老年血亏阴虚失眠证候大鼠模型的建立及酸枣仁汤干预作用研究

发布时间：2018-03-30 17:50

本文选题：睡眠剥夺　切入点：证候模型　出处：《湖北中医学院》2008年博士论文

【摘要】： 目的:本课题结合临床建立老年血亏阴虚型失眠证候动物模型,并将该复合型模型与传统单因素睡眠剥夺模型进行对照,遵循中医因-脉-证-治的思路进行评价:首先对“因”进行分析,即根据中医病因病机来评价造模因素的科学性;然后对“脉”进行观测,即观测模型较直接、直观、重要的中医体表特征,并确认这种模型的证候属性;接着对“证”进行探讨,即通过分子生物学等现代科技手段对体表特征所反映的老年血亏阴虚型失眠病理机制进行概括;最后用验方酸枣仁汤“治”来进行反证,探讨对应治法验方酸枣仁汤的作用及其可能机制,并建立基于代谢网络变化的中药复方系统生物学药效评价平台。方法:在对酸枣仁汤的促眠作用进行实验研究之后,将健康Wistar大鼠100只(雌雄各半、体重200g±20g)随机分成5组,每组20只:环境对照组;睡眠剥夺组(单因素模型组);证候模型组(复合因素模型组);酸枣仁汤大剂量组;酸枣仁汤小剂量组。其中环境对照组给予其它组睡眠剥夺时相似的水环境;单因素模型组采用自制多平台水环境法剥夺睡眠48h制备;复合因素模型组及酸枣仁汤大、小剂量组均采用颈背部皮下注射D-半乳糖6w、然后腹腔同时注射环磷酰胺和氢化可的松5d、接着用自制多平台水环境法剥夺睡眠48h制备复合模型,造模历时共7w;酸枣仁汤大、小剂量组在造模4w后开始同时灌胃给药3w,其它组均给予等容积生理盐水。指标检测具体方法如下: 1、通过同步检测体重、摄食量、腋温、及显微放大拍照爪、尾、口唇等部位,对各组实验大鼠的中医体表特征进行比较; 2、用组织化学法检测各组实验大鼠大脑皮质、海马部位自由基和一氧化氮合酶(NOS)水平的变化; 3、采用血液细胞分析仪检测各组实验大鼠血细胞计数的变化; 4、采用Morris水迷宫实验检测各组实验大鼠学习记忆能力的改变; 5、透射电镜下观察各组实验大鼠大脑皮质部位超微结构的病理改变; 6、采用高相液相法检测各组实验大鼠大脑皮质及下丘脑部位谷氨酸(Glu)及γ-氨基丁酸(GABA)含量的变化; 7、采用免疫组化染色和mRNA半定量RT-PCR法检测分析各组实验大鼠大脑皮质及海马部位γ-氨基丁酸A受体(GABA_AR)a_1和γ_2亚单位的表达改变; 8、采用免疫组化染色法检测各组实验大鼠大脑皮质部位星形胶质细胞的表达改变。结果: 1、与空白对照组比较,舒乐安定组、酸枣仁汤大、小剂量组均能显著延长阈上剂量戊巴比妥钠致小鼠睡眠时间(P＜0.01),显著增加阈下剂量戊巴比妥钠致小鼠入睡率(P＜0.01)。且酸枣仁汤大剂量组作用比小剂量组更明显,但无统计学意义(P＞0.05); 2、中医体表特征显示,与环境对照组比较,睡眠剥夺组出现失眠症状,表现为重度疲劳状态;与睡眠剥夺组比较,证候模型组在出现失眠症状的同时,出现面色无华,闭目,血呈暗红,嗜睡,食欲明显下降,四肢无力,毛蓬竖少光泽等血亏阴虚体表特征,且体重、摄食量显著下降(P＜0.01),而腋温的下降无统计学意义(P＞0.05);与证候模型组比较,酸枣仁汤大、小剂量组对血亏阴虚体表特征有改善作用,但无统计学意义(P＞0.05); 3、与证候模型组比较,其它各组大鼠大脑皮质、海马丙二醛(MDA)含量、超氧化物歧化酶(SOD)和NOS活性均明显降低(P＜0.01,P＜0.05)。且酸枣仁汤大、小剂量组呈现一定量效关系(P＜0.05); 4、与证候模型组比较,其它各组大鼠全血中红细胞计数、白细胞计数、血小板计数及血红蛋白含量显著升高(P＜0.01)。且酸枣仁汤大、小剂量组呈现一定量效关系(P＜0.05); 5、在定位航行实验中,与证候模型组比较,环境对照组和酸枣仁汤大、小剂量组大鼠实验4d及2d的潜伏期均显著缩短(P＜0.01)。在空间搜索实验中,各组平台象限的游泳距离占总距离百分比、池壁20%和40%区域游泳距离百分比的实验结果表现出与定位航行实验各组一致的趋势;与证候模型组比较,环境对照组和酸枣仁汤大、小剂量组跨过原平台位置次数明显升高(P＜0.01)。两种模型组间相比无明显差异(P＞0.05); 6、睡眠剥夺48h后,透射电镜下显示:睡眠剥夺组大鼠大脑皮质神经元细胞异染质轻微聚集,趋边排列,核周间隙轻微增宽,大部分线粒体中高度水肿,线粒体基质及线粒体脊消失;证候模型组大鼠大脑皮质神经元细胞间胞质水肿,连接增宽,可见单个神经元细胞异染质凝聚,趋边,呈现凋亡形态,细胞器消失;酸枣仁汤大、小剂量组能改善大鼠神经元细胞超微结构的病理改变; 7、与环境对照组比较,睡眠剥夺组大鼠大脑皮质及下丘脑部位氨基酸类神经递质Glu、GABA含量均明显增高(P＜0.01),但GABA增加更为明显,Glu/GABA比例减小;与环境对照组比较,证候模型组大鼠Glu、GABA含量均明显增高(P＜0.01),但Glu增加更为明显,Glu/GABA比例增大;与证候模型组比较,酸枣仁汤大、小剂量组大鼠大脑皮质及下丘脑部位氨基酸类神经递质Glu、GABA含量显著减少,但Glu减少更为明显,Glu/GABA比例减小,并呈现一定量效关系(P＜0.05); 8、与证候模型组比较,环境对照组和酸枣仁汤大、小剂量组大鼠大脑皮质及海马部位的GABA_ARa_1和γ_2亚单位免疫化学累积光密度明显较低(P＜0.01),皮质部位GABA_ARa_1和γ_2 mRNA的表达均显著下调(P＜0.01)。