【作者】 单丽囡；

【导师】 刘小虹；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2012， 博士

【摘要】 研究目的：本课题旨在从细胞学、分子生物学和病理学三方面观察益气法、活血法、化痰法及其不同组合治疗方案对COPD大鼠气道炎症和气道重塑的影响和干预作用,从而对比和评价各不同治疗方法的疗效,并初步探讨其可能的作用机理,为中医药治疗COPD提供有效可靠的实验理论依据。研究方法：雄性SPF级SD大鼠90只,根据体重,随机分成9组(n=9)。同时,采用香烟熏结合气管内滴注脂多糖的方法建立COPD大鼠模型。具体实验分组及用药如下：①益气法组：给予益气组中药汤剂灌胃②活血法组：给予活血组中药汤剂灌胃③化痰法组：给予化痰组中药汤剂灌胃④益气活血法组：给予益气活血组中药汤剂灌胃⑤益气化痰法组：给予益气化痰组中药汤剂灌胃⑥活血化痰法组：给予活血化痰组中药汤剂灌胃⑦益气活血化痰法组：给予益气活血化痰组中药汤剂灌胃⑧模型组：给予生理盐水灌胃⑨空白组：给予生理盐水灌胃给药时间及次数：各组从造模后第四天开始起给药,每日一次,直至造模结束第5天,用药共30天。给药结束后,取材。采用细胞计数和HE染色法测量不同组别之间BALF中白细胞数量以及中性粒细胞、巨噬细胞和淋巴细胞的百分比,同时应用ELISA方法测定BALF中细胞因子IL-8和TNF-a的含量；免疫组化方法测量肺组织u-PA、TGF-p1和Elastin蛋白表达；显微镜下观测肺组织结构变化,并结合图像分析仪测量支气管总管壁和平滑肌厚度的改变。研究结果：1.各组BALF炎性细胞和细胞因子的变化各组白细胞计数,巨噬细胞、中性粒细胞和淋巴细胞百分比,细胞因子IL-8和TNF-a的含量相比较,结果显示：白细胞计数,巨噬细胞、中性粒细胞百分比,细胞因子IL-8和TNF-a含量存在统计学意义(F=121.04, P=0.000<0.05; F=64.97, P=0.000<0.05; F=77.83, P=0.000<0.05; F=23.4, P=0.000<0.05; F=17.2, P=0.000<0.05),淋巴细胞不存在统计学意义(F=0.74,P=0.653>0.05)。各组之间白细胞计数、巨噬细胞、中性粒细胞百分比,细胞因子IL-8和TNF-α含量两两比较结果显示：模型组与空白组、益气活血化痰组、活血化痰组、益气活血组、益气化痰组和活血组比较；空白组同益气活血化痰组、活血化痰组、益气活血组、益气化痰组、化痰组、活血组及益气组相比较；益气活血化痰组同益气组、化痰组、活血组及益气化痰组相比较；益气活血组、活血化痰组同益气组、化痰组、活血组相比较；益气化痰组、活血组同益气组和化痰组相比较,白细胞计数,巨噬细胞百分比和中性粒细胞百分比,以及细胞因子IL-8和TNF-α含量,有明显差异,具有统计学意义(P<0.05)；但是益气活血化痰组、活血化痰组及益气活血组相比较,益气化痰组和活血组相比较,模型组、益气组及化痰组相比较,白细胞计数、巨噬细胞百分比和中性粒细胞百分比,细胞因子IL-8和TNF-α含量无明显差异(P>0.05)。2.各组肺组织病理结构改变和支气管总管壁及平滑肌厚度的变化HE染色观察各组形态学变化：正常对照组气道壁薄,气道平滑肌薄；气管粘膜上皮排列规整,无脱落、粘膜上皮纤毛完整,排列整齐、管壁平滑、官腔无狭窄；未见杯状细胞增生及鳞状上皮化生；肺泡形态正常,间隔完整；未见炎症细胞。模型组显示：支气管黏膜上皮纤毛粘连、倒伏,部分气道有脱落；上皮细胞变性、坏死；出现鳞状上皮化生；杯状细胞数量增加；气道粘膜下腺体肥大增生,出现浆液腺泡粘液腺化生,部分支气管黏膜及腺体出现萎缩;小气道管腔狭窄；支气管壁充血水肿,有淋巴细胞和浆细胞浸润,并有管壁淋巴小结形成；肺泡扩张,出现间隔变窄破裂；气道壁增厚,气道平滑肌的厚度和正常组比较增厚明显。除益气组和化痰组外,其余各治疗组与模型组相比较,肺组织结构较好,炎症浸润较轻,气道壁增厚、气道平滑肌厚度减轻。且改善程度同模型组相比较由低到高分别为：活血组、益气化痰组、益气活血组、活血化痰组、益气活血化痰组。图像分析结果显示：各组支气管总管壁厚度和平滑肌厚度,结果显示都存在统计学意义(F=133.6,P=0.000<0.05；F=65.6,P=0.000<0.05)。各组之间支气管总管壁厚度和平滑肌厚度两两比较结果显示：模型组、益气组和化痰组同空白组、益气活血化痰组、活血化痰组、益气活血组、益气化痰组和活血组比较；益气活血化痰组、活血化痰组、益气活血组、益气化痰组及活血组同空白组相比较；活血组同和益气活血组、活血化痰组及益气化痰活血组相比较；益气化痰组同益气化痰活血组相比较,前者支气管总管壁厚度和平滑肌厚度增厚,差异有显著性,具有统计学意义(P<0.05)；但是益气活血化痰组、活血化痰组及益气活血组相比较,益气化痰组和活血组相比较,模型组、益气组及化痰组相比较,支气管总管壁厚度和平滑肌厚度无明显差异(P>0.05)。3.各组肺组织u-PA、TGF-p1和Elastin表达的变化采用Kruskal Wallis Test统计方法分析各组肺组织u-PA、TGF-β1和Elastin蛋白表达的变化,结果显示：各组肺组织u-PA、TGF-p1和Elastin蛋白表达,有明显差异,存在统计学意义(P=0.000<0.05)。各组之间肺组织u-PA、TGD-β1和Elastin蛋白表达两两比较结果显示：模型组、益气组及化痰组与正常对照组、益气活血化痰组、活血化痰组、益气活血组、益气化痰组及活血组相比较；益气化痰组及活血组同空白组相比较；活血组同和益气活血组、活血化痰组及益气化痰活血组相比较；益气化痰组同益气化痰活血组相比较,u-PA和TGF-β1蛋白表达增强,Elastin蛋白表达降低,,差异有显著性,具有统计学意义(P<0.05)；但是益气活血化痰组、活血化痰组及益气活血组相比较；益气化痰组和活血组相比较；模型组、益气组及化痰组相比较,u-PA、 TGF-β1和Elastin蛋白表达无明显差异(P>0.05)。研究结论：1.具有益气活血化痰、活血化痰、益气活血、益气化痰及活血功效的中药可以抑制COPD大鼠BALF中性粒细胞聚集和巨噬细胞的增多和活化,减少细胞因子IL-8和TNF-α的分泌,达到缓解炎症反应的疗效。2.具有益气活血化痰、活血化痰、益气活血、益气化痰及活血功效的中药可以抑制COPD大鼠肺组织u-PA和TGF-β1表达的同时,增强Elastin表达。3.具有益气活血化痰、活血化痰、益气活血、益气化痰及活血功效的中药可以改善COPD大鼠支气管总管壁和平滑肌的厚度。4.具有益气活血化痰、活血化痰、益气活血、益气化痰及活血功效的中药可能是通过调节COPD大鼠肺组织u-PA、TGF-β1和Elastin的表达,达到干预和延缓COPD气道重塑的进程。5.以益气、活血、化痰为主的三种治疗方法中,活血法更具疗效。6.益气活血化痰组、活血化痰组和益气活血组的功效优于益气化痰组和活血组。更多还原

