补肺汤通过调控TLR2/NF-κB信号转导通路改善COPD气道重塑机制研究

【作者】 文秀华；

【导师】 王飞；

【作者基本信息】 成都中医药大学 ， 中医内科学， 2016， 博士

【摘要】 [目的]探索补肺汤对COPD稳定期大鼠气道重塑的影响,求证补肺汤是否能通过TLR2/NF-κB信号转导通路来调节COPD大鼠免疫功能及抑制其气道炎症,为补肺汤的临床应用提供实验依据,从而进一步论证将“补肺益肾”法作为COPD稳定期中医药主要治疗措施的可行性和重要性,深入理解“肺肾气虚”这一COPD稳定期中医证型和“免疫功能低下”及“气道炎症”等客观指标的相关性,加深对COPD稳定期本质——肺肾气虚的认识,促进中医证型客观化、标准化、规范化,对指导中医药治疗COPD的基础研究和临床应用均具有重要的意义。[方法]将84只大鼠随机分为7组：假手术组、模型组、中药补肺汤免煎颗粒剂高剂量组(简称免高组)、中药补肺汤免煎颗粒剂中剂量组(简称免中组)、中药补肺汤免煎颗粒剂低剂量组(简称免低组)、中药补肺汤饮片汤剂组(简称汤药组)、阳性对照西药孟鲁司特钠组(简称西药组),每组12只。除假手术组外其余各组均在实验开始后第1天和第25天气道注射脂多糖,第2—8天、23—24天、26—28天不做任何处理以待大鼠恢复体力,第9—22天、29—36天每天烟熏1次；第37—44天每天烟熏2次,第45—52天每天烟熏3次。假手术组在第1天和第25天气道注射等量生理盐水,不予被动吸烟。造模成功后,第二天开始灌胃给药。假手术组、模型组给予生理盐水(10 ml/kg·d),西药组给予孟鲁司特钠(1.0 mg/kg·d),免高组、免中组、免低组分别给予补肺汤免煎剂(27.8 g/kg·d、 13.9 g/kg·d、7.0 g/kg·d),汤药组给予补肺汤水煎剂(13.9 g/kg·d),连续给药32天。第33天开始将大鼠股动脉切开,放血处死,取右肺组织观察其病理学变化,并用免疫组化S-P法检测TLR2、TLR4、MyD88、NF-κB蛋白基因的阳性表达,用Western-Blot法检测肺组织中TLR2、MyD88、NF-κB的含量；取支气管肺泡灌洗液,用ELISA法检测支气管肺泡灌洗液中IL-10、IL-6、IL-8、 NF-κB、TNF-α炎性因子的水平。[结果]1.一般情况：假手术组大鼠伤口恢复好,瘢痕小,营养状况良好,精神正常,目光有神,皮毛色白而光泽度好,活泼好动,反应灵敏,呼吸平稳,进食、进水及大便正常,无咳嗽、喘息等表现,未见异常呼吸道分泌物,未闻及哮鸣音；而其余各组在造模过程中逐渐出现营养状况不佳,活动减少,精神不振,双目无神,反应迟钝,进食量少,大便稀溏,皮毛枯槁发黄,失去光泽,易脱落,并出现倒竖现象,有时口角见分泌物流出,可闻及咳嗽及哮鸣音；各治疗组在给药后上述情况均有所好转,其中以免高组最为明显,其次是免中组和西药组。2.病理学检查：假手术组大鼠气管黏膜纤毛柱状上皮细胞完整,呼吸道及肺泡上皮结构完整,纤毛排列整齐,各级气管、支气管可见少量散在的淋巴细胞浸润,肺泡组织形态均匀、肺泡间质厚薄适当,肺泡区极少见到巨噬细胞,支气管腔中几乎没有分泌物和炎症细胞。模型组大鼠黏膜上皮不完整,气管、支气管纤毛上皮部分剥脱、杯状细胞及腺体显著增生、肥大,支气管黏膜皱襞增多、变长,突入管腔；各级支气管黏膜层、黏膜下层可见以淋巴细胞、巨噬细胞为主的炎细胞浸润,此外尚有浆细胞和少量中性粒细胞,支气管平滑肌增厚明显,小支气管腔内可见黏液栓及大量中性粒细胞；小支气管和肺小动脉旁可见大量吞噬了烟尘颗粒的巨噬细胞(尘细胞),部分巨噬细胞崩解破坏；终末细支气管远端狭窄,呼吸性细支气管及肺泡管囊状扩张,肺泡腔不规则扩大,肺泡壁变薄、胀大、破裂或融合形成肺大疱,免高组、免中组、汤药组与模型组相比,上述病变有所减轻。3.用ELISA法检测炎性因子：与假手术组相比,模型组大鼠BALF中IL-1 β、IL-6、IL-8、NF-κB、TNF-α的含量均有显著差异。与模型组相比：免高组、免中组、汤药组、西药组大鼠BALF中IL-1β的含量有显著差异；免高组、西药组、免中组大鼠BALF中IL-6的含量有显著差异；免高组、汤药组、西药组大鼠BALF中工L-8的含量有显著差异；免高组、西药组大鼠BALF中NF-κB的含量有显著差异；免高组、免中组、汤药组、西药组大鼠BALF中TNF-α的含量有显著差异。4.用免疫组化SP法检测蛋白基因的阳性表达：与假手术组比较,模型组大鼠肺组织中NF-κB、TLR2、TLR4、MyD88的表达均有显著差异。与模型组相比：汤药组、西药组、免高组大鼠肺组织中NF-κB的表达有显著差异；汤药组、西药组、免高组、免中组大鼠肺组织中TLR2的表达有显著差异；汤药组、西药组、免高组、免中组大鼠肺组织中TLR4的表达有显著差异；汤药组、免高组、免中组大鼠肺组织中MyD88的表达有显著差异。5.用Western-Blot法检测蛋白含量：与假手术组相比,模型组MyD88、NF-κB、TLR2的含量均有显著差异。与模型组相比：西药组、汤药组、免高组、免中组MyD88的含量有显著差异；西药组、汤药组、免高组、免中组NF-κB的含量有显著差异；西药组、免高组、免中组TLR2的含量有显著差异。[结论]1.补肺汤免煎剂、汤剂均可同时抑制COPD大鼠肺组织中TLR2、TLR4两种Toll样受体的活性,通过MyD88依赖性经典途径,调节下游NF-κB信号通路,减少IL-1 β、IL-6、IL-8、NF-κB、TNF-α等炎性因子的释放,从而改善气道重塑,但以TLR2通路为主,TLR4通路起协同作用。2.补肺汤通过调节TLR2/NF-κB信号通路改善COPD气道重塑的效果具有剂量依赖性。更多还原