而与睡眠剥夺组比较,证候模型组表达相对较弱(P＜0.05); 9、与证候模型组比较,环境对照组和酸枣仁汤大、小剂量组大鼠睡眠剥夺48h后星形胶质细胞标志物—胶原纤维酸性蛋白(GFAP)在皮质部位表达明显较弱;与睡眠剥夺组比较,证候模型组表达亦相对较强。结论: 1、酸枣仁汤具有明显的镇静催眠作用,且大剂量作用强于小剂量; 2、模型大鼠的体表特征显示,D-半乳糖+环磷酰胺、氢化可的松+睡眠剥夺这一复合模型较接近临床老年血亏阴虚型失眠患者的表现,是一种可行、可靠的复合型动物模型; 3、老年大鼠的学习记忆能力和大脑皮质病理损伤与睡眠剥夺、血亏阴虚证候具有相关性。自由基水平增加、全血象减少是老年血亏阴虚型失眠的病理机制之一。反证验方酸枣仁汤可有效改善证候模型大鼠的学习记忆能力及修复脑损伤,其作用机理可能与抗自由基、补血作用有关; 4、大脑皮质及下丘脑部位Glu、GABA含量及Glu/GABA比值均升高可能是老年血亏阴虚型失眠的神经生物学病理机制。酸枣仁汤治疗老年血亏阴虚型失眠的作用机制可能与通过下调大脑皮质及下丘脑部位Glu、GABA的含量及Glu/GABA的比值来保护脑组织有关。 5、睡眠剥夺可明显上调大鼠脑内皮质及海马部位GABA_ARa_1和γ_2亚单位表达。酸枣仁汤治疗老年血亏阴虚型失眠的作用机制可能与下调大鼠大脑皮质及海马部位GABA_ARa_1和γ_2亚单位的表达有关。 6、睡眠剥夺可上调大鼠大脑皮质部位星形胶质细胞标志物—胶质纤维酸性蛋白(GFAP)的表达,提示星形胶质细胞为可兴奋细胞,且参与睡眠调节。大脑皮质部位GFAP的表达明显增强可能是老年血亏阴虚型失眠的又一病理机制。酸枣仁汤治疗老年血亏阴虚型失眠的作用机制可能还与下调大脑皮质部位GFAP的表达有关。
[Abstract]:Objective: This study combined with the clinical establishment of elderly blood deficiency and Yin insomnia syndrome animal model, and the composite model and the traditional single factor model of sleep deprivation group, traditional Chinese medicine for vein syndrome - treatment of evaluation: first of all, "because" analysis, namely according to the pathogenesis of scientific evaluation modeling of factors; then observe the "pulse", which is more intuitive, direct observation model, the Chinese surface important characteristics, and confirm the syndrome attribute of the model; then discusses the "syndrome", namely the elderly insomnia mechanisms with blood deficiency and Yin by molecular biology and other modern technologies of surface features the reflection was summarized; finally, the prescription szrt "cure" for evidence to the contrary, on the corresponding treatment prescription szrt effect and the possible mechanism, and the establishment of Chinese herbal compound system on the basis of metabolic network changes Biological efficacy evaluation platform.
