【作者】 刘雅；

【导师】 李晓辉；

【作者基本信息】 第三军医大学 ， 药理学， 2008， 博士

【摘要】 证是中医辨证论治的核心,证本质的深入阐释已经历史性地成为实现中医药学现代化的重要契机和突破口。中医证候众多,其中气虚血瘀证较为多见,与多种疾病相联系。近年来对气虚血瘀证的研究存在明显的局限性,这样一个证不能简单用血瘀证和气虚证来概括、等同。人类基因组计划的完成和后基因组时代的到来为全面认识证实质提供了可能的突破口。但目前关于气虚血瘀深层次的的分子机理的阐释仅仅是对个别基因的检测,不能从整体揭示证的实质。“芪归药对”是配伍理论丰富,临床应用甚广,颇具代表性的药对之一。该药对依据气血配伍原则组成,因其既能自成一方,又可作为基础要对配伍组方,而备受历代医家重视。但迄今为止,关于“芪归药对”相使及其益气活血效应的分子生物学基础等科学问题未见报道。本课题拟采用传统气虚致瘀动物模型,通过对气虚致瘀发展过程中动态基因表达谱的差异分析和比较研究,揭示气虚血瘀证的动态演变过程及其规律的分子基础;通过对证相关基因(群)功能的调控研究,初步阐明“芪归药对”益气活血效应的分子机理;同时,从“以药测证”的认识论角度反证气虚致瘀分子基础发现的合理性和科学性。方法:1.采用饥饿、过劳和寒凉复合因素复制大鼠气虚血瘀模型。实验周期4周。2.观察气虚血瘀发展中模型动物表征变化。动态监测气虚血瘀模型大鼠5、14、28天时的血液流变性、T淋巴细胞转化、T细胞亚型计数、肾上腺重量和肾上腺指数。3.全基因组芯片研究对照组大鼠和模型组大鼠5、14、28天时外周血细胞基因表达谱的变化。采用Gene Onology、GenMAPP2.1软件对比分析实验各时相点免疫功能差异表达基因生物学功能和信号通路。4.将SD大鼠分为对照组、气虚血瘀模型组、“芪归药对”高、中、低剂量组和黄芪组,造模方法同前。各剂量药对组分别灌胃给予相当于生药24、12、6g/kg体重的黄芪当归(5:1)水煎液,黄芪组灌胃给予相当于生药20g/kg体重的黄芪水煎液。5.动态监测“芪归药对”、黄芪组对气虚血瘀大鼠表征、血液流变性、T淋巴细胞转化和T细胞亚型计数、肾上腺重量的影响。6.采用实时荧光定量PCR检测实验5、14、28天时,“芪归药对”、黄芪对气虚血瘀大鼠外周血细胞IL-1β、TNF-α、HSP-70、NF-κB、p38MAPK和JNK六种因子mRNA的表达。7.观察“芪归药对”、黄芪对气虚血瘀大鼠胸主动脉病理变化的影响。8.采用实时荧光定量PCR检测实验28天时,”芪归药对”、黄芪对气虚血瘀大鼠胸主动脉组织中IL-1β、TNF-α、HSP-70、NF-κB、p38MAPK和JNK mRNA表达的影响;采用western blotting检测“芪归药对”、黄芪对气虚血瘀大鼠胸主动脉组织中NF-κB/p65和p-c-jun蛋白表达的影响。结果:1.模型动物表征与气虚血瘀临床表现一致。2.模型组大鼠全血粘度高切、中切与对照组相比无显著差异,全血粘度低切、全血还原粘度升高,28天时与对照组比较非常显著的升高(P<0.01),纤维蛋白原含量、血浆粘度升高,在14天、28天时与对照组相比明显增多(P<0.05或P<0.01);模型组大鼠RBC计数、RBC压积和RBC刚性指数在实验进程中均无明显变化,RBC聚集指数在5天与对照组比较有显著性的升高(P<0.05),28天时有非常显著的升高(P<0.01);RBC电泳时间延长,在14天、28天时与对照组比较差异显著或非常显著(P<0.05或P<0.01)。3.模型组大鼠CD4+T细胞计数减少,28天时与对照组相比差异显著(P<0.05);CD8+T、CD3+T细胞计数在5天时与对照组比较明显减少(P<0.05), 14、28天时与对照组相比减少的更为显著(P<0.01)。4.模型组大鼠外周血T淋巴细胞3H-TdR掺入量下降,与对照组相比有非常显著的差异(P<0.01)。5.对照组大鼠肾上腺重量在实验中一直增加,模型组大鼠肾上腺重量与对照组相比在第5天时有显著升高(P<0.05),14天时与正常组接近,28天显著低于正常组(P<0.05)。对照组大鼠肾上腺指数在实验中无明显改变,模型组大鼠肾上腺指数随造模时间延长显著或非常显著的增加(P<0.05或0.01)。与对照组比较非常显著的增加(P<0.01)。6.大鼠全基因组芯片结果显示差异表达基因生物学途径分类集中在细胞过程、代谢过程,生物调节;细胞组件分类主要集中在细胞组件、细胞器细胞器组件和大分子复合物;分子功能分类主要是结合分子、催化活性、酶调活性。实验14天时差异表达的基因数目最多。7.与免疫功能相关的差异表达基因共有212个,免疫功能差异表达基因主要与免疫应答相关,免疫应答差异表达基因参与众多免疫过程,如免疫效应过程、适应性免疫应答、体液免疫应答等。GenMAPP信号通路分析细胞因子和炎症反应通路变化最明显。8.与模型组比较,“芪归药对”高剂量组大鼠体重28天时非常显著的增加(P<0.01);大鼠游泳时间延长(P<0.05);与模型组比较28天时舌象评分非常显著的下降(P<0.01);低切全血粘度、全血还原粘度、纤维蛋白原含量下降,与模型组比较28天时差异显著(P<0.05);血浆粘度14天时开始非常显著的下降(P<0.01);RBC聚集指数与一直显著下降(P<0.05),RBC电泳时间缩短,5天、14天与模型组比较差异显著(P<0.05),28天差异非常显著(P<0.01);CD8+T细胞数量显著升高(P<0.05);外周血T淋巴细胞3H-TdR掺入量非常显著或显著性升高(P<0.01或P<0.05);肾上腺重量5天时非常显著的下降(P<0.01)。、大鼠体重、游泳时间、舌象评分与模型组比较均无明显差异。9.与模型组比较,黄芪组大鼠RBC电泳时间缩短,5天、28天与模型组比较差异显著(P<0.05);外周血T淋巴细胞3H-TdR掺入量14和28天时显著增加( P<0.05)。10.与模型组比较,“芪归药对”中、低剂量组大鼠体重、游泳时间、血液流变学、T细胞亚群计数、淋巴细胞转化和肾上腺重量均无显著差异。11.与对照组比较,模型组大鼠外周血细胞中IL-1β, HSP-70, NF-κB, p38MAPK和JNK mRNA表达较对照组显著升高(P<0.05或0.01),TNF-αmRNA表达在5、14天时较对照组非常显著升高(P<0.01)。与模型组比较,“芪归药对”高剂量组IL-1β, JNKmRNA表达非常显著下降(P<0.01);TNF-αmRNA表达在5、14天时非常显著降低(P<0.01);NF-κBmRNA表达28天时非常显著下降(P<0.01);HSP-70, p38MAPK mRNA表达在14、28天时非常显著下降(P<0.01)。黄芪组与模型组比较,IL-1β, JNKmRNA表达显著下降(P<0.05或0.01);TNF-αmRNA表达在5、14天时下降(P<0.05或0.01);HSP-70, NF-κBmRNA表达28天时非常显著下降(P<0.01);p38MAPK mRNA表达在14、28天时显著下降(P<0.05)。与黄芪组比较,“芪归药对”高剂量组IL-1β, JNKmRNA表达14天时显著下降(P<0.05或0.01)。12.模型组动脉内皮细胞有脱落,内膜明显增厚,中膜结构紊乱,平滑肌层明显增生;“芪归药对”组动脉内膜增生较模型组明显减轻,趋于正常动脉,黄芪组动脉内膜增生较模型组减轻但不明显,可见少量内皮细胞脱落。13.气虚血瘀大鼠动脉组织中TNFα、IL1、NF-κB、p38MAPK和JNKmRNA的表达均显著升高,与对照组差异非常显著(P<0.01), HSP-70 mRNA表达无变化。黄芪组、“芪归药对”组显著或非常显著降低TNFα、IL1、NF-κB、p38和JNKmRNA(P<0.05或0.01)的表达,对HSP-70 mRNA表达无影响。与黄芪组比较,“芪归药对”高剂量组IL-1和JNK mRNA表达显著降低(P<0.05或P<0.01)。