【作者】 赵迎盼；

【导师】 唐旭东；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2012， 博士

【摘要】 本研究主要分为两大部分,分别是IBS-D肝郁脾虚型病证症结合大鼠模型的建立与评价的初步研究和肠安I号方干预实验性IBS内脏高敏感大鼠的脑肠轴作用机制研究。1 IBS-D肝郁脾虚型病证症结合大鼠模型的建立与评价的初步研究目的：建立一种疾病-证候-症状相结合的IBS-D肝郁脾虚型大鼠模型。方法：(1)模型建立方法：采用新生母子分离+慢性束缚+番泻叶灌胃法复制IBS-D肝郁脾虚型病证症结合大鼠模型。(2)模型评价方法：以结直肠扩张的疼痛阈值代表内脏高敏感性,评价”疾病”模型的建立；以旷场实验和血清D-木糖水平评价肝郁脾虚”证型”的建立；以排便粒数和稀便率评价腹泻”症状”的建立。结果：(1)造模结束时,不同组间大鼠的体重变化存在统计学差异(F=10.132,p=0.000<0.05),与正常组比较,三因素组大鼠的体重增长量较少,存在统计学差异p<0.05)。(2)造模结束后,各组大鼠的疼痛阈值存在统计学差异(F=41.299,p=0.000<0.05),三因素组大鼠疼痛阈值明显降低,与正常组相比存在统计学差异(p<0.05)。(3)束缚结束后,各组大鼠总穿格数、站立次数、修饰次数存在统计学差异(p<0.05),与正常组相比,三因素组大鼠总穿格数、站立次数、修饰次数明显减少(p<0.05)。(4)各组大鼠血清D-木糖含量存在统计学差异(F=143.614,p=0.000<0.05),与正常组相比,三因素组大鼠血清D-木糖含量均显著下降(p<0.05)。(5)造模结束时,三因素组大鼠排便粒数与正常大鼠存在统计学差异(p<0.05)。结论：应用SD大鼠进行新生母子分离+慢性束缚+番泻叶灌胃的复合造模,造模结束时三因素组大鼠体重增长速度减缓,结直肠扩张时疼痛阈值降低,内脏敏感性增高；旷场实验穿格数、站立次数及修饰次数均明显降低；血清D-木糖含量明显降低,小肠吸收功能下降；排便粒数及稀便率均明显升高,而结肠黏膜组织HE染色未见明显异常,符合IBS-D肝郁脾虚型疾病特点,可能是一种较好的研究中医药治疗IBS疗效机制的动物模型,但仍需要进一步的深入研究。2肠安I号干预实验性IBS内脏高敏感大鼠的脑肠轴作用机制研究目的：研究肠安Ⅰ号方对IBS内脏高敏感模型大鼠的作用机理,验证肠安Ⅰ号方改善IBS内脏高敏感性是基于脑和肠的双重作用的假设。实验一、肠安Ⅰ号对IBS模型大鼠体重及内脏敏感性的影响方法：(1)模型的建立与评价：采用慢性束缚应激结合游泳致疲劳法复制IBS内脏高敏感大鼠,以体重变化、内脏敏感性评价和病理组织学作为模型评价指标。(2)肠安Ⅰ号的干预作用：采用体重变化量评估体重的变化；大鼠结直肠扩张时大鼠腹壁回撤反射表现出3级反应时的压力阈值为疼痛阈值,以疼痛阈值评估大鼠内脏敏感性的变化。结果：(1)造模结束时,不同组间大鼠的体重变化存在统计学差异(F=3.551,p=0.000<0.05),与正常组相比,模型组(p=0.019)大鼠的体重增长量明显减少；组间大鼠的疼痛阈值存在统计学差异(F=8.311,p=0.000<0.05),与正常组相比,模型组大鼠疼痛阈值明显下降(p<0.05)；各组大鼠结肠病理组织HE染色各组均未见明显改变。(2)灌药4、8、12天及治疗结束时,不同组间大鼠的体重变化均无统计学差异(p=0.730,0.804,0.137,0.444)。用药结束后,各组大鼠直肠扩张时的疼痛阈值存在统计学差异(x 2=30.933,p=0.000<0.05)；与正常组大鼠相比,模型组大鼠疼痛阈值明显降低(x 2=24.8472；p=0.0000<0.05)；与模型组相比,氟西汀组、肠安Ⅰ号高、中、低剂量组大鼠疼痛和阈值明显升高(x 2=8.7969,13.9562,12.7508,13.8110；p=0.0123,0.0009,0.0017,0.0010<0.05)。实验二、肠安Ⅰ号对IBS模型大鼠结肠5-HT的影响方法：采用结肠免疫组织化学染色法结合结肠粘膜积分光密度、积分光密度组织比的半定量分析评价大鼠结肠5-HT水平。结果：用药结束后,各组大鼠结肠5-HT水平存在统计学差异(χ2=53.144,p=0.000<0.05)。与正常组大鼠相比,模型组大鼠结肠5-HT水平明显升高(x 2=38.2112；p=0.0000<0.05)。与模型组相比,得舒特组、肠安Ⅰ号高、中剂量组结肠5-HT水平显著下降(x 2=13.9439,14.6154,15.8966；p=0.0009,0.0004,0.0007<0.05)。实验三、肠安Ⅰ号对IBS模型大鼠血清5-HT的影响方法：采用酶联免疫吸附法测定大鼠血清5-HT水平。结果：用药结束后,各组大鼠血清5-HT水平存在统计学差异(x 2=53.125,p=0.000<0.05)。与正常组大鼠相比,模型组大鼠血清5-HT水平升高(x 2=34.4853；p=0.0000<0.05)。与模型组相比,得舒特组、肠安Ⅰ号高、中剂量组大鼠血清5-HT水平显著下降(x 2=19.6384,16.0064,16.6231；p=0.0001,0.0003,0.0002<0.05)。实验四、肠安Ⅰ号对IBS模型大鼠海马5-HT1a、BDNF基因表达的影响方法：采用荧光定量实时PCR方法检测大鼠海马5-HT1a、BDNF mRNA基因表达水平。结果：(1)用药结束后,各组大鼠海马5-HTla mRNA相对表达量存在统计学差异(x 2=59.606,p=0.000<0.05)。与正常组大鼠相比,模型组大鼠海马5-HT1a mRNA表达水平升高(x 2=40.5840；p=0.0000<0.05)。与模型组相比,肠安Ⅰ号高、中、低剂量组大鼠海马组织5-HT1a mRNA表达水平显著下降(x 2=11.6823,25.7737,14.5150；p=0.0029,0.0000,0.0007<0.05)。(2)用药结束后,各组大鼠海马BDNF mRNA的相对表达量存在统计学差异(x 2=45.914,p=0.000<0.05)。与正常组大鼠相比,模型组大鼠海马BDNF mRNA表达水平升高(x 2=36.7966；p=0.0000<0.05)。与模型组相比,氟西汀组、肠安Ⅰ号高、中、低剂量组大鼠海马BDNF mRNA表达水平升高下降(x 2=17.2327,10.9142,10.2546,13.6571；p=0.0002,0.0043,0.0059,0.0011<0.05)。实验五、IBS模型大鼠内脏敏感性与结肠黏膜5-HT水平的相关性研究目的：观察大鼠内脏敏感性与结肠黏膜5-HT水平的相关关系。