【作者】 贺环宇；

【导师】 吴泰华；

【作者基本信息】 大连医科大学 ， 内科学， 2014， 博士

【摘要】 特发性肺间质纤维化(Idiopathic Pulmonary Fibrosis, IPF)是一种慢性、进展性、致死性，自诊断后中位生存时间少于3年的疾病，已成为呼吸系统疾病中严重威胁人民健康的重大疾病，目前发病机制尚不十分清楚，尚无有效治疗方法。近十年来，有关IPF的临床试验活动呈指数增长，众多学者对此病的重视，解决之迫切可见一斑。IPF不仅在发生环节其病因谱极为广泛和复杂，同时在进展过程中存在多个病理生理过程参与、多个信号通路活化，是免疫紊乱、炎症、纤维化的交织和不断进展的过程。近年来，大多数学者把多种原因引起的肺脏损伤作为研究IPF的出发点，同时在体内外实验中应用组织病理学及多种检测技术观察到在IPF病变的初期，是以下呼吸道急性炎症反应为主，包括中性粒细胞、单核巨噬细胞等炎症细胞浸润聚集，分泌多种具有生物活性的炎症因子，如肿瘤坏死因子-α（TNF-α），白介素（IL-1β和IL-6）等，同时又可刺激产生众多氧化产物，最终导致肺泡结构的紊乱，肺实质损伤，胶原纤维成分在间质内聚集及慢性增生，导致肺间质纤维化。近年来越来越多的证据也表明氧化应激（氧化/抗氧化失衡）与特发性肺纤维化的形成和发展过程密切相关，同时肺脏在解剖学上的结构位置及其主要的生理功能决定了肺是最易受到氧化应激攻击的靶器官之一。氧化应激（Oxidative Stress）是指体内氧化与抗氧化作用失衡，氧化作用占优势，可导致中性粒细胞的聚集浸润，分泌蛋白酶增加，产生许多的氧化中间产物，造成氧化损伤。正常情况下，机体内存在着抗氧化系统，如酶系的超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-Px）等，这些抗氧化酶能对体内产生的自由基作出迅速及时地调节，能把过多生成的自由基清除，以保证体内氧化/抗氧化作用平衡，对机体有保护作用。但如果在一些不明原因的刺激因素下，体内有超过清除能力的氧自由基生成，或者机体抗氧化能力下降，体内SOD和GSH-Px的活性降低，就会致使相应组织细胞的损伤，对肺脏而言，就会引起肺泡上皮细胞、内皮细胞及肺脏间质细胞等的损伤，引起这些细胞膜的脂质成分发生过氧化作用。我们在实验中也常用检测氧化应激过程中脂质过氧化的主要产物丙二醛（MDA）的生成量来判断体内发生脂质过氧化作用的程度，许多研究在体内外实验中发现经炎性刺激后动物体内或细胞培养液中MDA的生成量增加也佐证了这一观点，同时因自由基过多可损伤组织或细胞导致MDA的生成，是自由基生成情况的一个间接反映。同时，氧化/抗氧化系统失衡的另一种生物标记物--髓过氧化物酶（MPO），其水平也经实验证实在IPF患者的BALF中有明显地升高。同时，在肺纤维化复杂病变的发生发展中，多种不明原因的刺激可使诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）的活性被诱导，导致其合成的一氧化氮（NO）浓度较组成型NOS（constitutiveNOS，cNOS）的浓度明显增高，可引起NO的产生增多。同时，在纤维化病变发展过程中，异常增多的炎性介质可使体内环氧化酶-2（cyclooxygenase-2，COX-2）的活性增强，产生前列腺素（prostaglandin，PG），尤其是PGE2增多，进而也会导致炎症损伤，引起炎性循环。因此现使用抗氧化剂治疗特发性肺纤维化也正在逐渐受到众多专家和学者的重视和认同。近年来，转化生长因子-β1(Transforming growth factor β1，TGF-β1)在肺间质纤维化发生发展中的作用备受肯定，TGF-β1可以促进成-肌成纤维细胞的表型转化，而循环或肺组织中的成纤维细胞向肌成纤维细胞的表型转化也是现阶段肺间质纤维化发病机制中的研究热点，已被众多的学者认为是IPF发展过程中的重要病理过程，这种表型转化在细胞实验及动物组织病理学检测中都已被观察到。同时TGF-β1也可刺激许多种细胞及炎症因子的合成、分泌，引起细胞外基质沉积，其作用的实现是通过先与受体结合，而后主要通过下游的Smad2/3转导通路完成的，而我们之前课题研究也已证实TGF-β1受体的I型受体中的活化素受体样激酶5（activin receptor like kinase5，ALK5）在肺间质纤维化过程中有明显升高，这提示ALK5参与了肺间质纤维化的发展。医学界关于IPF治疗方法的研究一刻都没有停止过，多年来，对于其治疗方法经历了不同方向的探索，但由于IPF是一种病因及发病机制尚不十分明确的疾病，无法从根源着手去阻抑其发生发展，也无特效治疗方法。2011年IPF的诊治指南有了不同于2000年ATS/ERS共识的新意见，其将大多数治疗措施改为不同强度的推荐意见，仅N-乙酰半胱氨酸、吡菲尼酮、抗凝药物等少数药物获得“弱推荐”，主要因为它们有较好的安全性，同时少部分患者用药治疗后有部分疗效。而糖皮质激素等免疫抑制剂等药物因其对机体有较大的不良作用，在临床上的应用有很大的局限性，因此，开发有效的药物以控制肺间质纤维化的病情，对于提高患者生活质量或降低患者死亡率具有重要意义。现代药理学研究也证实多种中药单体成分均有不同程度抑制肺纤维化的作用。丹参作为一种经典传统中药，多年来被广泛应用于心血管病的治疗中，而丹参酮IIA（Tanshinone IIA）是从丹参中提取出的脂溶性成分之一，是近年来国内外学者对中药单体中研究较多的活性成分，其在丹参酮中含量最高，也是最为稳定的有效活性成分，其具有明确的分子结构，已有实验证实丹参酮IIA不仅具有清除氧自由基和抗炎的作用，且还具有抗肿瘤等多种药学活性。同时，丹参酮IIA还具有多种肺保护作用。Wang等人的研究表明丹参酮IIA可以减轻博莱霉素诱导的大鼠肺纤维化。然而，目前关于丹参酮IIA如何作用于肺间质纤维化，其相关的具体生物学机制尚不十分清楚。本课题拟采用气管内滴注博莱霉素的方法建立大鼠肺间质纤维化模型，探讨丹参酮IIA对大鼠肺间质纤维化的保护作用及对炎症反应及氧化应激的影响，并进一步探讨其对肺间质纤维化相关分子作用机制的影响。第一部分丹参酮ⅡA对肺间质纤维化大鼠组织形态学变化的影响目的：探讨丹参酮IIA对大鼠肺纤维化组织形态学变化的影响，以明确丹参酮IIA对肺间质纤维化有无保护作用。方法：清洁级SD大鼠，体重200-220g，雌雄不限，采用随机数字表法分为正常对照组（N组），丹参酮IIA组，纤维化组（模型组），丹参酮IIA+纤维化组，每组6只。各组干预28天，然后收集各组肺泡灌洗液后，处死所有大鼠，取肺组织，对肺组织进行石蜡包埋后行组织病理学检测，并计算肺组织的湿/干重比。结果：经丹参酮IIA干预性治疗的丹参酮IIA+纤维化组大鼠的肺湿/干重比明显低于纤维化模型组，病理组织学可见肺泡间隔纤维增生灶较少，累及病变范围较小，肺实质结构破坏较少，胶原总含量减少。结论：丹参酮IIA能降低肺间质纤维化大鼠细胞外基质的含量，减轻肺间质纤维化的程度，对肺间质纤维化有一定的保护作用。