【作者】 顾文；

【导师】 周春祥；

【作者基本信息】 南京中医药大学 ， 中医临床基础， 2013， 博士

【摘要】 过敏性哮喘是一种由多种炎性细胞参与的气道慢性炎症性疾病,发病机制复杂。中医认为属“哮证”范畴,痰饮在其发生发展过程中占据主导作用。仲景《金匮要略·痰饮咳嗽病脉证并治篇》中提出“病痰饮者,当以温药和之”的治疗痰饮的总则,对痰饮的内治和外治均具有指导意义。本研究通过建立过敏性哮喘动物模型,在温化痰饮原则指导下,运用穴位敷贴这一临床常用且疗效确切的外治手段进行干预,探讨穴位敷贴治疗哮喘的效应机制,希望能为外治法领域的基础研究拓宽一些思路。目的：通过观察以温化痰饮立法的穴位敷贴对过敏性哮喘豚鼠肺组织嗜酸性粒细胞凋亡情况、凋亡相关基因bax和bcl-2表达情况,血清白介素-4和y-干扰素的含量变化以及转录因子T-bet和GATA-3mRNA的表达情况的影响,初步探索穴位敷贴对过敏性哮喘气道炎症的作用机理。方法：将Hartley豚鼠30只随机分为正常组、模型组、穴位敷贴组、非穴位敷贴组、阳性对照组,每组6只。采用卵蛋白腹腔注射致敏后呼吸道吸入激发法制备哮喘豚鼠模型。于造模成功次日给药,穴位敷贴组取肺俞、膻中、定喘穴进行敷贴,敷贴药物选取白芥子、麻黄、细辛、甘遂按一定比例以生姜汁调和,每次敷贴4h,每日1次,连续敷贴10天；阳性对照组以氨茶碱药饼敷贴,取穴部位及敷贴方法同穴位敷贴组；非穴位敷贴组取肺俞、膻中、定喘穴旁开1cm处,敷贴方法同穴位敷贴组；正常组、哮喘模型组不予治疗。治疗结束后对各组豚鼠体重、行为表现进行观察,对肺组织切片进行HE染色观察病理改变；TUNEL法检测肺组织EOS凋亡,免疫组化法检测肺组织凋亡相关基因bax和bcl-2的表达；ELISA法检测血清IL-4和IFN-γ的含量,RT-PCR方法检测转录因子T-bet和GATA-3mRNA的表达情况。所测数据均以均值加减标准差(X±s)表示,采用SPSS18.0统计软件分析数据,组间数据比较采用方差分析,P<0.05为差异有统计学意义。实验结果1.穴位敷贴对各组豚鼠一般情况及病理学的影响穴位敷贴治疗后,穴位组、非穴位组体重较模型组有不同程度增加,行为学评分较模型组有明显降低。HE染色显示,模型组可见：气道粘膜充血水肿,大量炎性细胞浸润,穴位敷贴组、非穴位敷贴组、阳性对照组与模型组相比,上述改变明显减轻。2.穴位敷贴对哮喘豚鼠EOS凋亡的影响穴位组、非穴位组、阳性对照组EOS计数与模型组相比,均明显下降,三组EOS凋亡指数与模型组相比显著增加(P<0.01)；穴位组、非穴位组、阳性对照组bcl-2表达均较模型组下降,各组bax表达均较模型组上升,且统计学差异显著(P<0.01)。3.穴位敷贴对哮喘豚鼠免疫调节机制穴位组、非穴位组、阳性对照组血清IL-4含量较模型组显著下降,穴位组明显低于非穴位组(P<0.01)；穴位组、非穴位组、阳性对照组血清IFN-y含量较模型组显著上升,穴位组明显高于非穴位组(P<0.01)。结论1.以温化痰饮治法为指导,运用穴位敷贴治疗过敏性哮喘可以获得较好的疗效。2.穴位敷贴可以改善发作期哮喘豚鼠的症状,减轻气道炎症反应。3.穴位敷贴可以通过上调bax的表达并抑制bcl-2(?)勺表达,达到促进EOS凋亡的目的。4.穴位敷贴能够通过调节转录因子T-bet和GATA-3的表达,从而影响细胞因子IL-4和IFN-y的分泌情况,纠正Thl/Th2失衡状态。更多还原

