疏肝、健脾、疏肝健脾方对肝郁脾虚证模型动物外观表征及行为学的影响

【作者】 杨薇；

【导师】 谢鸣；

【作者基本信息】 北京中医药大学 ， 方剂学， 2010， 硕士

【摘要】 方剂是由多味中药配伍而成、具有特定功用和主治的一种防疾治病的工具。其配伍与其所主病证病机或病理环节之间具有高度的相关性或针对性,即“方证相关”。通常针对一个特定病证可能会有数个方剂起作用,但疗效存在差异,或同一方剂作用于不同病证会呈现不同的治疗效果。“方证相关”是中医辨证论治原理的核心,也是方剂学学理的重要逻辑基础。研究方与证之间的关系对揭示辨证论治原理具有重要的理论与实践意义。本课题采用慢性束缚+饮食失节+过度疲劳法复制出肝郁脾虚证动物模型,以模型动物外观行为作切入点,比较观察具有疏肝、健脾、疏肝健脾不同功效的柴胡疏肝散、四君子汤、柴疏四君汤对模型大鼠外观行为变化作用的异同,并探查模型的中枢递质及生化物的变化和三方对其变化的影响。论文包括文献综述和实验研究两部分。文献综述对肝郁脾虚证及柴胡疏肝散、四君子汤、柴疏四君汤三方的临床与实验研究概况进行了综述。实验研究观察了肝郁脾虚证大鼠模型外观行为表征及其中枢神经递质及有关生化物的变化和柴胡疏肝散、四君子汤、柴疏四君汤三方对该模型的相关作用。研究方法：将大鼠随机分为正常对照组、肝郁脾虚模型组(以下简称模型组)、柴疏四君汤组、四君子汤组、柴胡疏肝散组共5组,每组12只。后4组大鼠于每天8：00 am置于束缚器中限制3h,2：00pm置于盛有温水(22±1℃)的大塑料桶中游泳10 min,隔日喂食(隔日禁食,隔日足量给食),连续4周；正常对照组不施加任何刺激,自然饲养。于造模第15天,中药各组大鼠分别给予柴疏四君汤4.27g／kg、四君子汤4.53g／kg、柴胡疏肝散4.2g／kg灌胃,每天1次；模型组与对照组分别给予等量蒸馏水,连续14天。并于第7d、14d、21d、28d,分早、中、晚三个时间点观察并记录各组大鼠的行为状态、活跃程度、情绪反应、皮肤毛发、饮食状态、睡眠状态、大便状态等方面的变化；于每周第7天2：00-2：30pm测定各组大鼠的体重；于第14d、28d通过旷场实验记录大鼠5min内爬行格数、直立次数、修饰时间及中央格停留时间。于第29天8：00am断头处死,在冰盘上迅速剥离大鼠海马、下丘脑和脑干,置于液氮罐中保存,待测。采用高效液相色谱荧光法测定海马组织中Asp、Glu、GABA、Gly、Tau;采用放射免疫法测定下丘脑组织中cAMP、PGE2;采用酶联免疫法测定脑干组织中P450酶的活性及含量。统计学方法：所得数据均以χ±S表示,采用单因素方差分析(one-way ANOVA)进行检验后,多组间比较采用Student Newman Keuls Test检验。全部数据采用SPSS 13.0 for windows软件统计处理。研究结果：1.肝郁脾虚证模型大鼠外观表征的变化及相关方药的作用：造模1周后,模型组、柴疏四君汤组、四君子汤组、柴胡疏肝散组大鼠行为活动、情绪反应、皮肤毛发、饮食、睡眠与大便状态等均未有明显变化。模型组大鼠造模2周后即表现出活动减少、活跃程度减弱、情绪易怒、饮食减少、皮毛不顺泽、易醒、便干等；造模3周后开始出现动作迟缓、情绪不稳、饮食减少、皮毛不泽、倦卧、便溏；造模4周后,则出现明显的活动减少、扎堆甚至倦卧多睡,情绪由开始的易激惹状态转为情绪低落、争斗减少,饮食减少,皮毛枯乱,大便稀溏等。与模型组比较,3个中药组大鼠在给药1周后(造模3周后),活动增加,情绪趋于稳定,饮食有增,皮毛、睡眠与大便状态均见不同程度好转。给药2周后(造模4周后),柴疏四君汤组大鼠行为活动明显增加、反应敏捷、皮毛接近顺整、饮食增加、睡眠深浅适度、大便成形；四君子汤组大鼠在皮毛、饮食、睡眠与大便状态改善方面明显；柴胡疏肝散组大鼠在行为状态、活跃程度、情绪反应等方面均见明显好转。2.肝郁脾虚证模型大鼠体重的变化及相关方药的作用：实验期间,正常组大鼠体重明显增加,模型组与3个给药组大鼠体重增加较为缓慢,其中模型组、柴疏四君汤组和四君子汤组大鼠在第1周末体重出现负增长；模型组与柴胡疏肝散组大鼠在第4周末体重出现下降趋势。与正常组比较,模型组与柴疏四君汤组、四君子汤组、柴胡疏肝散组大鼠在第1w、2w、3w、4w末的体重均明显降低,差异具有显著性意义(P<0.05)。与模型组比较,3个给药组大鼠体重变化无显著性差异(P>0.05)；且3个给药组间大鼠体重变化无明显差异(P>0.05)。3.肝郁脾虚证模型大鼠自主行为的变化及相关方药的作用：与正常组比较,造模第14天时的模型组与3个中药组大鼠的爬行格数明显增加,差异具有显著性意义(P<0.01或P<0.05)；模型组、柴疏四君汤组大鼠直立次数增加,差异具有显著性意义(P<O.05)；模型组、四君子汤组大鼠修饰时间和中央格停留时间均延长,其中修饰时间差异具有显著性意义(P<0.05)。给药2周后(造模第28天)与正常组比较,模型组、四君子汤组与柴胡疏肝散组大鼠爬行格数增加,差异均有显著性意义(P<0.05),柴疏四君汤组大鼠爬行格数接近正常水平(P>0.05)；模型组与四君子汤组大鼠直立次数增加,差异有显著性意义(P<0.05),柴疏四君汤组与柴胡疏肝散组大鼠增加不明显(P>0.05)；模型组与3个中药组大鼠修饰时间、中央格停留时间均有延长,但差异无显著性意义(P>0.05)。与模型组比较,3个中药组大鼠的爬行格数和直立次数均有不同程度的降低,其中柴疏四君汤组爬行格数降低明显,差异有显著性意义(P<0.05)4.肝郁脾虚证模型大鼠中枢神经生化指标的变化及相关方药的作用：(1)与正常组比较,模型组大鼠海马组织中Asp(天冬氨酸)的含量呈升高趋势,Glu(谷氨酸)、GABA(Y-氨基丁酸)、Gly(甘氨酸)、Tau(牛磺酸)的含量呈下降趋势,但差异均无显著性意义(P>0.05)；下丘脑组织中PGE2和cAMP的变化均无显著性差异(P>0.05)；脑干P450酶活性与酶含量的变化均无显著性差异(P>0.05)。(2)与模型组比较,3个中药组大鼠的上述指标均无明显变化(P>0.05)。研究结论：采用慢性束缚+饮食失节+过度疲劳法制备的肝郁脾虚证模型大鼠表现出活动减少、扎堆多睡、情绪低落、争斗减少、饮食减少,皮毛枯乱,大便稀溏、体重减轻等,同时伴有自主活动增强的行为特征。经柴疏四君汤、四君子汤、柴胡疏肝散治疗2周后,可使模型大鼠外观表征呈现不同程度的改善,并对自主活动具有一定调节作用,其中以柴疏四君汤的干预效果最佳,柴胡疏肝散和四君子汤在改善模型大鼠外观表征中的行为状态、情绪反应和皮肤毛发、睡眠状态等方面各有侧重。结果表明,该模型具有类似于肝郁脾虚证的中医证候的一些特点,柴疏四君汤与该证模型具有更高的关联性,符合“疏肝健脾与肝郁脾虚证相应”的中医证治经验。本研究为肝郁脾虚证模型的中医证候学评价提供一定的研究思路,为理解“方证相关”的内涵及认识相关方剂的功用提供了一定的现代依据。更多还原