【Abstract】 Objective:To observe the intervention effect of Supplementing Qi, activating blood circulation, resolving phlegm and its combination regimens on airway inflammation and airway remodeling of COPD rats from the aspect of cytology, molecular biology and pathology. Through comparing and evaluating the efficacy of different treatment methods, we explore its possible mechanism action, and provide effective and reliable experimental basis for TCM on COPD.Method:90SD male rats were randomly divided into nine groups, meantime COPD rat model was established by intratracheal instillation of LPS and exposure to cigarette smoking daily. Specific subgroups and delivery are as follows:①Supplementing Qi group:Administrated by decoction of supplementing Qi②Activating blood circulation group:Administrated by decoction of activating blood circulation③Resolving phlegm group:Administrated by decoction of resolving phlegm④Supplementing Qi and activating blood circulation group:Administrated by decoction of Supplementing Qi and activating blood circulation⑤Supplementing Qi and resolving phlegm group:Administrated by decoction of Supplementing Qi and resolving phlegm⑥Activating blood circulation and resolving phlegm group:Administrated by decoction of activating blood circulation and resolving phlegm⑦Supplementing Qi, activating blood circulation and resolving phlegm group: Administrated by decoction of Supplementing Qi, activating blood circulation and resolving phlegm⑧The model group:Administrated by physiological saline⑨The normal group:Administrated by physiological salineEach group was administrated from the fourth day of molding, once a day, medication for30days.Using cell counting and HE staining method to measure white blood cell count and the percentage of neutrophil, macrophage and lymphocyte in BALF, ELISA method to measure the content of cytokine IL-8and TNF-α in BALF, immunohistochemical method to measure the protein expression of u-PA, TGF-β1and Elastin in lung tissue, to observe the pulmonary tissue structural changes under the microscope, and measure the thickness of bronchial duct wall and smooth muscle with image analyzer.Result:1. The changes of inflammatory cells and cytokines in BALFThere was significant difference in different groups in white blood cell count,the percentage of neutrophil, macrophage and lymphocyte, and the content of cytokine IL-8and TNF-α in BALF (F=121.04, P=0.000<0.05; F=64.97, P=0.000<0.05; F=77.83, P=0.000<0.05; F=23.4, P=0.000<0.05; F=17.2, P=0.000<0.05). There was no significant difference in different groups in lymphocyte (F=0.74, P=0.653>0.05). The comparison of each two groups in white blood cell count,the percentage of neutrophil, macrophage and lymphocyte, and the content of cytokine IL-8and TNF-α in BALF show:the model group comparison with control group, supplementing Qi, activating blood circulation and resolving phlegm group, activating blood circulation and resolving phlegm group, supplementing Qi and resolving phlegm group, supplementing Qi and activating blood circulation group, activating blood circulation group; the control group comparison with supplementing Qi, activating blood circulation and resolving phlegm group, activating blood circulation and resolving phlegm group, supplementing Qi and resolving phlegm group, supplementing Qi and activating blood circulation group, activating blood circulation group, resolving phlegm group, and supplementing Qi group; supplementing Qi, activating blood circulation and resolving phlegm group comparison with activating blood circulation group, resolving phlegm group, supplementing Qi group, and supplementing Qi and resolving phlegm group; supplementing Qi and activating blood circulation group,activating blood circulation and resolving