【作者】 杨晗；

【导师】 王飞；

【作者基本信息】 成都中医药大学 ， 中医内科学， 2013， 博士

【摘要】 肺纤维化(pulmonary fibrosis, PF)禹于纤维增殖性疾病的范畴,是多种原因所致的间质性肺疾病(interstitial lung disease,ILD)共有的病理基础过程,其病变主要累及肺间质,也可累及肺泡上皮细胞及肺血管,以肺成纤维细胞的异常增生和胶原蛋白为主的细胞外基质过度沉积为主要表现,至今发病机制不明,且没有理想的治疗方法。由于大多数肺部疾病最终转归为肺纤维化,并且随着污染、环境恶化等因素,肺纤维化当今在全球的发病率呈不断上升的趋势,已成为严峻的健康问题,因此对肺纤维化发生机制和有效药物的研究具有非常重要的科学价值。研究显示转化生长因子-β1(transforming growth factor-β1, TGF-β1)是致纤维化关键性的细胞因子,Smads通路介导的致纤维化作用与ERK通路介导的促增殖作用在纤维化的发生发展中均是不可或缺的。肺纤维化属中医“肺痿”、“肺痹”等范畴,其病机为气虚血瘀,故益气活血法是治疗肺纤维化的基本大法。我们在前期对益气活血法干预肺纤维化的研究中发现：补阳还五汤具有抑制实验性肺纤维化大鼠肺组织TGF-β1的过度表达,减少肺组织中羟脯氨酸含量,降低肺泡巨噬细胞肿瘤坏死因子含量的作用,提示补阳还五汤干预肺纤维化的作用可能与其调控TGF-β/Smad/ERK信号通路有关。基于此,我们以TGF-β/Smad/ERK信号通路为切入点,将研究靶点精准定位于肺纤维化形成的关键病理环节,探讨补阳还五汤拮抗肺纤维化的可能作用机制,以期拓展补阳还五汤的适用范围,为临床研究奠定基础。本研究以博莱霉素(bleomycetin, BLM)诱导肺纤维化大鼠模型为评价载体,从病理学、分子生物学角度探讨补阳还五汤调控TGF-β/Smad/ERK信号通路的作用机制。实验共分为四部分：1.补阳还五汤对博莱霉素所致肺纤维化大鼠肺指数及肺组织形态学的影响建立肺纤维化大鼠模型,通过补阳还五汤干预后,动态观察模型大鼠的一般情况(精神、摄食、毛色、活动、体重等)、肺指数,采集肺组织行HE染色和Masson染色,分析病肺组织肺泡炎及肺纤维化程度,探讨补阳还五汤抗肺纤维化的作用。结果：①-般情况：假手术组大鼠皮毛光泽,行动灵活,反应敏捷,食量正常；模型组大鼠食欲下降,毛发凌乱、稀疏、部分脱毛严重,对外界刺激反应慢、行动迟缓,体重增长缓慢；其余各给药组随实验时间的推进,活动减少、食量下降等症状有不同程度的改善,体重逐渐增加；②肺指数：与假手术相比,模型组大鼠各时间点肺指数增加明显⑦<0.01)；与模型组比较,各给药组大鼠肺指数有不同程度的下降(矿0.01)：与强的松对照组相比,补阳还五汤中剂量组第14d、28d、低剂量组第28d肺指数降低较明显p<0.05)；高剂量组三个时间点肺指数均高于强的松对照组,但与同期模型组相比仍较有明显的降低。③病理形态学：与假手术相比,模型组及各给药组大鼠可见肺泡结构有不同程度的破坏,肺间质增生,有炎细胞漫润和胶原纤维增生形成；与模型组相比,给药组各时间点的肺泡炎和肺纤维化程度均有不同程度的改善p<0.01);其中补阳还五汤中剂量组在中后期降低肺泡炎及纤维化程度方面明显优于同期强的松对照组p<0.05)。结论：博莱霉素诱导肺纤维化大鼠模型制备成功；补阳还五汤能够较好地控制模型大鼠肺指数的增长,抑制早期的肺泡炎和中后期的肺纤维化。2.补阳还五汤对博莱霉素所致肺纤维化大鼠血清纤维化指标PCⅢ、Ⅳ-C、HA、LN含量的影响建立肺纤维化大鼠模型,通过补阳还五汤干预后,采用放免法检测各组大鼠血清纤维化指标PCⅢ、Ⅳ-C、HA、LN的含量,验证补阳还五汤对肺纤维化的治疗作用。结果：①与假手术组相比,其它各组大鼠血清PCⅢ、Ⅳ-C、HA、LN的含量均逐渐升高,以模型组最为明显p<0.01)；②与模型组相比,各给药组大鼠血清PCⅢ、Ⅳ-C、HA、LN含量均有不同程度降低p<0.01);③与强的松对照组相比,补阳还五汤中剂量组第14d、28d以及补阳还五汤低剂量组第28d时,PCⅢ, Ⅳ-C、HA、LN含量下降明显p<0.05)。结论：经补阳还五汤治疗后的模型大鼠血清纤维化指标含量有所下降,提示补阳还五汤可以有效抑制病肺组织中胶原纤维的增殖以及细胞外基质的沉积,起到较好的抗肺纤维化作用。3.补阳还五汤调控肺纤维化TGF-β1/Smad通路机制研究建立肺纤维化大鼠模型,通过补阳还五汤干预后,采用免疫组化染色法(S-P法)检测不同时间点各组大鼠肺组织Col-Ⅰ、TPRⅠ、TpRⅡ、Smad3、Smad7的表达,逆转录聚合酶链反应(RT-PCR法)检测肺组织TGF-β1、Smad3、Smad7mRNA的含量,探讨补阳还五汤通过调控TGF-β1/Smad信号通路干预肺纤维化的作用机制。结果：①与假手术组相比,模型组大鼠肺组织Col-Ⅰ、TβPⅠ、TβRⅡ TGF-β1mRNA、Smad3mRNA表达水平明显上升,差异有统计学意义(p<0.01)；Smad7、Smad7mRNA表达下降(p＜0.01)；②与模型组相比,各给药组Col-Ⅰ、TβRⅠ、 TβRⅡ、TGF-β1mRNA、Smad3mRNA有不同程度的下降(p<0.01or p<0.05);Smad7? Smad7mRNA表达增高(p<0.01or p<0.05);③与强的松对照组相比,补阳还五汤中、低剂量组肺内Col-Ⅰ、TβRⅠ、TβRⅡ、TGF-β1mRNA、Smad3mRNA的表达降低(p<0.01or p<0.05), Smad7、Smad7mRNA的表达增高(p<0.05)。结论：补阳还五汤能够有效抑制TGF-β1/Smad信号通路中发挥效应的Col-Ⅰ、 TPRⅠ、TPRⅡ、TGF-β1、Smad3等因子的高表达,同时促进该通路中发挥负性调控效应的Smad7达增加,证实了补阳还五汤能够发挥较好的抗肺纤维化作用与其调控TGF-β1/Smad信号通路有关。4.补阳还五汤调控肺纤维化TGF-β1/ERK通路机制研究建立肺纤维化大鼠模型,通过补阳还五汤干预后,采用Western blot法检测不同时间点各组大鼠肺组织TGF-β1、ERK1/2、p-ERK的表达,探讨补阳还五汤通过调控TGF-β1/ERK通路干预肺纤维化的作用机制。结果：①与假手术组相比,模型组大鼠肺组织TGF-β1、ERK1/2、p-ERK表达水平明显增高,差异有统计学意义(p<0.01)；②与模型组相比,各给药组TGF-β1、ERK1/2、p-ERK有不同程度的下降,差异明显(p<0.01or p<0.05)；③与强的松对照组相比,补阳还五汤中剂量组在第14d、28d,低剂量组在第28d时TGF-β1、ERK1/2、p-ERK的表达明显降低(p<0.05)。结论：补阳还五汤可以有效抑制病肺组织TGF-β1、ERK1/2、p-ERK表达上调,提示补阳还五汤能够较好的调控肺纤维化中TGF-β1介导的ERK通路,进而抑制其产生的促增殖作用。综上所述,本研究采用博莱霉素气管滴注经典造模方法制备大鼠肺纤维化模型,以TGF-β/Smad/ERK信号通路为切入点,采用现代分子生物学等研究技术和手段,从动物水平探讨补阳还五汤干预肺纤维化的可能作用机制。研究结果表明：补阳还五汤可以有效改善模型大鼠肺泡炎症及肺纤维化程度,降低血清PCⅢ、 Ⅳ-C、HA、LN含量,抑制TβRⅠ、TβRⅡ、Smad3、CollagenⅠ、TGF-β1mRNA、 Smad3mRNA、ERK1/2、p-ERK蛋白在病肺组织中的过度表达,促进Smad7以及Smad7mRNA的活性,提示补阳还五汤可以有效调控TGF-β1/Smad介导的致纤维化作用,以及TGF-β1/ERK通路介导的促增殖作用,进而起到抗纤维化的作用。更多还原

