【作者】 刘闰红；

【导师】 王文健； 张新民； 陈伟华；

【作者基本信息】 复旦大学 ， 中西医结合临床， 2008， 博士

【摘要】 背景呼吸道变应性疾病包括变应性鼻炎和哮喘,是在变应原的刺激下,机体免疫平衡失调引发的气道慢性变应性炎症。呼吸道变应性疾病的发病率有逐年上升的趋势,严重影响人类的健康和生活质量,现代医学对其至今没有根治的方法。中医药治疗变应性鼻炎和哮喘有着悠久历史和肯定的临床疗效,从中医药中开发防治呼吸道变应性疾病的药物前景广阔。中药复方“别敏”在澳大利亚进行了严格的随机、双盲、对照临床试验,结果显示:“别敏”能明显改善变应性鼻炎患者鼻痒、喷嚏、流涕、鼻塞等临床症状;使发作次数减少、程度减轻,血清IgE下降,随访显示其有效性可长达一年。然而人们对“别敏”的作用机理却知之甚少。为了提高“别敏”的临床疗效,寻找其有效成分、探索它的免疫调节作用机制是十分必要的,本课题在这方面做了些相关的工作。第一部分中药复方“别敏”对呼吸道变态反应的免疫调节作用初探目的利用呼吸道变态反应小鼠模型,验证中药复方“别敏”对呼吸道变态反应的临床疗效,并初步探索其免疫调节机制。方法将96只BALB/C小鼠随机分为6组:正常组、模型组、“别敏”大、中、小剂量组及阳性药组。采用卵清蛋白(OVA)腹腔注射及滴鼻造成呼吸道变态反应小鼠模型。在最后一次滴鼻后,计数5分钟内喷嚏数及抓鼻数,以反映小鼠行为学变化;以ELISA法检测血清总IgE、OVA特异性IgE含量的变化;以~3H-TdR掺入法检测脾脏淋巴细胞增殖能力。结果一、呼吸道变态反应小鼠模型行为学、IgE、脾细胞增殖变化与正常组比较,模型组小鼠无论是喷嚏数还是抓鼻数都显著增加(P＜0.01);血清总IgE、OVA-sIgE浓度也比正常组显著升高(P＜0.01);在PHA或OVA刺激下,其脾淋巴细胞增殖刺激指数显著升高(P＜0.05或P＜0.01)。二、“别敏”对呼吸道变态反应小鼠的干预作用1“别敏”对呼吸道变态反应小鼠行为学的干预作用与模型组比较,“别敏”大、中、小剂量组小鼠喷嚏数均显著降低(P＜0.01或P＜0.05),“别敏”大、中剂量组抓鼻数显著下降(P＜0.05)。2“别敏”对呼吸道变态反应小鼠血清总IgE和OVA-sIgE水平的影响各用药组血清总IgE、OVA-sIgE浓度与模型组相比均有下降趋势,其中“别敏”大剂量组小鼠血清总IgE、OVA-SIgE浓度以及中剂量组OVA-SIgE浓度与模型组比较差异有显著性意义(P＜0.05)。3“别敏”对呼吸道变态反应小鼠脾淋巴细胞增殖的影响“别敏”大、中剂量组脾淋巴细胞在PHA或OVA作用下的刺激指数与模型组比较显著降低(P＜0.05)。小结致敏小鼠模型能模拟呼吸道变态反应的临床症状及免疫紊乱特征,可用于进一步的药效及机制研究。中药复方“别敏”对呼吸道变态反应的临床疗效能在致敏小鼠模型上得以验证,其免疫调节作用可能与降低血清IgE含量和抑制淋巴细胞增殖有关。第二部分黄芩苷、淫羊藿总黄酮(EF)对模型小鼠呼吸道变态反应的干预作用及T辅助细胞分化平衡的影响目的黄芩苷、EF是“别敏”的质量控制标准成分;课题组同期研究发现“别敏”中的七个有效成分中,黄芩苷具有降低致敏大鼠鼻粘膜血管通透性,EF能抑制变态反应状态下脾淋巴细胞异常增殖。为此,本实验进一步在整体动物模型水平上研究“别敏”有效成分黄芩苷、EF对呼吸道变态反应干预作用的免疫机制,尤其是对对T辅助细胞分化平衡的影响。方法BALB/C小鼠随机分为6组:正常对照组、模型对照组、黄芩苷组、EF组、黄芩苷加EF组、地塞米松组。在最后一次滴鼻后,计数5分钟內喷嚏数及抓鼻数,以反映小鼠行为学变化。HE染色观察肺、脾组织病理,计数肺组织单个视野嗜酸性粒细胞数目。ELISA法检测血清细胞因子IFN-γ、IL-4、IL-5、IL-10、TGF-β。流式细胞仪分析脾脏IFN-γ~+CD4~+、IL-4~+CD4~+细胞百分率,计算Th2/Th1比值,流式分析脾脏CD4~+CD25~+T、CD4~+foxP3~+T细胞百分率。应用免疫磁珠分离各组小鼠脾脏CD4~+CD25~+T细胞,在抗原OVA刺激下,加入抗原递呈细胞(APC)后,分别与模型组小鼠脾脏CD4~+CD25-T细胞混合培养,检测上清细胞因子IFN-γ、IL-4、IL-5含量,并用~3H-TdR掺入法检测细胞增殖,来反映各组小鼠脾脏CD4~+CD25~+T细胞对模型组小鼠CD4~+CD25~-T细胞的抑制功能。结果1、模型组小鼠行为学、肺组织病理、T辅助细胞亚群分化的变化与正常组比较,模型组小鼠无论是喷嚏数还是抓鼻数都显著增加(P＜0.01)。模型组肺组织病理见大量嗜酸性细胞、淋巴细胞浸润,细支气管平滑肌增厚,部分细支气管缩窄,上皮增生、脱落;单个高倍视野嗜酸性细胞数目显著增加(P＜0.01)。模型组小鼠血清IFN-γ显著降低,IL-4、IL-5显著升高(P＜0.01);IL-10、TGF-β含量显著下降(P＜0.01);模型组小鼠脾脏Th2细胞百分率及Th2/Th1比值显著增高(P＜0.01),CD4~+CD25~+T细胞、CD4~+foxP3~+T细胞比例显著下降(P＜0.05)。模型组小鼠脾脏CD4~+CD25~+T细胞对CD4~+CD25~-T细胞增殖及产生细胞因子的抑制能力减弱(P＜0.01)。2、黄芩苷、EF对呼吸道变态反应小鼠行为学及肺组织病理的干预作用与模型组相比,黄芩苷组喷嚏数显著降低(P＜0.05),抓鼻数无明显变化。肺组织病理显示黄芩苷组嗜酸性粒细胞浸润程度显著改善,而淋巴细胞浸润无明显改善;个别细支气管上皮增生、平滑肌增厚。EF组喷嚏数显著低于模型组(P＜0.05),抓鼻数显著下降(P＜0.01)。肺组织病理显示EF能同时降低嗜酸性粒细胞和淋巴细胞浸润程度。个别细支气管平滑肌增厚。黄芩苷加EF组喷嚏数显著低于模型组(P＜0.01),抓鼻数显著下降(P＜0.01)。肺组织病理显示嗜酸性粒细胞和淋巴细胞浸润程度都有降低,少见细支气管平滑肌增厚。