);与模型组比较,药物组、电针组和假手术组相对表达量高于模型组,差异有统计学意义(p0.05);与电针组比较,假手术组BDNF m RNA相对表达量高于电针组,差异有统计学意义(p0.05)。与正常组相比,模型组大鼠海马Trk B m RNA相对表达量高于正常组,差异有统计学意义(p0.05)。与正常组相比,模型组大鼠海马CREB m RNA相对表达量低与正常组,差异有统计学意义(p0.05);与模型组比较,药物组、电针组和假手术组相对表达量高于模型组,差异有统计学意义(p0.05);与电针组比较,假手术组CREB m RNA相对表达量高于电针组,差异有统计学意义(p0.05)。与正常组相比,模型组大鼠海马Bcl-2 m RNA相对表达量低于正常组,Bax m RNA相对表达量高于正常组,差异有统计学意义(p0.05);与模型组比较,药物组、电针组和假手术组Bcl-2 m RNA相对表达量高于模型组,Bax m RNA相对表达量低于模型组,差异有统计学意义(p0.05);与电针组比较,假手术组Bcl-2 m RNA相对表达量高于电针组,Bax m RNA相对表达量低于电针组,差异有统计学意义(p0.05)。结论:1.去势造模结合慢性不可预见温和刺激确立围绝经期抑郁症大鼠模型。2.基于“肾脑相济”理论的电针治疗能改善围绝经期大鼠的抑郁症状,实验结果与临床吻合,且为临床提供实验依据。3.假手术组和电针组行为学指标结果略有不同,说明“天癸竭”在围绝经期抑郁症的发病和转归中有着重要作用。4.围绝经期抑郁症大鼠“髓海空虚”的生物学基础可能是大脑海马神经元细胞数量减少和细胞结构破坏,且与海马神经元细胞凋亡增加、海马神经发生降低有关。5.电针能高提高大鼠海马内BDNF、Trk B和CREB的含量,促进海马神经发生,还能提高Bcl-2和Bax的比例,抑制神经元细胞凋亡,电针“补肾益精填髓”的作用是通过促进神经发生和抑制细胞凋亡实现的。6.模型组大鼠海马组织中Trk B m RNA相对表达量高但Trk B蛋白含量低,说明围绝经期抑郁症大鼠体内某些因素妨碍了Trk B基因的表达,这可能是慢性不可预见温和刺激造成海马中Trk B和BDNF含量减少的原因。
[Abstract]:Objective: in the kidney and brain of mercy "under the guidance of the theory, to observe the effect of" kidney tonifying the kidney essence of electro acupuncture therapy on the "Tiangui dried up" perimenopausal depression, and to explore its mechanism, provide guidance and experimental basis for the clinical treatment of perimenopausal depression. Materials and methods: 17 week old healthy 120 female SD rats were randomly divided into normal group, model group, drug group, EA group and sham group, 24 rats in each group.2 only a normal feeding behavior of.1. cage indexes: the rats in the observation before the start of the model of behavioral indicators, behavioral indicators of total mobile field test in the distance and open field center area of residence time; consumption of sugar sucrose preference test, total fluid consumption and sugar consumption rate; forced swimming test in rats in static time.2. preparation model of perimenopausal depression: normal rats do not accept made Die, normal feeding. Model group, drug group and EA group rats were bilaterally ovariectomized and sham operation group without excision of ovarian resection, only in ovarian and ovarian size near fat. Postoperative recovery of the rats for two weeks, after 5 consecutive days of vaginal smear, determine the operation group rats had no estrous cycle that success. After resection of ovariectomized rats in normal group fed normally does not accept the stimulus, model group, drug group, EA group and sham operation group rats were treated for 21 days chronic unpredictable mild stimulation (CUMS) stimulation. Including: swimming, water swimming, fasting water fasting, tail clamp restrict action, tilt, cage and night. Each stimulus appears randomly, and each stimulus in 21 days is not less than 3 times.21 days after stimulation, changes of indexes through the behavior of the rat model of perimenopausal depression determine the success of modeling.3. Acupuncture treatment: the normal rats were fed normally, do not receive treatment. EA group and sham operation group rats received acupuncture treatment for 28 days, according to the "kidney brain Jinan" theory, select Baihui, Shenshu, Sanyinjiao, acupuncture treatment 1 times daily, for 20 minutes. Rats in drug group with tricyclic antidepressant clomipramine hydrochloride as control with clomipramine hydrochloride intragastric gavage 1 times. To evaluate the changes of electroacupuncture after the end of treatment by behavioral indicators for treatment of perimenopausal depression rats.4. behavior indexes were observed after anesthesia, all the rats were killed at the rat brain, paraffin sections for observation the structure and morphology of hippocampal nerve cell changes; isolated rat hippocampus, adsorption method for detection of hippocampal brain-derived neurotrophic factor by enzyme linked immunosorbent assay (BDNF), tyrosine kinase receptor B (Trk B C), AMP response element binding protein White (CREB), B cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein (Bax) content, and hippocampuswere measured by real-time quantitative PCR BDNF, Trk B, CREB, m RNA and Bax Bcl-2 expression. Results: 1. before the start of the model rats in the open field the total moving distance and center area of residence time had no difference (P0.05). In the experiment, sugar consumption, total liquid consumption and sugar consumption rate had no difference (P0.05); forced swimming test in stationary time difference (P0.05).2. bilateral ovariectomy, except normal group and sham operation group, the rats had no vaginal smears of the estrous cycle of.3. after 21 days chronic unpredictable mild stimulation, open field test results: compared with normal group, the total moving distance of each group of rats were less than the normal group, the difference was statistically significant (P0.05). The rats in open field center stay The time was shorter than the normal group, the difference was statistically