【Abstract】 Prescription is a tool with a specific function and the main treatment for disease prevention, Which is made by a number of herbs compatibility. There is a a high degree of correlation or focused references between the compatibility their primary disease pathogenesis or pathological evidence. This is called "The correlation between formula and syndrome."Evidence for a specific disease may have a few formulas work, but the results are different, or the same role in different disease and syndrome prescriptions would be different treatment. "The correlation between formula and syndrome" is the core principle of traditional Chinese medicine dialectical thinking, but also to learn prescription based on the logic of justification. Study the relationship between the formula and the syndrome to reveal the syndrome differentiation theory has important theoretical and practical significance.this study in rats with chronic astricting+diet+excess fatigue to replicate animal model of liver depression and spleen deficiency.To act as entry points from the appearance of animal model to compare the observations of rats’the appearance of behavioral change on the role of the differences and similarities, with the Liver, Spleen, Liver Spleen different effects of chaihushugansan. Sijunzitang and chaishusijuntang.And then and the ascertain the central neurotransmitters and biochemical changes of the model animal and and the changes result in the different formulae.This dissertation contains of literature review and experimental research. Research evolutions of clinic and experiment about the model of stagnation of liver and deficiency of spleen (GYPX), chaihushugansan. Sijunzitang and chaishusijuntang are summed up.Experimental study observed stagnation and spleen deficiency syndrome in rats characterized by the appearance of behavior and central neurotransmitters and related biochemical changes in things and chaihushugansan. Sijunzitang and chaishusijuntang. relevant role of the models.Methods:Rats were randomly divided into normal control group, liver depression and spleen deficiency syndrome group (hereafter referred to as the model group), chaishusijuntang groups,SiJunzi tang group and chaihushugansan group. Each group has 12 annimals. the latter 4 groups of rats were placed in restraint devices every day 8:00 am restriction of 3 h,2:00 pm place filled with warm water (22±1℃) swimming in a large plastic bucket 10 min, every other day feeding (every other day ban food, enough to eat every other day) for 4 weeks; normal control group did not exert any stimulating natural feeding. At 15 days of modeling, the rats were treated with traditional Chinese medicine Chaishusijun Tang 4.27g/kg, Sijuzitang 4.53g/kg, ChauhuShugan San 4.2g/kg orally,1 time per day; model group with the control group were given distilled water for 14 days. And in the first 7d,14d,21d,28d, points early, middle and later time points the rats were observed and recorded changes in the behavior of state, activity level, emotional reactions, skin hair, food intake, sleep, stool state, etc.; In the first 7 days a week 2:00-2:30 pm Determination of body weight of rats in each group; in the first 14d,28d by open field test rats were recorded within 5min crawling cell number, number of rearing, modification time and Time on the central grid.8:00 am on the 29th day were decapitated, rapidly stripping the Ice hippocampus, hypothalamus and