第二部分丹参酮ⅡA对肺间质纤维化大鼠炎症反应和氧化应激的影响目的：观察丹参酮IIA对炎症反应和氧化应激的影响，进一步探讨丹参酮IIA改善肺间质纤维化的作用机制。方法：清洁级SD大鼠，体重200-220g，雌雄不限，采用随机数字表法分为正常对照组（N组），丹参酮IIA组，纤维化组（模型组），丹参酮IIA+纤维化组，每组6只。各组干预28天，收集各组大鼠肺泡灌洗液后处死，取肺组织，通过检测各组大鼠肺泡灌洗液中总蛋白含量、总细胞数，对中性粒细胞，巨噬细胞计数观察，ELISA检测各组大鼠BALF中相关炎症因子TNF-α，IL-1β和IL-6的表达水平，来探讨丹参酮IIA对肺炎症细胞及细胞因子的影响，通过试剂盒方法检测各组大鼠肺组织中的MDA含量、MPO的活性及NO和PGE2含量，运用实时荧光定量PCR及western blot技术检测各组大鼠肺组织中iNOS和COX-2的表达水平。结果：经博莱霉素干预28d后，纤维化模型组大鼠BALF中的总蛋白含量、总细胞数及中性粒细胞、巨噬细胞数量明显增加，同时炎症因子TNF-α、IL-1β和IL-6的含量也显著增加，肺组织内MDA的含量和MPO的活性较正常对照组明显增加或增强，同时也伴随有NO的过量产生和PGE2的增多。免疫印迹结果和实时荧光定量PCR结果显示在蛋白水平和mRNA水平上，iNOS和COX-2的表达量也是较正常对照组显著增加的。但在丹参酮IIA干预性治疗组，上述炎症细胞和炎症因子及产物明显降低或减少，这进一步解释了丹参酮IIA对肺间质纤维化的保护作用机制。结论：丹参酮IIA可通过降低肺间质纤维化过程中的炎症细胞和炎症反应因子，减弱氧化应激反应，降低iNOS和COX-2的表达，使肺纤维化缓解或减轻。第三部分丹参酮ⅡA对肺间质纤维化大鼠TGF-β1/ALK5/Smad2/3传导通路的影响目的：进一步探讨丹参酮IIA的保护作用是否与其抑制TGF-β1/ALK5/smad2/3信号转导通路的某个环节有关，为丹参酮IIA应用于治疗肺间质纤维化提供更多的理论基础。方法：清洁级SD大鼠，体重200-220g，雌雄不限，采用随机数字表法分为对照组（N组），丹参酮IIA组，纤维化组（模型组），丹参酮IIA+纤维化组，每组6只。各组干预28天，然后收集各组肺泡灌洗液后，处死所有大鼠，取肺组织，结合前期组织病理改变，应用ELISA，免疫印迹技术检测TGF-β1、ALK5和Smad2/3的表达。结果：在气管内滴注博莱霉素造模28d后，纤维化模型组大鼠血清及组织中的TGF-β1、ALK5及Smad2/3明显增加，与正常对照组相比，其差异有统计学意义（P<0.05），而在应用丹参酮IIA干预性治疗后，大鼠血清TGF-β1水平下降显著，同时ALK5及Smad2/3表达量也明显下降，与模型组比较，差异有统计学意义（P<0.05）。结论：丹参酮IIA可以抑制博莱霉素致肺间质纤维化大鼠中TGF-β1/ALK5/Smad2/3致纤维化信号通路。更多还原

【Abstract】 Idiopathic pulmonary fibrosis is a chronic, progressive and lethal disease ofunclear pathogenesis and no effective treatment, with a median survival of less thanthree years from diagnosis, it became a serious threat to people’s health and lives, hasalso been the difficulty and research focus of diagnosis and treatment of respiratorydiseases.IPF has complex etiology, and its pathophysiological processes involve theactivation of multiple signaling pathways, immune disorders, inflammation, fibrosis,and so on. In recent years, the researchers at home and abroad tend to consider that itspathogenesis of lung injury primarily is caused by a variety of reasons, earlypathological process is characterized by acute lower respiratory tract inflammation,including alveolitis, interstitial pneumonia, alveolar epithelial damage, fibroblastsproliferation, macrophages, neutrophils and other inflammatory cell infiltration, thesecretion of cytokines, inflammatory mediators and other biological active substances,such as tumor necrosis factor-α (TNF-α), interleukin (IL-1β and IL-6), etc., resultingmetabolic disorders of the extracellular matrix, the extracellular matrix deposition andexcessive fibrous tissue repair, and these processes eventually cause pulmonaryinterstitial structure disorder, lung parenchymal damage, resulting in chronic pulmonaryfibrosis, collagen deposition, alveolar structural changes, and ultimately pulmonaryfibrosis.In recent years, more and more evidences also indicate that oxidative stress isclosely related to the formation and development process of idiopathic pulmonaryfibrosis, the anatomical structure of lung and its main