肺痹汤对大鼠肺间质纤维化干预作用的实验研究

【作者】 关天宇；

【导师】 周平安；

【作者基本信息】 北京中医药大学 ， 中医内科， 2007， 博士

【摘要】 本研究探讨了肺痹汤治疗肺间质纤维化的理论依据及其对博莱霉素诱导肺间质纤维化大鼠的影响。此次实验在文献回顾和导师经验总结的基础上进行了理论研究，认为气虚血瘀，痰热互结，痹阻肺络是本病的主要发病机制。针对肺间质纤维化的病因病机，明确了肺间质纤维化的辨证思路。在临床上制定了益气活血、清热化痰、通络开痹的治疗大法。由于此法针对性强且标本兼治在临床收到满意疗效。实验研究通过对博莱霉素诱导的7天、14天、28天不同病程的肺间质纤维化大鼠体重、细胞外基质、细胞因子、形态学以及分子生物等不同指标的观察，探讨肺痹汤抗肺间质纤维化可能的作用机制。实验研究采用国际上公认博莱霉素诱发肺纤维化的方法制作肺间质纤维化的模型，选用健康的成年SD大鼠180只，清洁级，体重200—250g。在1.5％戊巴比妥钠(2.5ml·kg^-1)腹腔注射麻醉条件下，局部消毒后切开大鼠颈部皮肤，采用钝性剥离暴露气管，在肉眼直视下用注射器将博莱霉素A₅缓慢注入大鼠气管内，随后分层缝合并局部消毒。雄性SD大鼠180只，体重200±10g，随机分为博莱霉素（BLM）组（30只）、假手术组（30只）、中药大、中、小剂量组（90只）、阳性药物组（30只）。选用在临床中疗效较好的肺痹汤，根据人与动物剂量进行等效换算。并根据药物浓度的不同分为大、中、小3个剂量组，小剂量：中剂量：大剂量=1：2：4，中药小剂量组给药量为5ml／kg，中药中剂量组给予10ml／kg，中药大剂量组20ml／kg，每周灌胃6天。阳性药物组选用临床常规用药强的松，灌胃给予3.5mg／kg，加水稀释为含生药0.35mg／ml，每周灌胃6天。假手术组及模型组均给予生理盐水2ml灌胃，肺间质纤维化造模成功后第1天各治疗组即开始给药。6组动物同步于实验开始后的第7、14、28天分别随机处死10只。用1.5％戊巴比妥钠腹腔注射麻醉(2.5ml·kg^-1)后将大鼠固定，腹主动脉取血，分离血清，同时取出肺脏；观察并记录大鼠的体重；采用放免法检测血清中透明质酸、Ⅲ型胶原及白介素-6的含量。采用右肺组织进行病理检测，行HE染色比较肺泡炎及肺纤维化程度。左肺采用免疫组化法检测肺组织中TGF-β1表达；左肺组织匀浆后，采用免疫印迹杂交法（Western blot）检测大鼠肺组织中p38分裂原激活蛋白酶（p38MAPK）表达。研究结果显示：在各个时间点，肺痹汤各剂量组较模型组可以抑制P38MAPK磷酸化活性和下调Ⅲ型胶原透明质酸和IL-6细胞因子含量，改善肺泡炎及肺纤维化的程度，降低TGF-β₁在肺组织中的表达。其中7d、14d、28d时肺痹汤中剂量的降低作用较为显著，并与大、小剂量组比较具有差异性。说明肺痹汤能显著抑制肺间质纤维化大鼠基质重建的作用。本课题的创新之处在于用实验研究的方法探讨了肺痹汤对肺间质纤维化干预的作用。肺痹汤治疗肺间质纤维化是通过干预P38MAPK转导通路，抑制P38MAPK磷酸化活性和下调Ⅲ型胶原透明质酸和IL-6细胞因子含量，降低TGF-β₁在肺组织中的表达等作用实现的。同时，以上的创新也为对肺间质纤维化的进一步研究奠定了基础。更多还原

【Abstract】 The research explored the theory basis of Feibitang on treating the pulmonaryfibrosis and its effects to pulmonary fibrosis rats caused by bleomycin.On the basis of literature review and tutor clinical practice experiencesummary, got the idea that the main pathogenesis of the disease is theqi-deficiency and blood stasis, the stasis of phlegm and heat, obstruction of thelung collateral. Making clear that the thought of pulmonary fibrosis according tothe pathological factor and pathology. Setting up the treating principle thattonifying qi and promoting blood circulation, clearing heat and removing thephlegm, expel the obstruction from collaterals. It has satisfied effects in clinic forits strong pertinence and both treating the mark and root.The experimental research observed the height、extracellular matrix、cytokine、morphology and molecular biology of the pulmonary fibrosis rats indifferent course of disease which is 7d、14d、28d induced by bleomycin. Exploringthe probable mechanism of Feibitang in the pulmonary fibrosis treatment.Establishing the pulmonary fibrosis rats mode by the method of bleomycininducement. Choosing 180 SD rats（SPF）, height200—250g. Anaesthesia methodwas 1.5% sodium pentobarbital abdominal injection, cutting the neck skin of therats after local sterilization. Blunting peeling off the trachea and injecting thebleomycin A5 into the trachea slowly, then stitch up the wound. 180 SD male rats,height 200±10g, dividing into BLM group（30 rats）、sham operated group（30 rats）、experimental large、middle、small group（90 rats）、positive drugs group（30 rats）.Converting the Feibitang dosage between human and animals according to theequivalent dosage. And dividing three different dosage group according to theconcentration, small:middle:large=1:2:4. The concentration of the small dosage group is 5ml/kg, the middle group is 10ml/kg, the large group is 20ml/kg, 6d drugsfor a week. The positive drugs group choosed the common drugs prednisone inclinic, sham operated group and control group were given 2ml normal saline perday. All the group were given the drug after the first day of the success modeestablish of the lung interstitial fibrosis. The six group rats were killed since the 7,14、28 days of the experimental. Getting the serum and the lung of the rats by the1.5% sodium pentobarbital abdominal injection anaesthesia. Observing andrecording the rats height, detecting the amount ofⅢtype collagen, hyaluronicacid and IL-6 in the serum with the method of the radioimmunoassay. Doingpathology detection with the right lung tissue and comparing the degree ofalveolitis and pulmonary fibrosis with the method of HE staining. Observing theexpression of the TGF-β1 in the left lung tissue with the method of immunehistochemistry. Detecting the p38MAPK expression in the lung tissue with themethod of Western blotting.The result showed that the experimental group can restrain thephosphorylation activity of P38MAPK and down-regulate the amount ofⅢtypecollagen hyaluronic acid and IL-6, improve the degree of alveolitis and pulmonaryfibrosis, decrease the expression of TGF-β1 in the lung tissue compared with themode group at all times. The most obvious decreased function of the Feibitang isthe middle-dosage group at 7 days. 14, 28d, the Feibitang function ofmiddle-dosage group is most significant. It indicated that the Feibitang canrestrain the matrix reconstruction in the lung interstitial fibrosis rats significantly.The innovation of the project is exploring the function of Feibitang ininterfering the pulmonary fibrosis. By interfering the P38MAPK transductionpassway, restraining the phosphorylation activity of P38MAPK and down-regulating the amount ofⅢtype collagen hyaluronic acid and IL-6,decreasing the expression of TGF-β₁ in the lung tissue. Meanwhile, thisinnovation settled the basis of the further research of the pulmonary fibrosis.更多还原