3、黄芩苷、EF对T辅助细胞分化平衡的影响与模型组比较,黄芩苷组小鼠血清Th2类细胞因子IL-5水平显著降低(P＜0.01),T调节类细胞因子TGF-β水平显著升高(P＜0.01);脾脏Th1(IFN-γ~+CD4~+)细胞百分率、Th2(IL-4~+CD4~+)细胞百分率、Th2/Th1比值以及CD4~+CD25~+T、CD4~+foxp3~+T细胞比例与模型组比较无显著变化(P＞0.05);黄芩苷组小鼠脾脏CD4~+CD25~+T细胞能抑制CD4~+CD25~-T细胞的增殖,但与模型组CD4~+CD25~+T细胞对CD4~+CD25~-T细胞的抑制能力比较无显著差异(P＞0.05)。与模型组比较,EF能显著升高血清Th1类细胞因子IFN-γ水平(P＜0.05),降低血清Th2类细胞因子IL-4、IL-5水平(P＜0.05),增加脾脏Th1(IFN-γ~+CD4~+)细胞百分率(P＜0.01),降低Th2(IL-4~+CD4~+)细胞百分率(P＜0.05),降低Th2/Th1比值(P＜0.05);EF组T调节类细胞因子IL-10、TGF-β含量均显著升高(P＜0.01);自然发生的T调节细胞CD4~+CD25~+T、CD4~+foxp3~+T细胞比例显著升高(P＜0.05);CD4~+CD25~+T细胞对CD4~+CD25~-T细胞增殖和产生细胞因子的抑制能力增强(P＜0.01或P＜0.05)。与模型组比较,黄芩苷加EF能显著升高血清Th1类细胞因子IFN-γ水平(P＜0.05),降低血清Th2类细胞因子IL-4、IL-5水平(P＜0.05),增加脾脏Th1(IFN-γ~+CD4~+)细胞百分率(P＜0.05),降低Th2(IL-4~+CD4~+)细胞百分率(P＜0.05),降低Th2/Th1比值(P＜0.01)。与模型组比较,黄芩苷加EF组T调节类细胞因子IL-10、TGF-β含量显著升高(P＜0.01);自然发生的T调节细胞CD4~+CD25~+T、CD4~+foxp3~+T细胞比例显著升高(P＜0.05);与模型组比较,黄芩苷加EF组小鼠脾脏CD4~+CD25~+T对CD4~+CD25~-T细胞增殖和产生细胞因子的抑制能力增强(P＜0.01)。小结黄芩苷与EF均能显著减轻呼吸道变应性炎症,但作用侧重点不同。黄芩苷能降低血清Th2细胞因子IL-5水平,升高血清T调节类细胞因子TGF-β水平,显著改善肺组织嗜酸性粒细胞浸润程度。EF能调节Th1/Th2平衡,升高血清T调节类细胞因子IL-10和TGF-β,提高自然发生的T调节细胞比例,增强其抑制功能;能同时降低肺组织嗜酸性粒细胞和淋巴细胞浸润程度。黄芩苷加EF体现出两者的综合抗变应性炎症作用,使T辅助细胞分化平衡更趋于正常状态。第三部分黄芩苷、EF对T辅助细胞株D10G4.1分化的调节作用及机制目的建立变态反应状态下的T辅助细胞模型,在细胞水平上观察黄芩苷、EF对T辅助细胞分化及细胞因子生成的调节作用及机制。方法在ATCC细胞株D10.G4.1培养介质中添加不同浓度IL-2,应用XTT法检测细胞活力,确定培养体系中IL-2浓度。用ELISA法检测培养上清液中细胞因子、流式细胞染色检测细胞表面标志及胞内细胞因子,半定量PCR法检测胞内foxp3mRNA表达,以鉴定该细胞株基本特性。在T辅助细胞系D10.G4.1细胞培养体系中,加入变应原伴清蛋白(CA)和抗原递呈细胞(APC),建立变态反应状态下的T辅助细胞模型。采用~3H-TdR掺入法检测细胞增殖能力,ELISA法检测培养上清IFN-γ、IL-4、IL-5、TGF-β水平。培养的D10.G4.1细胞在CA和APC存在条件下,分为模型组,黄芩苷低、中、高剂量组,EF低、中、高剂量组,黄芩苷加EF低、中、高剂量组,地塞米松组,其中各药低、中、高剂量组用药浓度分别为相应药物0.5ug/ml、5ug/ml、50ug/ml。采用~3H-TdR掺入法检测细胞增殖能力,ELISA法检测培养上清IFN-γ、IL-4、IL-5、TGF-β水平;对于模型组、黄芩苷中剂量组、EF中剂量组、黄芩苷加EF中剂量组、地塞米松组,采用半定量PCR法检测胞内foxp3m RNA表达。结果一、D10.G4.1细胞特性:1.D10.G4.1细胞表面表达CD4、CD25,静止(未受抗原刺激)状态下,细胞培养上清可检测到一定量的IFN-γ、IL-4、IL-5和TGF-β,胞内有少量foxp3mRNA表达。2.D10.G4.1细胞在CA刺激下(APC存在),IL-4和IL-5分泌增加(P＜0.01),IFN-γ分泌无变化(P＞0.05),TGF-β分泌减少(P＜0.01),向Th2极性偏移。D10.G4.1在CA刺激下不具有调节细胞活性,对同源小鼠CD25~-T细胞无抑制功能(P＞0.05)。二、黄芩苷、EP对D10.G4.1细胞增殖、分化的影响1.黄芩苷高剂量对D10.G4.1细胞增殖有显著抑制作用(P＜0.01)。黄芩苷高剂量能明显降低D10G4.1细胞培养上清IL-4水平(P＜0.05),在低、中、高剂量能显著降低培养上清内IL-5水平(P＜0.05或P＜0.01),而对IFN-γ无改变(P＞0.05),黄芩苷低、中剂量明显升高TGF-β水平(P＜0.05)。2.EF低、中、高剂量对D10.G4.1细胞增殖有显著抑制作用(P＜0.05或P＜0.01)。EF低、中、高剂量均能显著降低培养上清内IL-4及IFN-γ水平(P＜0.05或P＜0.01),升高TGF-β水平(P＜0.01),中、高剂量能明显降低培养上清内IL-5水平(P＜0.05)。3.黄芩苷加EF低、中、高剂量均能显著抑制D10G4.1细胞增殖(P＜0.05或P＜0.01)。黄芩苷加EF能明显降低培养上清内IL-4、5及IFN-γ水平(P＜0.05或P＜0.01),升高TGF-β水平(P＜0.01)。4.黄芩苷中剂量组foxp3mRNA表达与模型组比较无明显变化。EF中剂量组foxp3mRNA表达明显增加(P＜0.05),黄芩苷加EF中剂量组foxp3mRNA表达显著增加(P＜0.05)。三、黄芩苷、EF对D10.G4.1细胞凋亡的影响黄芩苷在50ug/ml以下没有明显的促D10.G4.1细胞凋亡作用。