physiological function determinesthat the lung is one of the most susceptible to oxidative stress target organs. OxidativeStress refers to an imbalance between oxidation and antioxidation, which tends tooxidation, resulting in neutrophil infiltration and protease, intermediate oxidationincreased.Under physiological conditions, the antioxidant enzyme system in the body,such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), can adjust and scavenge free radicals timely. But if there is excessive oxygen free radicals or thebody’s ability is too low not to remove oxygen free radicals, the activity of SOD andGSH-Px is reduced, which can cause lung tissue damage and cell membrane lipidperoxidation, and MDA as a main product of lipid peroxide can increase. So in thelaboratory the determination of the amount of MDA can be used to reflect the degree oflipid peroxidation in vivo, indirectly indicate the production of free radicals.Anotherkind of biomarkers of oxidative stress, myeloperoxidase, its level in BALF of patientswith IPF also has obviously increased. Under complex pathophysiological processes,the induced nitric oxide synthase is induced by a variety of stimulus factors,which leadto the concentration of nitric oxide significantly increase. At the same time, theabnormal increase of inflammatory mediators can make cyclooxygenase-2enhanced,then prostaglandin increase, especially PGE2, which can lead to inflammatory lesions.So many experts and scholars gradually accept that antioxidant can be a new way fortreating idiopathic pulmonary fibrosis.Myofibroblast phenotype transformation is an important pathological process ofpulmonary fibrosis, and transforming growth factor-beta1is the key factor of thepulmonary fibrosis progression. TGF-β1can promote myofibroblast phenotypetransformation, stimulate cytokines synthesis and secretion, and extracellular matrixaccumulation. TGF-β1signal mainly through its receptor and Smad2/3transductionpathways, and our previous research has also confirmed that activin receptor likekinase-5in the process of pulmonary fibrosis significantly increased, this suggestsALK5involved in the development of pulmonary fibrosis.Medical research on IPF treatment never stop, over the years, its treatment hasexperienced different direction of exploration, but IPF is a kind of disease of etiologyand pathogenesis is not very clear, idiopathic pulmonary fibrosis lack of effectivetreatment methods at present. The diagnosis and treatment guide of IPF in2011replacesthe ATS/ERS IPF consensus in2000, which changed the most treatment methods intodifferent strength of recommendations, only N-acetylcysteine, pirfenidone,anticoagulation drugs, etc. get weak recommendations, mainly because of their goodsecurity. And immunosuppressants such as corticosteroids and other drugs, because ofits adverse effects to the body, there is a lot of limitations for application in clinic.Therefore, developing an effective