【Abstract】 Pulmonary fibrosis (PF) is a kind of the disease of fiber hyperplasia as a common pathological basis process of interstitial lung disease (ILD) caused by various reasons, its diseased tissues involve pulmonary interstitium, alveolar epithelial cells and ulmo-nary vascular, characterized with over reproduction of lung fibroblast (LFB) and excess deposition of extracellular matrix (ECM) contained collagen protein. As result of its pathogenesis is not thoroughly clear so far, there is no perfect therapeutic method in clinic. Since most of lung diseases could transform into pulmonary fibrosis as their final outcome, And as the air pollution, environment deterioration and other factors, the incidence rate of PF is increasing constantly in the world, it is becoming a severe threat to human health. Therefore, it is a very important value in scientific to study the pathogenesis of PF and effective medicine to treat this disease. Research shows that transforming growth factor-β1(TGF-β1) is the key cytokine of leading to PF, the effect of causing fibrosis of TGF-β1/Smad signal path-mediated and the effect of promoting proliferation of ERK signal path-mediated are indispensable to the development of PF. PF is under the category of "lung atrophy","lung impediment" in traditional Chinese medicine, its mechanism due mostly to qi deficiency with blood stasis, so we believes that tonifying qi and activating blood is the basically therapeutic method. Our preliminary research on the effect of tonifying qi and activating blood’ method on PF found that Buyanghuanwu decoction can efficiently regulate expression levels of TGF-β1in the lung tissues, reduce the hydroxylproline (HYP) in bronchoalveolar lavage fluid (BALF) and tumor necrosis factor-α (TNF-α) content in alveolar macrophage (AM) of pulmonary fibrosis rats, which suggests we inhibitory mechanism of Buyanghuanwu decoction on pulmonary fibrosis could be associated with regulating the TGF-β/Smad/ERK signal path. Based on the above study, we use the key point of TGF-β/Smad/ERK signal path, and make our target of research accurately position in the key pathological links of PF’ formation, to discuss the associated mechanism of action of Buyanghuanwu decoction in the pulmonary fibrosis, in order to wide the application range, and lay the foundation for the clinical study.In this study, we apply BLM to induce the PF model on rats as an appraisal carrier, to explore the mechanism of Buyanghuanwu decoction regulating the TGF-β/Smad/ERK signal path from the perspective of pathology and molecular biology. The experimental research consists of four parts as following:1. The effects of Buyanghuanwu decoction on lung index and pathomorphology in the lung tissue of PF rats by BLM.Established PF model rats, on the second day after the models were successfully made, treated with Buyanghuanwu decoction. At different points in time, though observing general status of PF rats (such as the condition of mental, diet, hair, behavioral activity, and weight, etc), the changes of lung index, to measure alveolar catarrh and pulmonary fibrosis degrees in lung tissue dyed with HE and Masson, discussed the effect of Buyanghuanwu decoction on PF.Results:①General status:The rats of sham operation group’s behavioral activity, glossiness of hair and diet were unchanged; model group had the phenomena of less eating, messy or sparse hair, even hair removal, movement decreasing, slow increase of weight, other medicated groups’s general status have