【Abstract】 [objective]Exploring the influence of tonifying lung decoction on the stable period of rats with COPD trachea remodeling, verifying whether tonifying lung decoction can regulate immune function and inhibit trachea inflammation through TLR2/NF-κB signal transduction pathway of rats with COPD, providing experimental basis for the clinical application of tonifying lung decoction, thus, demonstrating the feasibility and importance of taking "strengthening lung and benefiting kidney" method as the main treatment method of traditional Chinese medicine (TCM) for the stable period of COPD further, understanding the correlation between the syndrome type of TCM——"qi deficiency of lung and kidney" and objective indicators, such as "immunocomprosised" and "trachea inflammation" deeply, deepening the understanding of essence of the stable period of COPD——"qi deficiency of lung and kidney", promoting objectification, standardization and normalization of syndrome types of TCM, showing significance to guide basic research and clinical application of the TCM therapy of COPD.[methods]84 rats were divided into 7 groups:control group, model group, the high dose of tonifying lung decoction of TCM granules group (hereinafter referred to as granules high group), the middle dose of tonifying lung decoction of TCM granules group (hereinafter referred to as granules middle group), the low dose of tonifying lung decoction of TCM granules group (hereinafter referred to as granules low group), the tonifying lung decoction of TCM liquid group (hereinafter referred to as liquid group), positive control western medicine Montelukast group (hereinafter referred to as western medicine group), with 12 in each group randomly. All the rats except for those in control group were injected lipopolysaccharide (LPS) into tracheas at the first day and the 25th day from the beginning of the experiment, didn’t accept any processing in the 2-8th days, the 23-24th days, and the 26-28th days for the rats restoring physical strength, were smudged 1 time per day in the 9-22nd days and the 29-36th days,2 times per day in the 37-44 days, and 3 times per day in the 45 to 52 days. The rats in control group were injected the same amount of normal saline (NS) into tracheas at the first day and the 25th day, and didn’t have passive smoking. After the success of modeling, gavage administration began from the second day. Control group and model group were given NS (10ml/kg·d), western medicine group was given Montelukast (1.0 mg/kg·d), granules high group, granules middle group and granules low group were given respectively tonifying lung decoction of TCM granules (27.8 g/kg·d、13.9 g/kg· d、7.0 g/kg·d), and liquid group was given tonifying lung decoction of TCM liquid (13.9 g/kg·d), for 32 days in a row. All rats were incided in the femoral artery from the 33rd day, bled to death, took the right lung tissue to observe the pathological change.detect TLR2, TLR4, MyD88, and NF-κB positive expression of protein genes by immunohistochemical SP method, and detect the content of TLR2, MyD88, and NF-κB in the lung tissue by Western Blot method, took the bronchoalveolar lavage fluid (BALF), and detect the level of IL-1 β、IL-6、IL-8、NF-κB、TNF-α inflammatory factors in BALF by ELISA method.[results]1. General situation:the rats in control group recovered well in the wound, were with little scar, good nutritional status, normal mental, eloquent eyes, white and lustrous fur and smooth breathing, were active and responsive, their eating, drinking and defecation were normal, they were without symptoms such as coughing and gasping, etc, without abnormal secretions in the respiratory tract, without wheezing sound; while the rest of the groups appeared gradually during the process of modeling poor nutritional status, decreased activity, depression, dull eyes, slowing to respond, less eating, loose stool, gloomy, vertical and easy to fall off fur, secretions discharged from the corners of the mouth sometimes, and the sound of coughing and wheezing could be heard; each treatment group was improved in the above situation after the treatment, among which the most obvious group was granules high group, followed by granules middle group and western medicine group.2. Pathological examination:the rats in control group