brain stem, placed in liquid nitrogen tanks stored, tested. High Performance Liquid Chromatography and Fluorescence Detection hippocampus Asp, Glu, GABA, Gly, Tau; measured by radioimmunoassay in the hypothalamus of cAMP, PGE2; by enzyme-linked immunosorbent assay in the brain stem P450 enzyme activity and content.Statistical Methods:The data were to described an x±S, After the single factor analysis of variance (one-way ANOVA) to test, the more groups were analyzed using Student Newman Keuls Test test. All data using SPSS 13.0 for windows statistical processing software.Results:1. Syndrome Model of rats and related changes in appearance characterized the role of herbs:1 week after modeling, model group, chaishusijuntang group, SiJunzitang group, chaihushugansan group’s behavioral activity, emotional reactions, skin hair, diet, sleep and defecate state all did not change. Model rats 2 weeks after modeling showed reduced activity, reduced the level of activity, mood irritability, food intake, fur ring true Chak, easy to wake up, then dry, etc.; 3 weeks after modeling,the model group began to slow down the movement, emotions stability, food intake, fur is not Chak, tired of lying, loose stools; 4 weeks after modeling, the significant reduction of activities, get together or even tired of lying to sleep, emotional state from the beginning of the irritability to depression, the struggle to reduce, food intake, coat dry mess, loose stool and so on. Compared with model group, the rats in the administration of Chinese medicine after 1 week (3 weeks after modeling), activity, mood stabilization, food has increased, fur, sleep and bowel movements were seen in varying degrees of improvement in the state. After 2 weeks of administration (4 weeks of modeling), chaishusijuntang group significantly increased behavioral activity, responsive, smooth fur close to the whole, food increased, the depth of sleep, moderate, formed stool;sijuzitang grope, the rats’fur, food, sleep and improvement of stool condition obviously; rats of chaihushugansan in behavioral state, activity level, emotional response and so on were see significant improvement.2. Syndrome Model of body weight change and related prescriptions in Action: During the experiment, body weight of the normal rats was significantly increased,while the body weight in model group and three treated group gained weight more slowly, in which the model group, chaishusijuntang Group and sijunzitang group body weight had a negative growth in the first weekend. The weigh of the model group rats, chauhushugansan group showed a downward trend,after 4 weeks.Compared with normal group, the body weight of model group and chaishusijuntang group, SiJunzitang group, chaihushugansan group were significantly decreased at the end of 1w,2w,3w,4w. the difference was significant (P <0.05). Compared with model group, three treated group no significant changes in body weight difference (P> 0.05); and three dose groups no significant difference in body weight change (P> 0.05).3. Syndrome Model of autonomous behavior in rats and related changes in the role of herbs:Compared with normal group, he number of crawling cells of