significant (P0.05.4.) after 21 days chronic unpredictable mild stimulation, sucrose test results: compared with normal group, the rats sugar consumption rate is less than the normal group, the difference was statistically significant (P0.05). Compared with the model group, the sham operation group sucrose consumption rate is higher than the model group, the difference was statistically significant (P0.05.5.) after 21 days chronic unpredictable mild stimulation, forced swimming test results: compared with the normal group, the rats in the rest time were higher than the normal group, the difference was statistically significant (P0.05.6.) after 28 days of acupuncture and drugs after treatment, the experimental results of open field: compared with the normal group, the total moving distance of the rats in the model group than the normal group, the center area of the residence time is less than the normal group, the difference was statistically significant (P0.05). Compared with the model group, drug group and EA group The sham operation group and the total moving distance is greater than the model group, the open field center area of the residence time was more than the model group, the difference was statistically significant (P0.05.7.) after electroacupuncture and medication 28 days after treatment, in the experimental results: compared with the normal group, the rats were less than the normal rate of sugar consumption group, was statistically significant the difference (P0.05). Compared with the model group, sham operation group, electroacupuncture group and drug group sugar consumption rate is higher than the model group, the difference was statistically significant (P0.05). Compared with the electroacupuncture group, sham operation group, sugar consumption rate is higher than the acupuncture group, the difference was statistically significant (P0.05.8.) after electroacupuncture and drug 28 days after treatment, forced swimming test results: compared with the normal group, the rest time of rats in the model group than the normal group, the difference was statistically significant (P0.05). Compared with the model group, drug group, EA group and sham operation group the rest time less than model group, The difference was statistically significant (P0.05.9.) after electroacupuncture and medication after 28 days of treatment, observed in the paraffin sections of rats under light microscope, normal rat hippocampal CA3 pyramidal cell layer and dentate gyrus granular cell layer number of cells, rows, neuron structure is complete, the nucleus and the cytoplasm uniform staining and Nissl staining. Clear neuronal cytoplasm contained abundant Nissl bodies, purple blue, the morphology of neurons axon dendritic cells. The number of cells in hippocampal CA3 region of rats in the model group, the cell gap increases, loose arrangement is uneven, and even cell layer disruption, Nissl staining of neuronal nuclei disappeared, cytoplasm atrophy appeared empty, axon dendritic structure is not complete, or even disappear. The cell structure between EA group and drug group, between two sham operation group.10. after electric acupuncture and drug treatment after 28 days of sea The horse tissue ELISA results: compared with normal group, the content of BDNF protein in hippocampus of rats in the model group decreased significantly, the difference was statistically significant (P0.05); compared with the model group, drug group, EA group and sham operation group were higher than those in the model group, the difference was statistically significant (P0.05); compared with the electroacupuncture group numerical, sham operation group was higher than the normal group, the difference was statistically significant (P0.05). Compared with the normal group, model group of rat hippocampal Trk B protein content decreased significantly, the difference was statistically significant (P0.05); compared with the model group, drug group, electric acupuncture group and sham operation group were higher than those in the model group, there are statistically significant difference (P0.05); compared with the electroacupuncture group, sham operation group, the value is less than the normal group, the difference was statistically significant (P0.05). Compared with the normal group, decrease the content of CREB protein in hippocampus of rats in the model group, the difference was statistically significant (P0.05); compared with the model group, drug group, EA group and sham operation group were higher than those in the model group, the difference was statistically significant (P0.05); compared with the electroacupuncture group, sham operation group was higher than that in the EA group, the difference was statistically significant (P0.05). Compared with the normal group, the content of Bcl-2 protein in hippocampus of rats in model group were lower than the normal group, the content