EF在100 ug/ml以下有抑制D10.G4.1细胞凋亡作用。小结D10.G4.1细胞是具有Th2分化潜能的活化T辅助细胞,是研究变态反应状态下T辅助细胞分化及药物作用的可靠细胞模型。黄芩苷、EF均能抑制D10.G4.1细胞的Th2分化趋势,促进抑制性细胞因子分泌。黄芩苷以抑制IL-5显著,EF能促进细胞抑制性转录因子Foxp3的表达。黄芩苷加EF体现黄芩苷和EF的综合作用,更有利于转化变态反应状态下T辅助细胞的分化极向偏移状态,调整T辅助细胞分化平衡。结论1.本研究在动物模型上验证了中药复方“别敏”的临床试验结果,证明了免疫调节是“别敏”治疗呼吸道变态反应疾病的重要药效学基础,其作用可能与降低血清总IgE、特异性IgE含量和抑制变态反应淋巴细胞增殖有关。这为进一步研究药效成分的作用和机制奠定了基础。2.本研究结果显示:在整体动物模型水平上,黄芩苷与EF均能显著减轻呼吸道变应性炎症,两者合用具有与“别敏”类似的抗变态反应作用,表明黄芩苷与EF可能是“别敏”的主要有效成分。3.本研究结果显示:黄芩苷与EF对致敏状态下的T辅助细胞亚群及相应的细胞因子有广泛的调节作用,但两者作用各有侧重,两者合用能使T辅助细胞分化更趋于正常平衡状态,体现了中医扶正祛邪的治疗原则和配伍用药的优势。4.本研究结果显示:黄芩苷与EF均能能抑制T辅助细胞系D10.G4.1细胞的Th2极化特性,促进调节性细胞因子的分泌,调整T辅助细胞分化的平衡,进一步验证了“别敏”发挥临床疗效具有免疫学基础。创新性1.本研究发现:淫羊藿总黄酮(EF)不仅能明显改善致敏小鼠的行为学变化,抑制肺部嗜酸性细胞、淋巴细胞浸润,还能调节Th1/Th2平衡,增加T调节细胞数量和功能,为此我们提出:EF具有抗变态反应作用。2.本研究发现:黄芩苷能降低IL-5水平,升高TGF-β,抑制气道嗜酸细胞浸润,为黄芩苷具有抗变应性反应作用提供了进一步的依据。3.本研究发现:T辅助细胞系D10.G4.1细胞不仅能分泌Th2细胞因子,而且能分泌Th1和调节性细胞因子,能表达抑制性转录因子FOXP3,是具有Th2分化潜能的T辅助细胞。首次提出了活化的T辅助细胞具有极向转化的潜力,否定了治疗中单纯抑制某类细胞的对立思维模式。4.本研究发现:黄芩苷和EF对D10.G4.1细胞具有不同程度的极向转化作用。与整体研究结果类似,黄芩苷主要降低IL-5水平,升高TGF-β,EF在降低IL-4、IL-5,升高TGF-β同时还能促进FOXP3表达。证实了黄芩苷和EF对T辅助细胞的极向转化的能力。5.本研究在整体动物模型水平上,通过行为学、组织病理T辅助细胞亚群以及其相应的细胞因子的含量和功能的检测,系统地研究了黄芩苷和EF的抗变态反应作用,隐约地感觉到黄芩苷和淫羊藿总黄酮之间具有一定的协同作用。如果这一现象被进一步证实,这将在中药有效成分水平体现了中医学扶正祛邪的治疗原则和中药方剂配伍的绝妙之处。更多还原

【Abstract】 BackgroundRespiratory allergy is chronic airway inflammation which is caused by antigen-induced immunological imbalance.In addition to IgE-induced mast cell degranulation,lymphocyte and eosinophils and other inflammatory cells and cytokines all contribute to chronic airway inflammation.The imbalance of T lymphocytes differentiation subsets, such as Th2 polarize and Treg（regulatory r cell） dysfunction,play an important role in the process of asthma and allergy.In treating respiratory disease,glucocorticoids andβ₂-adrenergic receptor stimulators and histamine antagonist may improve symptoms in the short term,but its side-effects can not be ignored.Therefore,it is necessary to explore safer and effective methods for treating respiratory allergy.Traditional Chinese medicines can be effective in treating allergic diseases based on symptom-specific treatment.A randomized,double blind, placebo-controlled,clinical trial was conducted to evaluate the effectiveness of the herbal formulation Biminne in the management of perennial allergic rhinitis.The results showed that Biminne significantly improved some of the symptoms,reduced serum IgE,and showed some potential long-term benefits in the treatment of allergic rhinitis. Our team previously observed that Biminne significantly prolonged the incubation period of asthma