肝郁证雌性大鼠性腺轴的变化及柴胡疏肝散的干预作用

发布时间：2018-04-17 00:11

  本文选题：柴胡疏肝散 + 雌鼠　； 参考：《北京中医药大学》2017年硕士论文

【摘要】：目的:"方证相关"蕴含方药与病证之间存在某种对应的规律,中医方剂的研究应考虑到证候的选择性。中医证候通常具有跨系统和动态演变的特点,证候模型复制中需要注意跨系统多指标的综合评价。男女有异,生理有别,相同证候在不同性别患者身上存有差异,但目前基于性别差异的证候模型的系统探查及评价还很少见。本课题以女性肝郁证为切入点,侧重从性腺轴的角度,对束缚肝郁雌鼠涉及神经-内分泌的现代内涵进行探查;同时基于方证相关的理论,选择针对肝郁证的代表方柴胡疏肝散,观察该方对雌性肝郁模型大鼠的调节作用,为疏肝-肝郁证关联的现代生物学内涵提供理解。论文包括文献综述和实验研究两个部分,其中文献综述主要围绕中医肝脏与生殖系统、肝郁证与女性生殖疾病的关系进行相关文献的搜集与整理,提出女性肝郁证神经内分泌病生理的概念。实验研究探查了束缚肝郁模型雌鼠的中医证候和神经内分泌功能状态及柴胡疏肝散的相关作用。方法:大鼠随机分为正常对照组、肝郁模型组(以下简称模型组)、柴胡疏肝散大剂量组(柴大组)、柴胡疏肝散小剂量组(柴小组),每组10只。后三组按慢性应激束缚法造模连续4周,正常对照组不予处理。造模第2周末(造模第14天),柴大组和柴小组分别按相当于药材1.26g/kg和0.31g/kg的剂量(相当于人用量的2和0.5倍)给予柴胡疏肝散灌胃,每日1次,连续2周;模型组和正常对照组大鼠给予等量蒸馏水。于造模第27天,对各组大鼠阴道涂片进行观察,择动情前期/动情间期(实验第28-30天),禁食禁水12h,腹腔麻醉,经腹主动脉取血,分离血清与血浆;断头后低温下迅速取下丘脑、垂体、双侧卵巢等组织,分别于10%中性福尔马林中固定和液氮中速冻后置于-80℃冰箱保存,样本待测。观测指标:1)动情周期,阴道涂片观测造模前和给药前后的大鼠动情周期;2)记录造模期间大鼠的体重和外观行为(行为状态、活跃程度、情绪反应、睡眠状态、饮食状态、皮肤毛发),实验第2周末和第4周末做糖水偏嗜实验,4周末做旷场实验;3)放免法和酶联法测定下丘脑-垂体-肾上腺/甲状腺/性腺轴的相关指标。各组数据以x±s表示,采用SPSS 20.0软件处理,组间比较采用单因素方差分析(one-way ANOVA),用SNK法检验;非正态分布资料采用非参数统计分析。计数资料采用百分率表示,组间差异使用Fisher精确检验。所有图表均用GraphPad Prism 6 软件制作。结果:(1)证候学指标:①一般外观行为的变化:与正常组比较,模型大鼠造模第一周开始出现多动兴奋、烦躁易怒、大便干结,2周后逐渐出现扎堆少动,行动迟缓,饮食减少,大便干燥,毛发不荣;第2-4周的体重均见不同程度的降低(p0.05或p0.01)。第2周和第4周糖水偏嗜实验中糖水偏嗜度分别增加和降低(p0.01);第4周旷场实验中的中央区停留时间、穿格次数、直立次数均降低(p0.01)。与模型组比较,柴大组大鼠活动状态、情绪睡眠、饮食大便趋于适度;第3-4周体重均见明显升高(p0.05或p0.01)。第4周糖水偏嗜度增加(p0.01),旷场实验中的中央区停留时间、穿格次数、直立次数均见增加(p0.01);柴小组大鼠的上述指标无明显变化(p0.05)。与柴小组比较,柴大组大鼠第4周上述指标均有显著差异(p0.05或p0.01)。②实验室关联指标的变化:与正常组比较,模型组大鼠血中5-HT、DA、NE明显降低,E和β-EP明显升高(p0.01或p0.05)。与模型组比较,柴大组大鼠血中5-HT和NE明显升高,E和β-EP明显降低(p0.01或p0.05);柴小组大鼠血中DA明显升高(p0.01)。与柴小组比较,柴大组大鼠血中5-HT和NE显著升高、E显著降低(p0.01或p0.05)。(2)下丘脑-垂体-肾上腺皮质轴指标:与正常组比较,模型组大鼠下丘脑CRH、血清ACTH、CORT均显著升高(p0.01)。与模型组比较,柴大组大鼠下丘脑CRH、血清ACTH、CORT均显著降低(p0.01);柴小组大鼠CRH、ACTH明显降低(p0.01或p0.05),CORT呈降低趋势p0.05)。与柴小组比较,柴大组大鼠下丘脑CRH、血清CORT均显著降低(p0.01或p0.05)。(3)下丘脑-垂体-甲状腺轴指标:与正常组比较,模型组大鼠下丘脑TRH、血中T3、T4升高,TSH降低,差异均有统计学意义(p0.01或p0.05)。与模型组比较,柴大组大鼠下丘脑TRH、血中T3、T4降低,TSH升高,差异均有统计学意义(p0.01);柴小组大鼠下丘脑TRH、血中T3、T4降低,TSH升高,仅T4差异具有统计学意义(p0.05)。与柴小组比较,柴大组大鼠下丘脑TRH、血中T3、T4降低,TSH升高,差异均有统计学意义(p0.01或p0.05)。(4)下丘脑-垂体-性腺轴指标:①动情周期的变化:实验期间,正常组大鼠动情周期稳定,模型组大鼠的动情周期紊乱,呈现"前-中-后-间"延长或滞迟,实验第4周大鼠动情周期紊乱率为100%。与模型组比较,柴大组和柴小组大鼠动情周期紊乱率分别为20%和80%,其中柴大组紊乱率显著降低(p0.01)。与柴小组比较,柴大组大鼠动情周期紊乱率明显下降p0.05)。②下丘脑-垂体-性腺轴激素的变化:与正常组比较,模型组大鼠血中GnRH、FSH、LH、PRL、P、T、E2均显著升高p0.01或p0.05)。与模型组比较,柴大组大鼠血中GnRH、FSH、LH、PRL、P、T均显著降低(p0.01),E2有降低趋势;柴小组大鼠血中FSH、LH、PRL、P、E2均显著降低(p0.01),GnRH、T仅有降低趋势。与柴小组比较,柴大组大鼠血中GnRH和FSH降低显著(p0.01),其余指标差异无统计学意义(p0.05)。结论:慢性束缚法建立的雌性肝郁模型大鼠出现与中医证候相关的外观行为和实验室指标的变化,并伴有肾上腺皮质轴、甲状腺轴、性腺轴的功能亢进及动情周期紊乱,提示女性肝郁证存在神经-内分泌多系统的功能失调。中医肝郁证的治疗代表方-柴胡疏肝散对该雌性肝郁模型大鼠的中医证候和上述神经内分泌系统相关指标均有良好的改善作用,大剂量作用明显优于小剂量。该研究结果为认识女性肝郁证的现代病理生理内涵和中医方证相关(疏肝方-肝郁证关联)及柴胡疏肝散的临床应用提供了一定的实验证据。
[Abstract]:Objective: "there are some corresponding rules between the formula" contains prescriptions and syndromes of traditional Chinese medicine formula, consideration should be given to the selective syndrome. TCM syndrome usually has the characteristics of cross system and dynamic evolution of syndrome model replication should pay attention to in the cross system of multi index comprehensive evaluation. Men and women are different. Don't have the same physiological syndrome, there are some differences in the different gender of patients, but the current system of exploration and evaluation of syndrome model based on gender difference is still very rare. The female liver depression syndrome as the starting point, mainly from the gonadal