方法：相关性分析采用线性相关分析的方法,若数据符合双变量正态分布,使用Pearson相关系数r；若不符合,采用Spearman相关系数rs。p<0.05为差异有统计学意义。结果：对所有大鼠的分析发现,疼痛阈值与结肠黏膜5-HT水平呈线性负相关关系(rs=-0.435,p=0.000),内脏敏感性与结肠5-HT水平呈正相关关系。实验六、IBS模型大鼠内脏敏感性与血清5-HT水平的相关性研究目的：观察大鼠内脏敏感性与血清5-HT水平的相关关系。结果：对所有大鼠的分析发现,疼痛阈值与血清5-HT水平呈线性负相关关系(rs=-0.484,p=0.000),内脏敏感性与血清5-HT水平呈正相关关系。实验七、IBS模型大鼠内脏敏感性与海马BDNF mRNA基因表达水平的相关性研究目的：观察大鼠内脏敏感性与海马BDNF mRNA基因表达水平的相关关系。结果：对所有大鼠的分析发现,疼痛阈值与海马BDNF mRNA基因表达水平呈线性负相关关系(rs=-0.550,p=0.000),内脏敏感性与海马BDNF mRNA基因表达水平呈正相关关系。实验八、IBS模型大鼠内脏敏感性与海马5-HT1a mRNA基因表达水平的相关性研究目的：观察大鼠内脏敏感性与海马5-HT1a mRNA基因表达水平的相关关系。结果：对所有大鼠、模型组及用药组大鼠的分析发现,疼痛阈值与海马5-HT1a mRNA基因表达水平呈线性负相关关系(rs=-0.629,-0.773,-0.324,p=0.000,0.015,0.030),内脏敏感性与海马5-HT1a mRNA基因表达水平呈正相关关系。结论：肠安I号的作用靶点部位涉及”脑-肠”两部分,可下调血清及结肠黏膜5-HT水平,下调海马组织中BDNF和5-HT1amRNA的表达；大鼠内脏敏感性的改变与血清及结肠黏膜5-HT水平、海马BDNF和5-HT1a呈线性相关关系,内脏敏感性的下调是以血清及结肠黏膜5-HT水平、海马BDNF和5-HT1a水平的下调为内在机制的,至于其相互间的复杂作用关系可能需要进一步的深入研究。更多还原

【Abstract】 There are two parts in this paper, including preliminary study of establishing a new disease-syndrome-symptom integrated IBS animal model and exploration of brain-gut axis mechanism of Chang’anyihao in treatment of IBS animal model.1 Preliminary study of establishing a new disease-syndrome-symptom integrated IBS animal modelObjective:To establish a new disease-syndrome-symptom integrated animal model of IBS-D due to syndrome of liver stagnation and spleen deficiency.Methods:(1)Model establishment:we combined mother-infant separation, chronic restraint and senna gavage to establish a new animal model of IBS-D due to syndrome of liver stagnation and spleen deficiency. (2)Model evaluation:we used pain threshold indicating visceral hypersensitivity to evaluate "disease" model; open field test and serum D-xylose level to evaluate "syndrome" model; defecation grain number and loose stool rate to evaluate "symptom" model.Results:(1)In the end, rat body weight change was significantly different (F=10.132, p=0.000<0.05). Compared with normal group, body weight gain of the three-factor rats was significantly decreased (p<0.05). (2)In the end, the pain threshold in different groups was significantly different (F=41.299,p=0.000<0.05), pain threshold of the three-factor rats was significantly declined compared with the normal group (p<0.05). (3)In the end, there was significant difference in open field test between groups (p<0.05). Compared with the normal group, total cross number, standing number and decoration number of three-factor rats were significantly dropped (p<0.05). (4)In the end, the serum D-xylose level of three-factor rats significantly went down (F=143.614, p=0.000<0.05). (5)In the end, defecation grain number and loose stool rate of the three-factor rats significantly went up (p<0.05). Conclusion:we combined mother-infant separation, chronic restraint and senna gavage and