drug is of great significance for patients withpulmonary fibrosis. Modern pharmacology research also confirmed that a variety of traditional Chinese medicine monomer has some extent to inhibit the action of thepulmonary fibrosis. Salviae miltiorrhizae BGE. is a chinese traditional medicine.Tanshinone IIA is a kind of alcohol soluble ingredient, which extracted from salviaemiltiorrhizae BGE.. It has definite molecular structure, highest levels in Tanshinones,the most stable and effective active ingredient, most biological drug effect of salviaemiltiorrhizae BGE.. Recent studies have also shown tanshinone IIA has manypharmaceutical activities, including clear oxygen free radicals, anti-inflammatory,anti-tumor and lung protection. Wang et al. have shown that tanshinone IIA can reducepulmonary fibrosis in rats. However, its biological mechanism in pulmonary fibrosis isnot very clear.In this study, we adopt the rat model of bleomycin-induced pulmonary fibrosis toexplore protective effect of tanshinone IIA for pulmonary fibrosis, and to further studyits molecular mechanism.Part IThe effect of Tanshinone IIA on histomorphology of rats with pulmonary fibrosisObjective: To explore the effect of tanshinone IIA on pulmonary fibrosis in rats, inorder to make clear whether tanshinone IIA is beneficial for pulmonary fibrosis.Methods: Sprague-Dawley rats, weighing200-220g, male and female unlimited, weredivided into four groups by random number table method: control group (N group),tanshinone IIA group, fibrosis group(model group), tanshinone IIA+fibrosis group,with six rats in each group.28days after the intervention, collected alveolar lavage fluidof each group, then put to death all the rats, and collected lung tissue. We calculated andcompared the histopathology and wet/dry weight ratio of lung among groups.Results: The wet/dry weight ratio of tanshinone IIA+fibrosis group was significantlylower than that in the model group, the histopathology showed that fiber foci in alveolarseptum, lesions, structural damage to the lung parenchyma, total collagen content wereless.Conclusion: Tanshinone IIA could reduce the content of extracellular matrix ofbleomycin-induced pulmonary fibrosis, lower the degree of fibrosis, which play theprevention and treatment role for pulmonary fibrosis. Part IIThe effects of Tanshinone IIA on inflammation and oxidative stress of rats withpulmonary fibrosisObjective: To observe the effects of Tanshinone IIA on inflammation and oxidativestress of rats with pulmonary fibrosis, and to further explore the mechanism oftanshinone IIA on pulmonary fibrosis.Methods: Sprague-Dawley rats, weighing200-220g, male and female unlimited, weredivided into four groups by random number table method: control group (N group),tanshinone IIA group, fibrosis group(model group), tanshinone IIA+fibrosis group,with six rats in each group.28days after the intervention, collected alveolar lavage fluidof each group, then