Methods: after the experiments in the sleep promoting effect of Suanzaoren Decoction, the 100 healthy Wistar rats (Ci Xiong female, weight 200g + 20g) were randomly divided into 5 groups, 20 rats in each group: control group; sleep deprivation group (univariate model group); syndrome model group (model group compound factors); Suanzaoren Decoction high dose group; Suanzaoren Decoction low dose group. The control group was given the other group environment when sleep deprived similar water environment; single factor model group using the self-made multi platform water environment law 48h sleep deprivation preparation; multiple factors model group and szrt of large and small dose group were treated with neck subcutaneous injection of D- galactose 6W, then intraperitoneal injection of cyclophosphamide and hydrocortisone and 5D, then using the self-made multi platform water environment law of sleep deprivation model prepared by 48h model, for a total of 7W; szrt of large and small dose groups also began intragastric administration of 3W in 4W after modeling, the The group was given equal volume of saline. The specific methods were as follows:
1, by measuring the body weight, food intake, axillary temperature, and micromagnifying photo claw, tail, lip and other parts, the body surface characteristics of the experimental rats in each group were compared.
2, the changes in the level of free radicals and nitric oxide synthase (NOS) in the cerebral cortex, hippocampus and hippocampus were detected by histochemical method.
3, blood cell analyzer was used to detect the changes of blood cell count in each group.
4, the Morris water maze test was used to detect the changes in the learning and memory ability of the rats in each group.
5, under the transmission electron microscope, the pathological changes of the ultrastructure of the cortex of the cerebral cortex of the rats were observed.
6, the changes in the content of glutamic acid (Glu) and gamma aminobutyric acid (GABA) in the cerebral cortex and hypothalamus of the rats were detected by high phase liquid phase.
7, immunohistochemical staining and mRNA semi quantitative RT-PCR were used to detect the expression of GABA A receptor (GABA_AR) a_1 and gamma _2 subunits in each group of rats.
8, the expression of astrocytes in the cerebral cortex of each group was detected by immunohistochemical staining.
Result:
1, compared with the blank control group, diazepam group and szrt of large and small dose group could significantly extend the threshold dose of pentobarbital induced sleep time in mice (P < 0.01), significantly increased the subthreshold dose of sodium pentobarbital induced sleep rate (P < 0.01). Szrt large agent the amount of group was more obvious than low-dose group, but no statistical significance (P > 0.05);
2, the Chinese surface characteristics showed that, compared to environmental control group, sleep deprivation group appear insomnia symptoms, severe fatigue performance of the state; and the sleep deprived group, syndrome model group in insomnia symptoms appear at the same time, lusterless complexion, eyes closed, blood is dark, lethargy, weakness, appetite decreased significantly. Peng hair less shiny blood deficiency such as vertical surface characteristics, and the body weight, food intake decreased significantly (P < 0.01), but no significant decline in axillary temperature (P > 0.05); compared with syndrome model group, Suanzaoren Decoction, small dose group has good effect on blood deficiency of surface characteristics, but no statistical significance (P > 0.05);
3, compared with the syndrome model group, cerebral cortex of other rats hippocampus malondialdehyde (MDA) content, superoxide dismutase (SOD) and NOS activity were significantly decreased (P < 0.01, P < 0.05). Szrt of large and small dose group showed a dose effect relationship (P < 0.05);
4, compared with the syndrome model group, red blood cell count other rats'whole blood, white blood cell count, platelet count and hemoglobin content increased significantly (P < 0.01). Szrt of large and small dose group showed a dose effect relationship (P < 0.05);
5, in the navigation experiment, compared with syndrome model group, control group and szrt, experimental 4D and 2D low dose group rat latency shortened significantly (P < 0.01). In the search space in the experiment, each platform quadrant swimming distance percentage of the total distance, the wall of the pool 20% the 40% area and swimming distance percentage of the experimental results show the positioning navigation experiments consistent trend; compared with syndrome model group, control group and szrt of large and small dose groups across the original platform location of significantly increased (P < 0.01). The two groups showed no significant difference (P > 0.05);