successfully established a new disease-syndrome-symptom integrated animal model of IBS-D due to syndrome of liver stagnation and spleen deficiency.2 Exploration of brain-gut interaction mechanism of Chang’anyihao in treatment of IBS animal modelObjective:To explore the brain-gut axis mechanism of Chang’anyihao in treatment of IBS animal model. Experiment 1.Chang’anyihao on body weight and visceral sensitivityMethods:(1)Model establishment and evaluation:we combined chronic restraint stress and forced swimming to replicate IBS visceral hypersensitivity rat model and used weight change, visceral sensitivity and histopathological biopsy as evaluation indicators. (2)Chang’anyihao in treatment:we used body weight change to assess weight gain and pain threshold to assess changes in visceral sensitivity.Conclusions:(1)In the end, weight changes between groups were significantly different (F=3.551,p=0.000<0.05). Compared with the normal group, body weight gain in the model group was significantly reduced (p=0.019). There were significant differences in pain threshold between groups (F=8.311,p=0.000<0.05). Compared with the normal group, pain threshold of the model group was significantly decreased (p<0.05). Histopathological HE staining in each group showed no significant changes. (2)On the 4th,8th and 12th day and at the end of treatment, the weight changes in different groups were not statistically different (p=0.730,0.804,0.137,0.444). In the end, there were significant differences in pain threshold between different groups (X 2=30.933,p=0.000<0.05). Compared with the normal group, pain threshold of the model group significantlty decreased (x 2=24.8472;p=0.0000<0.05). Compared with the model group, pain thresholds of fluoxetine group, Chang’anyihao high, medium and low-dose groups were significantly increased (x2=8.7969,13.9562,12.7508, 13.8110;p=0.0123,0.0009,0.0017,0.0010<0.05).Experiment 2.Chang’anyihao on colon 5-HT levelMethods:We used immunohistochemical staining method to evaluate 5-HT level in rat colon.Results:In the end,5-HT levels in rat colon were significantly different between groups (x2=53.144,p=0.000<0.05). Compared with the normal group, the 5-HT level in model group significantly went up (x2=38.2112;p=0.0000<0.05). Compared with the model group,5-HT levels of Schutte group, Chang’anyihao high and medium-dose groups significantly decreased (X 2=13.9439,14.6154,15.8966;p=0.0009,0.0004,0.0007<0.05).Experiment 3.Chang’anyihao on serum 5-HT level of IBS rat modelMethods:We used ELISA to detect serum 5-HT level.Results:In the end, serum 5-HT levels were significantly different between groups (x2=53.125,p=0.000<0.05). Compared with the normal group,5-HT level in the model group significantly went up (x2=34.4853;p=0.0000<0.05). Compared with the model group,5-HT