put to death all the rats, and collected lung tissue. The total proteincontent and cell number, the neutrophils, macrophages count in BALF were detected,the expression of TNF-α, IL-1β and IL-6in BALF of each group was detected byELISA to explore the effect of tanshinone IIA on inflammatory cells and cytokines, thecontent of MDA, NO and PGE2and the activity of MPO were detected by kits, theiNOS and cox-2expression level were detected by real-time fluorescent quantitativePCR and western blot.Results:28days after the bleomycin administration, the total protein content and thenumber of neutrophils, macrophages in BALF obviously increased, and in the meantimethe expression of TNF-α, IL-1β and IL-6in BALF also increased significantly, thecontent of MDA and the activity of MPO in lung tissue obviously increased or enhanced,compared with control group, also accompanied by the generation of NO and PGE2increasing. Western blot and real-time fluorescent quantitative PCR results showed thatthe expression of iNOS and cox-2also increased significantly at the protein level andmRNA level. But in tanshinone IIA-treated group, the inflammatory cells and cytokinesreduced obviously, which could further explain the protection mechanism of tanshinoneIIA on pulmonary fibrosis.Conclusion: Tanshinone IIA can relieve pulmonary fibrosis by reducing theinflammatory cells and cytokines, mitigating oxidative stress and reducing theexpression of iNOS and cox-2in the process of pulmonary fibrosis. Part IIIThe effects of Tanshinone IIA on TGF-β1/ALK5/smad2/3signal transductionpathway of rats with pulmonary fibrosisObjective: To further explore whether the protective effect of tanshinone IIA wasrelated to its inhibition of TGF-β1/ALK5/smad2/3signal transduction pathway, andprovide more theoretical basis for application of tanshinone IIA in treatment ofpulmonary fibrosis.Methods: Sprague-Dawley rats, weighing200-220g, male and female unlimited, weredivided into four groups by random number table method: control group (N group),tanshinone IIA group, fibrosis group(model group), tanshinone IIA+fibrosis group,with six rats in each group.28days after the intervention, collected alveolar lavage fluidof each group, then put to death all the rats, and collected lung tissue. the expressionlevel of serum TGF-β1,lung ALK5and Smad2/3were detected by ELISA and westernblotting.Results:28d after the endotracheal bleomycin administration in model group, weobserved that the level of serum TGF-β1,lung ALK5and Smad2/3increasedsignificantly, compared with control group, there was significant difference(P<0.05),but after tanshinone IIA interventional therapy, the serum TGF-β1level decreasedsignificantly, and the level of lung ALK5and Smad2/3decreased,the difference wasstatistically significant compared with model group (P<0.05).Conclusion: Tanshinone IIA can inhibit TGF-β1/ALK5/smad2/3signal transductionpathway of rats with pulmonary fibrosis.更多还原