phlegm group comparison with activating blood circulation group, resolving phlegm group, supplementing Qi group; supplementing Qi and resolving phlegm group and activating blood circulation group comparison with resolving phlegm group and supplementing Qi group, there was significant difference (P<0.05). But comparison among supplementing Qi, activating blood circulation and resolving phlegm group, activating blood circulation and resolving phlegm group, and supplementing Qi and activating blood circulation group; supplementing Qi and resolving phlegm group and activating blood; the model group, resolving phlegm group, and supplementing Qi group was no significant difference (P>0.05).2. The thickness changes of bronchial duct wall and smooth muscleThe morphologic changes of lung:the control group was normal, while the model group was showed significant pathological changes in the lung. While the other group except for resolving phlegm group, and supplementing Qi group, was improved in different level. The level of improvement from high to low was supplementing Qi, activating blood circulation and resolving phlegm group, activating blood circulation and resolving phlegm group, supplementing Qi and resolving phlegm group, supplementing Qi and activating blood circulation group, activating blood circulation group.The statistical results show:There was significant difference in different groups in the thickness of bronchial duct wall and smooth muscle (F=133.6, P=0.000<0.05; F=65.6, P=0.000<0.05). The comparison of each two groups in the thickness of bronchial duct wall and smooth muscle show:the model group, supplementing Qi group and resolving phlegm group comparison with control group, supplementing Qi, activating blood circulation and resolving phlegm group, activating blood circulation and resolving phlegm group, supplementing Qi and resolving phlegm group, supplementing Qi and activating blood circulation group, activating blood circulation group; the control group comparison with supplementing Qi, activating blood circulation and resolving phlegm group, activating blood circulation and resolving phlegm group, supplementing Qi and resolving phlegm group, supplementing Qi and activating blood circulation group, activating blood circulation group; activating blood circulation group comparison with supplementing Qi and activating blood circulation group, activating blood circulation and resolving phlegm group, supplementing Qi, activating blood circulation and resolving phlegm group; supplementing Qi and resolving phlegm group comparison with supplementing Qi, activating blood circulation and resolving phlegm group, there was significant difference (P<0.05). But comparison among supplementing Qi, activating blood circulation and resolving phlegm group, activating blood circulation and resolving phlegm group, and supplementing Qi and activating blood circulation group; supplementing Qi and resolving phlegm group and activating blood; the model group, resolving phlegm group, and supplementing Qi group was no significant difference (P>0.05).3. The changes of protein expression of u-PA, TGF-β1and Elastin in lung tissue The statistical results show:There was significant difference in different groups in the protein expression of u-PA, TGF-β1and Elastin of lung tissue (P=0.000<0.05). The comparison of each two groups in the