14.模型组大鼠动脉组织中NF-κB/p65和p-c-jun蛋白表达非常显著的升高(P<0.01);“芪归药对”高剂量组大鼠动脉组织中NF-κB/p65和p-c-jun蛋白表达减少,(P<0.01);黄芪组大鼠动脉组织中NF-κB/p65和p-c-jun蛋白表达减少,与模型组相比差异显著(P<0.05);“芪归药对”降低NF-κB/p65、p-c-jun蛋白表达的能力更强,与黄芪组比较差异非常显著或显著(P<0.01或P<0.05)。结论1.长期饥饿、过劳、寒凉导致大鼠气虚,现代医学表征为毛色枯槁、体重下降、运动能力减低;随后出现舌象紫黯、鼠尾绀凉以及粘(全血粘度低切、全血还原粘度、血浆粘度的升高)、凝(纤维蛋白原含量增加)、聚(红细胞电泳时间延长、红细胞聚集指数增加)的血液流变学特点。2.气虚血瘀证动态演变过程中始终存在以T细胞数量、T细胞转化能力以及HPA轴免疫调节功能异常为主的免疫功能失衡。3.气虚致瘀证发展过程中涉及不同的生物学功能。气虚血瘀证是功能基因群而非单一基因的异常表达。4.淋巴细胞、补体、细胞因子等众多的免疫功能基因参与了气虚致瘀证的发展、变化,共同调控了免疫系统功能。5.“芪归药对”有效改善气虚血瘀模型动物表征和粘、凝、聚的血流状态;明显促进T细胞和肾上腺功能。6.“芪归药对”通过抑制NF-κB、p38MAPK和JNK多条免疫应答信号转导通路,进而调节IL-1β, TNF-α等细胞因子的表达来防治气虚血瘀证。更多还原

【Abstract】 As the root of treatment based on syndrome differentiation, treating principle and prescription, syndromes has been considered as a breakthrough for the modernization of TCM research, in which the essence of syndrome is of uttermost important. Within numerous syndromes in Chinese medicine, Qi deficiency and blood stasis syndrome is very important and universal. Although many researches have been reported, its essence is still unclear and it is not as simply as Qi deficiency or blood stasis. After the completion of Human Genome Project, the arrival of post genome era provides an opportunity to study the syndrome of TCM thoroughly. Due to the complexity of syndrome and the limitation of individual gene, it is very necessary to explore the essence of syndrome from the genome level. Astragalus and Angelica Compatibility (AAC) is one of typical couples with widely theoretical and clinical uses. The formation of this herb couple is guided under relationship of Qi and blood, and they can be used singly or with other herbs. But so far, the molecular biology mechanism about the effect of AAC on tonifying Qi and controlling blood circulation has not been reported. This study was designed to explore the dynamic evolution and molecular basis of Qi deficiency and blood stasis through gene expression profile on the animal model. Mechanism about the effect of AAC on tonifying Qi and controlling blood circulation was explained through modulation the functional genes.Methods1. The animal model of Qi deficiency and blood stasis was established through hungered, fatigued, and cold stimulated in rats. The whole experimental period was four weeks.2. The changes of hemorheological, T-lymphocyte transformation, T cell subtypes count, adrenal gland weight and adrenal gland weight index were observed to characterize Qi deficiency and blood stasis syndrome at 5th, 14th and 28th day during the development of this syndrome. 3. Gene expression profiles of syndrome development were detected using Affymetrix’s GeneChip Rat Genome 230 Arrays and analyzed with Gene Ontology and GenMAPP for the biology function and the signal pathway.5. Effects of AAC on hemorheology, T-lymphocyte transformation, T cell subtypes count and adrenal gland weight were determined at 5th, 14th and 28th day.6. Effect of AAC on mRNA expressions of IL-1β, TNF-α, HSP-70, NF-κB, p38MAPK and JNK in blood cells of rats with Qi deficiency and blood stasis were measured with real-time fluorescent quantitative-PCR at 5th, 14th and 28th day. And that in artery wall were determined at the 28th day.8. Effect of AAC on the pathologic alteration in rat artery wall with Qi deficiency and blood stasis were observed.9. Effect of AAC