different improvements with time, increased gradually weight;②Lung index:at all time points, a significant increase in lung index of model group were higher than that of sham operation group (p<0.01); other medicated groups had different decreases in lung index by comparison with model group (p<0.01); compared with the prednisone group, the lung index of Buyanghuanwu decoction middle-dose group showed an obviously decreased tendency on the14th and28th day (p<0.05), this condition of that in Buyanghuanwu decoction low-dose group on the28th day (p<0.05), Buyanghuanwu decoction high-dose group than in prednisone group the lung index of some high at there time points, but were significantly lower than the same period in the model group.③Pathomorphology:compared with the sham operation group, the model group and each medicated groups were mainly featured with destroyed alveoli, thickened alveoli septum and the deposition of collagen fibers on different level; the medicated groups could improve the alveolar catarrh and pulmonary fibrosis on pathomorphology in varying degree by comparison with the model group (p<0.01); the effect of reducing alveolar catarrh and pulmonary fibrosis of Buyanghuanwu decoction middle-dose group had an advantage over that of the prednisone group on the14th and28th day (p<0.05).Conclusion:The PF rat model induced by BLM was successfully duplicated; Buyanghuanwu decoction could obviously control the growth of lung index, and alleviate alveolar catarrh and pulmonary fibrosis. 2. The effects of Buyanghuanwu decoction on fibrosis index (PCⅢ、Ⅳ-C、HA、 LN) in blood serum of PF rats by BLM.Established PF model rats, on the second day after the models were successfully made, treated with Buyanghuanwu decoction.At different points in time, tested the contents of PCⅢ、Ⅳ-C、HA、LN in blood serum of experimental rats by radioimmanoassay (RIA) method, to prove the therapeutic effect of Buyanghuanwu decoction on PF.Results:①Compared with the sham operation group, the contents of PCⅢ、 IV-C、HA、LN in blood serum of other groups were gradually increased, and that of the model group was the most obvious (p<0.01);②Each medicated group had different decreases in the contents of PCⅢ、Ⅳ-C、HA、LN in blood serum by comparison with model group (p<0.01);③Compared with the prednisone group, the contents of PCⅢ、Ⅳ-C、HA、LN of Buyanghuanwu decoction middle-dose group obviously decreased on the14th and28th day (p<0.05), as well as that of Buyanghuanwu decoction low-dose group on the28th day (p<0.05).Conclusion:Buyanghuanwu decoction could effectively reduce the contents of PCⅢ、Ⅳ-C、HA、LN in blood serum of experimental rats by BLM, which proved that buyanghuanwu decoction could effectively control the reproduction of collagen fibers and excess deposition of ECM in lung tissue of PF, and played an anti-fibrosis effect.3. Research on the mechanism of regulation of Buyanghuanwu decoction in PF based on TGF-β1/Smad signal pathEstablished PF model rats, on the second day after the models were successfully made, treated with Buyanghuanwu decoction. At different points in time, tested the expressions of Col-1、TβRⅠ、TβRⅡ、Smad3、Smad7in lung tissue by immunohistochemical staining, measured the contents of TGF-β1、Smad3、Smad7mRNA in lung tissue by reverse transcription polymerase chain reaction (RT-PCR), to discuss the Buyanghuanwu decoction’s mechanism of anti-lung fibrosis