axis angle, to detect the connotation of the modern female rat liver bound to neuroendocrine at the same time; formula based on the theory of choice for the party on behalf of Ganyu Chaihushugansan, regulated by the side of the female Ganyu model rats, to provide an understanding of modern biology - liver stagnation of the liver Qi connection of culvert. The paper includes two parts: literature review and experimental research, the literature review mainly focuses on the Chinese medicine liver and reproductive system, collect and collate relevant literatures on the relationship between liver stagnation syndrome and female genital diseases, female Ganyu concept of neuroendocrine disease physiology. Experimental study on exploration of the shackles of Ganyu model female rats of TCM syndrome and nerve endocrine function and Chaihushugansan related. Methods: the rats were randomly divided into normal control group, depression model group (hereinafter referred to as the model group), CHSGS group of large dose (Chai Dazu), CHSGS group of small dose (Chai Xiaozu), 10 rats in each group. The three groups according to the chronic stress binding method modeling for 4 consecutive weeks, normal control group were not treated. Modeling the second week (fourteenth day of modeling), Chai Da group and group Chai respectively according to the equivalent of 1.26g/kg and 0.31g/kg medicine dose (equivalent to the amount of 2 and 0.5 people Times) give CHSGS gavage, 1 times daily for 2 weeks; the model group and normal control group rats were given distilled water. On the twenty-seventh day of modeling, the rats vaginal smears were observed, choose proestrus / diestrus (the 28-30 day), fasting 12h, abdominal cavity anesthesia, abdominal aortic blood, serum and plasma separation; after decapitation under low temperature rapidly from the hypothalamus, pituitary, ovary and other tissues, respectively, to the preservation of frozen in liquid nitrogen fixation and placed in a -80 C refrigerator 10% neutral formalin in the sample. Observation index: 1) the estrous cycle, vaginal smear observation made and before the model before and after Administration of the estrous cycle of rats; 2) to record the weight and appearance and behavior during the modeling rats (behavior, active, emotional reactions, sleep, diet, skin and hair), experiment second and fourth weeks for sucrose preference test, 4 weekend to do field test; 3 ) related indexes of hypothalamic pituitary adrenal / thyroid / gonadal axis radioimmunoassay and ELISA method. All the data were expressed by X + s, using SPSS 20 software, compared with the single factor analysis of variance (one-way ANOVA), with SNK test method; non normal distribution data collected for statistical analysis non parametric. Count data expressed by the percentage difference between the two groups using the Fisher exact test. All charts are used GraphPad Prism 6 software. Results: (1) syndrome index: the change of general appearance behavior: compared with the normal group, model rats in the first week with excitement, irritability after 2 weeks, dry stool, gradually appear to get together to move less, slow, eating less, dry stool, hair glory; different degrees of reduction were seen in article 2-4 week weight (P0.05 or P0.01) for second weeks and fourth weeks. Sucrose preference test in sucrose preference degree were increased 鍜岄檷浣，

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