on NF-κB/p65 and p-c-jun protein expressions in rat artery wall with Qi deficiency and blood stasis were detected by western blotting.Result1. Animal model characterization was in line with clinical performance of Qi deficiency and blood stasis syndrome.2. In model group, whole blood viscosity at high shear rate and middle shear rate had no obvious difference compared with that of control group. Whole blood viscosity at low shear and whole blood reduced viscosity increased significantly at 28th day (P<0.01); plasma viscosity and fibrinogen content increased at 14th, and 28th day (P<0.01); RBC count, hematocrit and RBC rigid index were no obviously changed. RBC aggregation index and electrophoresis time were all increased at 14th and 28th day compared with that of control group(P<0.01).3. Compared with control group, CD4+T count decreased significantly at 28th day (P<0.05); CD8+T and CD3+T count decreased significantly at 5th day (P<0.05), 14th and 28th day (P<0.01).4. 3H-TdR incorporation in T lymphocytes in model group decreased markly compared with control group (P<0.01).5. Adrenal gland weight in control group was always increasing. Adrenal gland weight in model group increased significantly at 5th day (P<0.05), but that decreased significantly at 28th day compared with control group (P<0.05). 6. Rat Gene Chip was used for examination of gene expression profiles in rats with Qi deficiency and blood stasis syndrome and normal rat. Differential expression genes focused on cellular process, metabolic process and biological regulation according to biological process. Differential expression genes focused on cell, organelle, and organelle part according to cell component, which focused on binding, catalytic activity according to cell function. The number of differential expression gene was more at 14th day.7. There were 212 differential expression genes associated with immune function, which mostly focused on immune response. Analyzed by GenMAPP software indicated that the change of cytokines and inflammation response was obvious.8. In AAC high dose group, weight increased at 28th day (P<0.01), swimming time extended (P<0.05), and tongue texture score decreased significantly at 28th day (P<0.01); whole blood viscosity low shear, whole blood reduced viscosity and fibrinogen content all decreased significantly at 28th day(P<0.05), plasma viscosity decreased obviously at the beginning of 14th day(P<0.01), RBC aggregation index always decreased significantly (P<0.05), RBC electrophoresis time shorten (P<0.05 or 0.01); CD8+T count is increased obviously (P<0.05); 3H-TdR incorporation in T lymphocytes increased significantly (P<0.05 or 0.01); adrenal gland weight decreased obviously at 5th day (P<0.05); however there was no significant difference between AAC high dose group and model group in CD3+T, CD4+T countand CD4+T/CD+8T.9. In stragalus group,RBC electrophoresis time shorten at 5th and 28th day (P<0.05); 3H-TdR incorporation in T lymphocytes increased significantly at 14th and 28th day (P<0.05).10. Compared with model group, rat weight, swimming time, tongue texture score, hemorheoligy, T subtypes counts, 3H-TdR incorporation and adrenal gland weight in AAC middle and low dose group all did not change significantly; in stragalus group, there was only RBC electrophoresis time shorten obviously at 5, 