model group and the three Chinese medicine groups significantly increased (P<0.01 or P <0.05) in the first 14 days of modeling, the difference; The rats of model group and chaishusijuntang vertical had increased, the difference was significant (P<0.05); model group, sijunzitang group modification time and the central grid residence time were extended, of which modifiede time’difference was significant (P<0.05). After 2 weeks of administration (At the 28th day), compared with the normal group, the number of rats crawling cells increased model group, sijunzitang group and chaishusijuntang group, the differences were significant (P<0.05), the number of rats crawling cells of chaishusijuntang close to the normal level (P> 0.05); The model group and sijunzitang group vertical increased in the number, the difference was significant (P<0.05), chaishusijuzitang group and chaihushugans group didn’t increased obviously (P> 0.05); The modification time and time of he central cell residence of model group and three Chinese medicine group, were extended, but the difference was not significant (P> 0.05). Compared with model group, crawling cell number and were s number of rearing of 3 Chinese medicine groups significantly lower. the number of crawling cells of chaishusijuntang group reduced significantly, the difference was significant (P<0.05).4. Syndrome Model of central nervous and related biochemical changes in the role of prescriptions:(1) Compared with with the normal group, hippocampus Asp (aspartic acid) of model group content tended to increase Glu (glutamic acid), GABA (y-aminobutyric acid), Gly (glycine), Tau (taurine) were decreased, but the differences were not significant (P> 0.05); PGE2 and cAMP in the hypothalamus changed not significantly different (P> 0.05); brainstem P450 enzyme activity and enzyme content in no significant difference (P> 0.05). (2) Compared with model group, three Chinese medicine group had no significant change in the above index (P> 0.05). Conclusions:The chronic restraint+diet+excess fatigue Disloyalty prepared Syndrome Model of rats showed reduced activities, get together to sleep, depression, fighting less, eating less, fur dry mess, loose stool, weight loss and so on, accompanied by the enhanced spontaneous activity behavior.2 weeks after the treat with chaishusijuntang, sijunzitang, chaihushugansan, rats can characterize the appearance of varying degrees of improvements and self-regulate certain activities.Then intervention effct of chaihusijuntang group is best,chauhushugansan group and sijuzitang has had play respective role in the behavioral state,emotional reactions and the skin hair, sleep and so on. These results show that the model is similar to the stagnation and spleen deficiency syndromes in some features, chaishusijuntang and this model has a higher correlation, consistent with "Shuganjianpi corresponding spleen deficiency and stagnation" in TCM experience.This study Syndrome Model of Syndrome Evaluation study provides some ideas for evaluating TCM Syndrome.lt also can provided some of modern basis for understanding the meaning"evidence related parties" and function of the relevant formulas.更多还原