【Abstract】 Allergic asthma is a chronic inflammatory disease of the airways which involves a variety of inflammatory cells and has complicated pathogenesis. Chinese traditional medicine considers it as a "xiao" category. Phlegm retention plays a dominant role in the development of its process. Zhongjing proposed the general principle that when one has phlegm retention, one should be treated with warm herbs, which gave lots of guidances to both internal and externaltreatments. Under this guidance, we discuss the effect mechanisms of acupoint application on established animal model of asthma, hoping to broaden the field of basic research for external treatment.Objective By observing eosinophil apoptosis, expressions of apoptosis-related genes bax and bcl-2in lung tissue of allergic asthma guinea pigs, changes of serum interleukin-4and IFN-y contents, as well as the expressions of transcription factors T-betmRNA and GATA-3mRNA, we will preliminarily explore the effcet mechanisms of point application to airway inflammation in allergic asthma.Methods30Hartley guinea pigs were randomly divided into normal group, model group, acupoint Application group, non-acupoint application group and positive control group, six for each group. Inhalation and intraperitoneal injection of ovalbumin sensitization were carried out to estabilish guinea pig model of allergic asthma. The second day of successful modeling, animals in point application group were treated by placing Chinese medicine at the point Feishu xue、danzhong xue and Dingchuan xue for4hours a day,lasting for10days in all. Animals in positive control group were treated with aminophylline applicator in the same way as point application group. Animals in non-acupoint applicator group were treated with drugs at the places lcm next to Feishu, danzhong and Dinchuan. Points applicator group.Normal group and asthma model group were not treated. At the end of treatment, we observed animals’weight variation, behaviors and HE staining pathological changes of lung tissue sections.we used Tunel assay to test EOS apoptosis, immunohistochemical assay to test the expressions of apoptosis re-lated genes bax and bcl-2in lung tissue.We detected the content of serum IL-4and IFN-y by ELISA method and adopted RT-PCR to detect expressions of transcription factors T-bet and GATA-3mRNA. All data were shown in the form of(x±s) and analyzed with one-way ANOVA by SPSS18.0,a=0.05.Results1. Influences on general situation and pathological changes in each group of guinea pigsCompared with the model group, weights of acupoint application acupoint group and non-acupoint group increased to different degrees, and the behavioral score significantly reduced HE staining showed that in model group, bronchial mucosa epithelial cells fell off, goblet cells increased and inflammatory cells infiltrated, which took a turn for the better in acupoint application group, non-acupoint applicator group and positive control group.2. Influences on EOS apoptosis in guinea pigsCompared to model group, EOS count in positive control group, acupoint group and non-acupoint group, were significantly decreased, but EOS apoptotic index of these three groups showed a remarkable increase.The expression of bcl-2in acupoint group, non-acupoint group and positive control group decreased compared with model group, and the bax expression rose with statistically significant difference.3. Immune adjustment mechanisms of acupoint application to allergic asthmaIn acupuncture point group, non-acupuncture group and positive control group, the serum IL-4contents dropped significantly compared with model group.In acupuncture point group, non-acupuncture group and positive control group, the serum IFN-gamma increased significantly compared with the model group, and that of acupuncture group was more higher than acupuncture group.Conclusions1. Guided by the phlegm treatment, using acupoint sticking therapy for allergic asthma can achieve good effects.2. Acupoint application can improve the symptoms of the acute episode of asthma, and relieve the airway inflammation.3. Acupoint application can increase the expression of bax and suppress that of bcl-2, to achieve the goal of promoting EOS apoptosis.4. Acupoint application can correct the Thl/Th2imbalance state by regulating the expressions of transcription factors T-bet and GATA-3, which affect the secretion of IL-4and IFN-γ.更多还原