attack in allergic guinea pigs,and reduced the nose membrane permeability in allergic Brown Norway rats.The Biminne formulation is effective in treating allergic disease with its effect observed both in patients and respective animal models,However its mode of action is as yet unknown.The aim of this study is to explore the immunomodulating effect of the formulation Biminne and its components in the management of allergy,and use animal or cell models to identify its effective components.If this aim is achieved,we can use the effective components to form new formulations for more effective treatment.For this end,our team designed a series of experiments.This study is part of a larger study aimed at exploring the immunomodulating effect of the formulation Biminne,and looks at its components Baicalin and EF in treating respiratory allergic diseases,especially in regulating T helper cell differentiation。PARTⅠA Study of the immunomodulating effect of the traditional Chinese formulation Biminne in treating respiratory allergyObjective To test the effect of the formulation Biminne in treating mice model of airway allergy,and investigate its immunomodulating mechanism.Methods 96 BALB/C mice were randomly divided into 6 groups: normal group,model group,Biminne high-dose group,medium-dose group, low-dose group and Bidouyan group.The respiratory allergic model was induced by intraperitoneal and intranasal administration of ovalbumin （OVA） into mice.The occurrences of sneezing and nasal rubbing was counted; the proliferative capacity of splenocyte was tested with ³H-TdR and the levels of serum total IgE and OVA specific IgE were measured with ELISA.Results Biminne showed a positive effect in treating allergic rhinitis.The amount of sneezing and nasal rubbing increased markedly after allergy-induction modeling（P＜0.01）,the stimulation index（SI） of splenocyte proliferation stimulated by PHA and OVA elevated（P＜0.05 and P＜0.01 respetively）,and the levels of serum total IgE and OVA specific IgE increased significantly（both P＜0.01）.However the elevation in serum total and OVA specific IgE were able to be reduced by Biminne（P＜0.01 and P＜0.05 respectively）.Conclusion Respiratory allergy mice model could mimic clinic the symptoms and immuno-imbalance status of the disease,and could be used in the study on the effect and mechanism of formulations and components. Biminne has been shown in previous studies to be effective in the management of allergic rhinitis,and this study shows its immuno-modulating effect may well be related to inhibiting the