were with complete tracheal mucosa ciliated columnar epithelium cells, respiratory tract and alveolar epithelial, regular cilia, a few scattered lymphocytes infiltrating in all levels of trachea and bronchi, uniform alveolar, proper thickness of alveolar interstitial, rarely macrophages in alveolar area, hardly any secretions and inflammatory cells in the bronchial lumen. The rats in model group were with incomplete mucosa epithelial, part stripped tracheal and bronchial ciliated epithelium, significantly hyperplasia and hypertrophy of goblet cells and glands, increased and extended bronchial mucosal fold into the lumen; inflammatory cells infiltration in all levels of bronchial mucosa and submucosa, mainly lymphocytes and macrophages, in addition, plasma cells, and small amounts of neutrophils, thickening of bronchial smooth muscle, mucous plugs and numerous neutrophils in the small bronchial lumen; there were a lot of macrophages which engulfed dust particles (dust macrophages) beside small bronchi and pulmonary artery, part macrophages collapse and destroyed; far-end of terminal bronchiole was narrow, respiratory bronchioles and alveolar ducts were cystic dilatation, alveolar space irregular expand, alveolar wall became thinning, swelling, and ruptured, or fused, so pulmonary bulla formed. Compared with model group, these lesions alleviated in granules high group, granules middle group and liquid group.3. Detecting inflammatory factors by ELISA method:compared with control group, the contents of all of IL-1β、IL-6、IL-8、NF-κB and TNF-a in BALF of rats in model group have significant difference. Compared with model group:the content of IL-1β in BALF of rats in granules high group, granules middle group, liquid group and western medicine group has significant difference; the content of IL-6 in BALF of rats in granules high group, western medicine group and granules middle group has significant difference; the content of IL-8 in BALF of rats in granules high group, liquid group and western medicine group has significant difference; the content of NF-κB in BALF of rats in granules high group and western medicine group has significant difference; the content of TNF-a in BALF of rats in granules high group, granules middle group, liquid group and western medicine group has significant difference.4. Detecting protein gene positive expression by immunohistochemical SP method:compared with control group, the expression of all of NF-κB,TLR2, TLR4 and MyD88 has significant differences in model group. Compared with model group:the expression of NF-κB has significant differences in liquid group, western medicine group and granules high group; the expression of TLR2 has significant differences in liquid group, western medicine group, granules high group and granules middle group; the expression of TLR4 has significant differences in liquid group, western medicine group, granules high group and granules middle group; the expression of MyD88 has significant differences in liquid group, granules high group and granules middle group.5. Detecting the contents of protein by Western Blot method:compared with control group, the contents of all of MyD88、NF-κB、TLR2 in model group have significant difference. Compared with model group:the content of MyD88 in western medicine group, liquid group, granules high group and granules middle group has significant difference; the content of NF- κB in western medicine group, liquid group, granules high group and granules middle group has significant difference; the content of TLR2 in western medicine group, granules high group and granules middle group has significant difference.[conclusions]1. Both tonifying lung decoction of TCM granules and liquid can inhibit activity of TLR2 and TLR4, which are the two kinds of toll-like receptors in lung tissue of COPD rats at the same time, regulate downstream NF-κB signal transduction pathway by classic way of MyD88 dependence, reduce the release of inflammatory factors such as IL-1β、IL-6、IL-8、 NF-κB、TNF-α, etc, thus, improve trachea remodeling, but which is mainly through TLR2 pathway and probably with joint effect of TLR4 pathway.2. Tonifying lung decoction has the dose dependency to improve trachea remodeling of COPD through regulating TLR2/NF-κB signal transduction pathway.更多还原