6, sleep deprivation after 48h transmission electron microscope showed that sleep deprivation rat cerebral cortex neurons of heterochromatin margination array, slight aggregation, nuclear week gap widened slightly, the majority of mitochondrial highly edema, mitochondrial matrix and mitochondrial crista disappeared; syndrome model group rat cortical neurons cell cytoplasm edema, connecting widened, visible single neurons with different chromatin condensation, margination, showed apoptotic morphology, cell disappeared; szrt of large and small dose group could improve the ultrastructure of neurons pathological changes in rat;
7, with comparison to the control group, sleep deprivation group rat cerebral cortex and hypothalamus of amino acid neurotransmitters Glu, GABA content increased significantly (P < 0.01), but GABA was significantly increased, Glu/GABA ratio decreased; and in comparison to the control group, the rats in group Glu syndrome model, GABA significantly increased (P < 0.01), but Glu was significantly increased, the proportion of Glu/GABA increased; compared with syndrome model group, Suanzaoren Decoction and small dose group rat cerebral cortex and hypothalamus of amino acid neurotransmitters Glu, GABA were significantly reduced, but Glu reduced more significantly, the ratio of Glu/GABA decreased, and a certain dose effect relationship (P < 0.05);
8, compared with the syndrome model group, control group and szrt, GABA_ARa_1 and gamma subunit _2 immunohistochemistry in small dose group rat cerebral cortex and hippocampus of the accumulated optical density was significantly lower (P < 0.01), the expression of cortex GABA_ARa_1 and gamma mRNA _2 were significantly lower (P < 0.01). Compared to the sleep deprived group, syndrome model group showed weaker expression (P < 0.05);
9, compared with the syndrome model group, control group and szrt of large and small dose groups of rats after sleep deprivation 48h astrocyte marker glial fibrillary acidic protein (GFAP) expression was significantly weaker in the cortex; and the sleep deprived group, the expression of syndrome model group is relatively strong.
Conclusion:
1, acid zizzizen decoction has obvious sedative and hypnotic effect, and the effect of large dose is stronger than that of small dose.
2, show the surface characteristics of the rat model, D- galactose + cyclophosphamide and hydrocortisone + sleep deprivation, this composite model is closer to the elderly patients with blood deficiency and Yin insomnia, is a feasible and reliable animal model of compound;
3, the ability of learning and memory and cerebral cortex injury in aged rats with sleep deprivation, associated with Yin deficiency syndrome in blood. The level of free radicals increased, whole blood reduction is one of the pathological mechanism of insomnia in elderly patients with blood deficiency and Yin. But the ability of learning and memory and improve the repair of brain injury syndrome model rats disproving prescription Suanzaoren Decoction and its mechanism may be related to anti free radical effect on blood;
4, cerebral cortex and hypothalamus of Glu were increased, GABA content and Glu/GABA ratio might be a neurobiological pathological mechanism of insomnia in elderly blood deficiency and Yin. The mechanism of Suanzaoren Decoction in the treatment of senile insomnia, blood deficiency and Yin may be related to the cerebral cortex and hypothalamus through down-regulation of Glu contents and Glu/GABA ratio of GABA to protect the brain.
5, sleep deprivation could significantly increase the cortex and hippocampus in rat brain GABA_ARa_1 and gamma subunit _2 expression. The mechanism of Suanzaoren Decoction in the treatment of senile insomnia, blood deficiency and Yin may be associated with the down-regulation of expression in rat cerebral cortex and hippocampus GABA_ARa_1 and gamma subunit _2.
6, sleep deprivation can be parts of the cerebral cortex increased rat astrocyte marker glial fibrillary acidic protein (GFAP) expression, suggesting that astrocytes to excitable cells and is involved in the regulation of sleep. The expression of GFAP in cerebral cortex increased and a part of the pathogenesis may be blood loss in elderly insomnia Yin. The mechanism of Suanzaoren Decoction in the treatment of senile insomnia, blood deficiency and Yin may be associated with the down-regulation of GFAP expression in brain cortex.

【学位授予单位】：湖北中医学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R-332;R285.5

【引证文献】