proliferation of splenocyte and reducing the level of serum IgE which were increased by allergy.PARTⅡEffects of Baicalin and EF in the treatment of respiratory allergy and regulation of T helper cell differentiation in mice model of airway allergyObjective To test the effect of Baicalin and EF in the treatment of a mice model of airway allergy,and investigate its immunomodulating effect on T helper cell differentiation,including regulating Th1/Th2 balance and the number and function of T regulatory cells.Methods BALB/C mice were randomly divided to 6 groups:normal group, model group,Baicalin group,EF group,Baicalin plus EF group and Dexamisone group,with 12 mice in each.The respiratory allergy model was induced by the intraperitoneal and intranasal administration of ovalbumin （OVA） into mice.The occurrences of sneezing and nasal rubbing were counted.The lung and spleen tissues were fixed in buffered formalin for histopathology examination.Lung eosinophils per HP were counted under microscope.Serum cytokines IFN-γ、IL-4、IL-5、IL-10、TGF-βwere tested using ELISA.Spleen IFN-γ⁺CD4⁺ T,IL-4⁺CD4⁺T,CD4⁺CD25⁺T,and CD4⁺foxp3⁺T cells were determined using flow cytometry.Spleen CD4⁺CD25⁺T cells were isolated by magnetic separation.Under the stimulation of OVA,CD4⁺CD25⁺T cells of each group and APC co-cultured with and CD4⁺CD25^-T cells from model group,the level of IFN-γ,IL-4,IL-5 in culture supernatants were tested by ELISA,ceils proliferation was determined by ³H-TdR,to reflect the depressing function of CD4⁺CD25⁺T cells。The OVA-induced-CD4⁺CD25⁺T cells from each group were mixed with the antigen provider ceils（APC）,and then co-cultured with the CD4⁺CD25^-T cells from the model group.The level of IFN-γ,IL-4,IL-5,TGF-βin the culture supernatants were tested by ELISA. Ceils proliferation was determined by ³H-TdR,to show the suppression function of CD4⁺CD25⁺T cells。Results 1、Behaviors,lung tissue histopathology,T cell differentiation of the allergic model miceCompared with normal control group,The level of sneezing and nasal rubbing increased markedly after allergy-induction modeling（P＜0.01）. Examination of lung sections under light microscopy revealed an infiltration of eosinophils and lymphocytes into the airway of OVA-challenged mice in the immunized group.The percentage of Th2 cells and ratio of Th2/Th1 in the model group up-regulated significantly（P＜0.01）,while the percentage of CD4⁺CD25⁺T and CD4⁺foxP3⁺T cells downregulated（P＜0.05）.Meanwhile the level of IL-4andIL-5 increased significantly（P＜0.01）;but the level of serum IFN-γ,IL-10 and