levels of Schutte group, Chang’anyihao high and medium-dose groups significantly decreased (x 2=19.6384,16.0064,16.6231;p=0.0001,0.0003,0.0002<0.05).Experiment 4. Chang’anyihao on hippocampal 5-HT1a and BDNF gene expressionMethods:We used fluorescent quantitative real-time PCR to detect 5-HTla and BDNF mRNA gene expression level in rat hippocampal.Results:(1)In the end,5-HT1a mRNA expression levels were significantly different between groups (x2=59.606, p=0.000<0.05). Compared with the normal group,5-HTla mRNA expression level in the model group significantly went up (x2=40.5840;p=0.0000<00.05). Compared with the model group,5-HTla mRNA expression levels of Chang’anyihao high, medium and low-dose groups significantly decreased (x2=11.6823,25.7737,14.5150; p=0.0029,0.0000,0.0007<0.05). (2)In the end, BDNF mRNA expression levels were significantly different between groups (x2=45.914,p=0.000<00.05). Compared with the normal group, BDNF mRNA expression level in the model group significantly went up (x2=36.7966;p=0.0000<0.05). Compared with the model group, BDNF mRNA expression levels of fluoxetine group, Chang’anyihao high, medium and low-dose groups significantly decreased (x2=17.2327,10.9142,10.2546,13.6571;p=0.0002,0.0043,0.0059,0.0011<0.05).Experiment 5.Correlation between visceral sensitivity and colon 5-HT levelObjective:To investigate the relationship between visceral sensitivity and colon 5-HT level.Methods:We used Spearman correlation to assess the correlations between two parameters, differences were considered significant at p<0.05.Results:There was a linear negative correlation between pain threshold and colon 5-HT level (rs=-0.435,p=0.000).Experiment 6.Correlation between visceral sensitivity and serum 5-HT levelObjective:To investigate the relationship between visceral sensitivity and serum 5-the HT level.Results:Pain threshold and serum 5-HT level showed a linear negative correlation (rs=-0.484, p=0.000).Experiment 7.Correlation between visceral sensitivity and 5-HT1a mRNA level in rat hippocampalObjective:To investigate correlation between visceral sensitivity and BDNF mRNA expression level in rat hippocampal.Results:Pain threshold and 5-HT1a mRNA level in rat hippocampal showed a linear negative correlation (rs=-0.550, p=0.000).Experiment 8. Correlation between visceral sensitivity and BDNF mRNA level of rat hippocampalObjective:To investigate the correlations between visceral sensitivity in rats and hippocampal BDNF mRNA gene expression level.Results:Pain threshold and 5-HT1a mRNA level in rat hippocampal showed a linear negative correlation in all rats, model rats and treated rats (rs=-0.629,-0.773,-0.324,p=0.000,0.015, 0.030).Conclusion:Chang’anyihao can reduce serum and colon 5-HT levels and lower BDNF and 5-HT1a mRNA expression in rat hippocampus.更多还原