though regulating TGF-β1/Smad signal path.Results:①Compared with the sham operation group, the expressions of Col-Ⅰ、 TβRⅠ、TβRⅡ、TGF-β1mRNA、Smad3mRNA in lung tissue of model group were obviously increased (p<0.01); the expressions of Smad7、Smad7mRNA were gradually decreased (p<0.01);②Each medicated group had different decreases in the expressions of Col-Ⅰ、TβRⅠ、TβRⅡ、TGF-β1mRNA、Smad3mRNA in lung tissue by comparison with model group (p<0.01or p<0.05), the expressions of Smad7、 Smad7mRNA of medicated groups had different increases (p<0.01or p<0.05);③The expressions of Col-Ⅰ、TβRⅠ、TβRⅡ、TGF-β1mRNA Smad3mRNA of buyanghuanwu decoction were lower than that of the prednisone group (p<0.01or p<0.05); on the contrary, the expressions of Smad7、Smad7mRNA showed a increased tendency,, with statistical significance (p<0.05).Conclusion:Buyanghuanwu decoction could effectively regulate the cell factors’ expression levels of exerting effect in TGF-β1/Smad signal path, those were Col-Ⅰ、TβRⅠ、TβRⅡ、TGF-β1、Smad3, meanwhile promote the expressions of Smad7,which played an opposite role to regulate the signal path, which proved that the therapeutic effect of Buyanghuanwu decoction in PF were associated with regulating the TGF-β/Smad signal path.4. Research on the mechanism of regulation of Buyanghuanwu decoction in PF based on TGF-β1/ERK signal pathEstablished PF model rats, on the second day after the models were successfully made, treated with Buyanghuanwu decoction. At different points in time, tested the expressions of TGF-β1、ERK1/2、p-ERK in lung tissue by Western blot method, to discuss the Buyanghuanwu decoction’s mechanism of anti-lung fibrosis though regulating TGF-β1/ERK signal path.Results:①Compared with the sham operation group, the expressions of TGF-β1、ERK1/2、p-ERK in lung tissue of model group were significantly increased (p<0.01);②Each medicated group had different decreases in the expressions of TGF-β1、ERK1/2、p-ERK in lung tissue by comparison with model group (p<0.01or p<0.05);③Compared with the prednisone group, the expressions of TGF-β1、 ERK1/2、p-ERK of Buyanghuanwu decoction middle-dose group obviously decreased on the14th and28th day (p<0.05),as well as that of Buyanghuanwu decoction low-dose group on the28th day (p<0.05).Conclusion:Buyanghuanwu decoction could effectively suppress the excess expressions of TGF-β1、ERK1/2、p-ERK, which proved that Buyanghuanwu decoction could regulate the expressions of the key cell factors in TGF-β1/ERK signal path, and had a good effect of controlling proliferation.In conclusion, we established animal model of PF by a classic method though tracheofistulization with BLM, based on TGF-β/Smad/ERK signal path, to describe the effect and mechanism of Buyanghuanwu decoction on PF from integral level using the methods and techniques of molecular biology. The results prove that Buyanghuanwu decoction could obviously alleviate alveolar catarrh and pulmonary fibrosis in lung tissue of experimental rats by BLM, reduce the contents of PCⅢ Ⅳ-C、HA、LN in blood serum,suppress the excess expressions of Col-Ⅰ、TβRⅠ、 TβRⅡ、TGF-β1、Smad3、ERK1/2、p-ERK,meanwhile promote the viabilities of Smad7, which proved that Buyanghuanwu decoction could efficiently regulate the effect of causing fibrosis of TGF-β1/Smad signal path-mediated and the effect of promoting proliferation of ERK signal path-mediated, and had a good effect of anti-lung fibrosis.更多还原