TGF-βdecreased significantly（P＜0.01）.The function of CD4⁺CD25⁺T cells decreased in depressing CD4⁺CD25^-T ceils（P＜0.01）.2、The effect of Baicalin and EF treatment on behaviors and lung tissue histopathology in respiratory allergic miceCompared with the model control group,the number of sneezes in five minutes in the Baicalin group decreased significantly（P＜0.05）.Lung tissue histopathology revealed that the infiltration of eosinophils was also reduced.In the EF group sneezing and nasal rubbing numbers decreased significantly（P＜0.05 and P＜0.01 respectively）.Lung tissue histopathology revealed that the infiltration of eosinophils and lymphocytes were also reduced.In the Baicalin plus EF group,sneezing and nasal rubbing levels decreased significantly（P＜0.01）.Lung tissue histopathology revealed that the infiltration of eosinophils and lymphocytes were reduced. 3、The regulating effect of Baicalin and EF on helper T cell differentiationBaicalin decreased the level of Th2 cytokine IL-5 significantly （P＜0.01）,and increased the level of regulatory T cytokine TGF-β（P＜0.01）。In the Baicalin group,spleen CD4⁺CD25⁺T cells showed suppression of the CD4⁺CD25^-T cell proliferation,but did not reach statistical significance compared to the model group.EF increased the level of Th1 cytokine IFN- significantly（P＜0.05）, decreased the level of Th2 cytokine IL-4 and IL-5（P＜0.05）,up-regulated the percentage of spleen Th1（IFN-γ⁺CD4⁺） cells（P＜0.01）,down-regulated the percentage of Th2（IL-4⁺CD4⁺） cells（P＜0.05）,reduced the ratio of Th2/Th1（P＜0.05）.Compared to the model group,the level of regulatory T cytokine IL-10 and TGF-βincreased significantly（P＜0.01）,the percentage of nTreg（CD4⁺CD25⁺T,CD4⁺foxP3⁺T） increased significantly（P＜0.05）,and the depression function of CD4⁺CD25⁺T versus CD4⁺CD25^-T increased（P＜0.01 and P＜0.05 respectively）。In the Baicalin plus EF group,the level of Thl cytokine IFN-γincreased significantly（P＜0.05）,the level of Th2 cytokine IL-4 and IL-5 decreased（P＜0.05）,the percentage of spleen Th1（IFN-γ2+CD4⁺） cell was up-regulated（P＜0.05）,the percentage of Th2（IL-4⁺CD4⁺） cells was down-regulated（P＜0.05）,and the ratio of Th2/ Th1 reduced（P＜0.01）. Compared to the model group,the level of regulatory T cytokine IL-10 and TGF-βincreased significantly（P＜0.01）,the percentage of nTreg （CD4⁺CD25⁺T,CD4⁺foxP3⁺T） increased significantly（P＜0.05）,and the depression function of CD4⁺CD25⁺T versus CD4⁺CD25^-T increased（P＜0.01）.Conclusion Both Baicalin and EF could reduce the allergic respiratory inflammation reaction,but their key functions are different. Baicalin was able to reduce serum Th2 cytokine IL-5 level,increase T regulatory cytokine TGF-β,and reduce the infiltration of eosinophils in the lung tissue。