28th day (P<0.05).11. In model group, TNF-α, IL1-β, NF-κB, HSP-70, p38MAPK and JNK mRNA expression in blood cell increased significantly compared with control group(P<0.05 or 0.01). In stragalus group, mRNA expression of IL1-βand JNK decreased significantly (P< 0.05 or 0.01); TNF-αdecreased at 5th, 14th day (P<0.05 or 0.01); p38MAPK decreased at 14th, 28th day (P<0.05); HSP-70 and NF-κB decreased at 28th day (P<0.01) compared with control group. In AAC high dose group, mRNA expression of IL1-βand JNK decreased significantly (P< 0.01); TNF-αdecreased at 5th, 14th day (P< 0.01); HSP-70, p38MAPK decreased at 14th, 28th day (P<0.01); NF-κB decreased at 28th day (P<0.01). Moreover, IL1-βand JNK mRNA expression in AAC high dose group decreased significantly compared with stragalus group(P<0.05 or 0.01).12. Pathological changes of artery in model group showed that vascular endothelium was injuryed and neointimal hyperplasia. Preventment and treatment of AAC high dose group was more obvious than stragalus group.13. In model group, TNF-α, IL1-β, NF-κB, p38MAPK and JNK mRNA expression in artery increased compared with control group, excluded HSP-70 (P< 0.01), and that in AAC high dose group and stragalus group decreased significantly (P<0.05 or 0.01). Moreover, IL1-βand JNK mRNA expression in AAC high dose group decreased significantly compared with stragalus group(P<0.05 or 0.01).14. In model group, NF-κB/p65 and p-c-jun protein expression in artery increased compared with control group (P< 0.01), and that in AAC hig hdose group and stragalus group decreased significantly (P<0.05 or 0.01) compared with model group. Compared with stragalus group, the effects of AAC high dose group was more obvious (P<0.05 or 0.01).Conclusion1. Hungered, fatigued, and cold stimulated in rats could induced Qi deficiency, then blood stasis coulde be resulted from and resulted in Qi deficiency.2. Changes of T-lymphocyte subtypes counts, T-lymphocyte transformation and modulation of PHA axis were involved in Qi deficiency and blood stasis. Immune system disorder was run through whole process of Qi deficiency and blood stasis.3. Rats gene profile indicated differentially expressed genes categories included different bioglogy pathway, molecular function and molecular component. Qi deficiency and blood stasis was abnormal expression of functional gene groups.4. The immunological profile of Qi deficiency and blood stasis syndrome was closely related to immune network disorder, including depression of T amd B cell activation, abnormal expression of complement system and cytokine genes, and inflammation activation.5. AAC could obviously prevent and treat Qi deficiency and blood stasis syndrome through improving symptoms,hemorheology, T-lymphocyte subtypes counts, T-lymphocyte transformation and modulation of PHA axis.6. The effects of AAC on Qi deficiency and blood stasis syndrome was brought from the regulating of cytokine network at multi-link and multi-target, NF-κB, p38MAPK and JNK signal transduction pathways included. Through which the immune system and whole body reached to a functional balance status. Supported by the adminicular evidence of“syndrome differentiation through drug effects”, the relationship between immune disorder of Qi deficiency and blood stasis syndrome and cytokine disorder was reasonable and scientific.更多还原