protein expression of u-PA, TGF-β1and Elastin of lung tissue show:the model group, supplementing Qi group and resolving phlegm group comparison with control group, supplementing Qi, activating blood circulation and resolving phlegm group, activating blood circulation and resolving phlegm group, supplementing Qi and resolving phlegm group, supplementing Qi and activating blood circulation group, activating blood circulation group; supplementing Qi and resolving phlegm group and activating blood circulation group comparison with the control group; activating blood circulation group comparison with supplementing Qi and activating blood circulation group, activating blood circulation and resolving phlegm group, supplementing Qi, activating blood circulation and resolving phlegm group; supplementing Qi and resolving phlegm group comparison with supplementing Qi, activating blood circulation and resolving phlegm group, there was significant difference (P<0.05). But comparison among supplementing Qi, activating blood circulation and resolving phlegm group, activating blood circulation and resolving phlegm group, and supplementing Qi and activating blood circulation group; supplementing Qi and resolving phlegm group and activating blood; the model group, resolving phlegm group, and supplementing Qi group was no significant difference (P>0.05).Conclusion:1. The management of supplementing Qi, activating blood circulation and resolving phlegm group, activating blood circulation and resolving phlegm, Supplementing Qi and resolving phlegm, Supplementing Qi and activating blood circulation, activating blood circulation can inhibit accumulation, increase and activation of neutrophil and macrophage, reduce secretion of cytokine IL-8and TNF-α of BALF, and then relieve inflammation of COPD rat.2. The management of supplementing Qi, activating blood circulation and resolving phlegm group, activating blood circulation and resolving phlegm, Supplementing Qi and resolving phlegm, Supplementing Qi and activating blood circulation, activating blood circulation can inhibit the expression of u-PA and TGF-β1,meanwhile enhance the expression of elastin in COPD rat lung tissue.3. The management of supplementing Qi, activating blood circulation and resolving phlegm group, activating blood circulation and resolving phlegm, Supplementing Qi and resolving phlegm, Supplementing Qi and activating blood circulation, activating blood circulation can reduce thickening trend of bronchial duct wall and smooth muscle. 4. Tthe management of supplementing Qi, activating blood circulation and resolving phlegm group, activating blood circulation and resolving phlegm, Supplementing Qi and resolving phlegm, Supplementing Qi and activating blood circulation, activating blood circulation can intervent and delay COPD airway remodeling process, which maybe through the regulation of the expression of u-PA, TGF-β1and elastin in COPD rat lung tissue.5. Activating blood circulation group was effective comparing with resolving phlegm group and supplementing Qi group.6. The effect of supplementing Qi, activating blood circulation and resolving phlegm group, activating blood circulation and resolving phlegm group, supplementing Qi and activating blood circulation group is better than supplementing Qi and resolving phlegm group and activating blood circulation group.更多还原