EF could regulate Th1/Th2 balance,increase serum T regulatory cytokine IL-10 and TGF-β,and up-regulate the percentage and suppressing function of nTreg.EF was shown to reduce the infiltration of eosinophils and lymphocytes in the lung tissue.The Baicalin plus EF group showed the total anti-inflammation effects of the two components,and improved normal immunity status of helper T cell differentiation. PARTⅢThe immunomodulating effect and mechanism of Baicalin and EF on the differentiation of T helper cell line D10G4.1Objective To establish an allergic model of T helper cell,and study the immunomodulating effect and mechanism of baicalin and EF on the differentiation of T helper cell line D10G4.1.Methods Different concentrations of IL-2 were added to the culture supernants of T cell lineage D10.G4.1.The cell viability was tested by XTT to ensure the IL-2 addition concentration in the culture supernatants. Cytokines in the culture supernatants were examined using ELISA,cell membrane markers and cytokines in the cell were examined by flow cytometry, and foxP3mRNA expression was examined by semi-quantitation RT-PCR,to determine the characteristics of D10.G4.1.Antigen conalbumin（CA） and antigen presenting cell（APC） was added to the culture supernants of D10.G4.1,to establish an allergic T helper cell model.Cell proliferation was tested by ³H-TdR,with the level of IFN-γ、IL-4、IL-5、TGF-βin the supernatants examined using ELISA。In the presence of CA and APC,D10.G4.1 cultures were divided into: model group,Baicalin high dose group,medium dose group and low dose group, EF high dose group,medium dose group and low dose group,Baicalin plus EF high dose group,medium dose group and low dose group,and dexamethasone group.Cell proliferation was tested by ³H-TdR,with the level of IFN-γ,IL-4,IL-5 and TGF-βin the supernatants were examined by ELISA, and foxP3 mRNA expression was examined by semi-quantitation RT-PCR.ResultsⅠcharacteristics of D10.G4.11.Cell marker CD4 and CD25 express on the D10.G4.1 cell membrane. Without antigen stimulation,a certain level of IFN-γ,IL-4,IL-5 and TGF-βcould be tested out in the supernants,and foxP3mRNA expression was positive.2.At the present of CA and APC,D10.G4.1 culture supernatants IL-4 and IL-5 level increased,TGF-βlevel decreased,IFN-γlevel didn’t change,showed Th2 polarization.But D10.G4.1 have no regulatory T cell activity and no depressing function to CD4⁺CD25^-T cell.