of Bax was higher than that of normal group the difference was statistically significant (P0.05); compared with the model group, drug group, EA group and sham operation group Bcl-2 was higher than that in the model group, the Bax content is lower than the model group, the difference was statistically significant (P0.05). Comparison with the normal group, the ratio of Bcl-2/Bax in model group rats than the normal group, the difference was statistically significant (P0.05); compared with the model group, drug group, EA group and sham operation group Bcl-2/Bax proportion is greater than the model group, the difference was statistically significant (P0.05); compared with the electroacupuncture group, sham operation group Bcl-2/ Bax proportion is greater than the acupuncture group, there was statistically significant difference The meaning of (P0.05).11. after electroacupuncture and medication after 28 days of treatment, the result of real-time fluorescence quantitative PCR in hippocampus: compared with normal group, RNA m BDNF in hippocampus of rats in the model group the relative expression amount is less than the normal group, the difference was statistically significant (P0.05); compared with the model group, drug group, EA group and sham operation group the relative expression quantity is higher than the model group, the difference was statistically significant (P0.05); compared with the electroacupuncture group, sham operation group BDNF m relative expression of RNA was higher than the acupuncture group, the difference was statistically significant (P0.05). Compared with the normal group, RNA B m Trk in hippocampus of rats in the model group the relative expression quantity is higher than the normal group, there are statistically significant difference (P0.05). Compared with the normal group, the RNA m CREB in hippocampus of rats in the model group is relatively low expression with the normal group, the difference was statistically significant (P0.05); compared with the model group, drug group, EA group and sham operation group relative expression quantity is higher than the model group, the difference was statistically Statistically significant (P0.05); compared with the electroacupuncture group, sham operation group CREB m relative expression of RNA was higher than the acupuncture group, the difference was statistically significant (P0.05). Compared with the normal group, the RNA m Bcl-2 in hippocampus of rats in the model group the relative expression of Bax m were lower than the normal group, the relative expression level of RNA is higher than the normal group, there are statistically significant difference (P0.05); compared with the model group, drug group, EA group and sham operation group Bcl-2 m RNA relative expression quantity is higher than the model group, the relative expression of RNA Bax m lower than that of model group, the difference was statistically significant (P0.05); compared with the electroacupuncture group, sham operation group Bcl-2 m relative expression of RNA was higher than that of EA group, Bax m RNA relative expression is lower than the acupuncture group, the difference was statistically significant (P0.05). Conclusion: 1. ovariectomized model combined with chronic unpredictable mild stimulation to establish perimenopausal depression rat model of.2. based on the "kidney brain mercy" theory of electroacupuncture therapy can improve perimenopausal The menstrual period in rats with depressive symptoms, are in good agreement with the experimental results and clinical, and provide experimental basis for clinical.3. sham operation group and electroacupuncture group behavioral indexes were slightly different, "Tiangui dried up" at the onset of perimenopausal depression and prognosis has important biological basis of the role of.4. in perimenopausal depression rats' spinal cord the sea empty "may be the number of cells in hippocampal neurons of brain damage and reduce the cell structure, and increase and apoptosis of hippocampal neurons, hippocampal neurogenesis is related to the decrease of.5. EA can improve the high in rat hippocampus BDNF, Trk content B and CREB, to promote neurogenesis in the hippocampus, Bcl-2 and Bax can improve the proportion of inhibitory neurons cell apoptosis, effect of acupuncture of" kidney tonifying the kidney essence "is through the promotion of high volume but Trk B m RNA Tr relative expression of neurogenesis and cell proliferation and apoptosis.6. in hippocampus of rats in model group K B protein content is low, indicating that some factors in the perimenopausal depression rats interfere with the expression of Trk B gene, which may be the reason why the Trk B and BDNF contents in hippocampus are reduced due to chronic unpredictable mild stimulation.

【学位授予单位】：辽宁中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R245

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