ⅡEffect of Baicalin and EF on D10.G4.1 proliferation and differentiation1.Baicalin depressed D10.G4.1 proliferation significantly at the concentration of 50ug/ml.Baicalin decreased supernatant IL-4 levels, markedly decreased IL-5 levels,increased TGF-βlevels,but didn’t change IFN-γlevels.2.EF depressed D10.G4.1 proliferation significantly at the concentration of 0.5-50ug/ml.EF decreased supernatants IL-4,IL-5 and IFN-γlevels,while increasing TGF-βlevels.3.Baicalin plus EF decreased supernatants IL-4 IL-5and IFN-γlevels, increased TGF-βlevels.Conclusion:D10.G4.1 is a T helper cell line that contains the potential for Th2 polarization.It is useful to employ this cell line to study the differentiation of allergic T helper cells and the drugs to modulate allergic T helper cell differentiation.Baicalin could depress the Th2 differentiation polarization of D10.G4.1,in particular the depression IL-5 levels.EF could depress the Th2 differentiation polarization of D10.G4.1,and improve some regulatory characteristics.Baicalin combined with EF showed the total effects of the two components, transforming the allergic Th2 polarization of D10.G4.1 and regulating the helper T cell differentiation to normal immunity status.Conclusion1、Our study confirmed the clinical treatment effect of Chinese herbal formulation Biminne in respiratory allergy model,and immuno-modulation act as an important effective mechanism,reducing serum total IgE and OVA-sIgE and suppressing allergic lymphocyte proliferation may be one of Biminne’ s mode of action.This work formed foundation for further study of the effect and mechanism of other components.2、On the animal model level,baicalin and EF have the similar effect as Biminne on treating allergy,manifest that baiclin and EF are important effective components of Biminne.Both baiclin and EF could release respiratory allergic inflammation,regulate the allergic helper T cells and responding eytokines widely.But baiclin and EF focus on different aspects,the two together may make the T helper cell differentiation tending to normal.3、Baiclin and EF could suppress Th2 polarization characteristics of T helper cell line D10.G4.1,increase regulatory cytokines secretion, modulate the balance of T helper cell differentiation.The cell lineage experiment conformed further that Baiclin and EF could transform the allergic Th2 polarization.4、Use Baiclin and EF together,may suppress unnormal products,and promote normal immuno-modulation,embody the FuZhengQuXie regular of traditional Chinese medicine.更多还原