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莪术油干预肺纤维化上皮细胞转分化的实验研究
【作者】 杨长福；
【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2011， 博士
【摘要】 研究背景及目的肺纤维化是指由多种病因引起的具有相似病理过程的一类弥漫性肺间质疾病,常发生在肺泡,早期有肺泡结构破坏和弥漫性肺泡炎,继后出现细胞外基质过度沉积,并逐渐演变为弥漫性肺间质纤维化,是肺脏损伤的结果。病人多死于进行性加重的肺纤维化所致的呼吸衰竭。在临床上又以特发性肺纤维化(IPF)最为常见。随着污染、环境恶化及药物等因素的影响,发病人数有逐年增加的趋势,但其作用机制尚不清楚,肺泡上皮细胞发生了上皮细胞间质转化可能是肺纤维化的机制之一。本实验室在前期研究中医药防治器官纤维化中发现莪术具有抗肺纤维化的药理活性。莪术油是莪术中有效提取部位,研究表明其能抑制体外培养细胞的增殖和凋亡。但是莪术油抗肺纤维化的细胞和分子调控机理目前还不清楚。为阐明莪术油对IPF的作用及可能的作用机制,本研究以博来霉素诱导的特发性肺纤维化小鼠和人肺Ⅱ型上皮细胞A549为模型,研究莪术油对特发性肺纤维化小鼠和A549细胞增殖的作用,并初步探讨了其抑制肺纤维化的机制。研究方法及结果1药物中成份鉴定及含量测定1.1方法本实验以莪术油为研究对象,利用气相色谱-质谱联用技术(GC-MS)对其化学成份进行分析。莪术油样品在GC-MS仪进样后,利用Nist98标准质谱图库及相关标准品进行对照,鉴定其结构,所得结果以相对保留时间和峰面积所占百分比表示。1.2结果共鉴定色谱峰23个,其中单峰面积占总峰面积大于10%的峰有三个单峰面积占总峰面积在5-10%的峰有三个；在23个化合物中有广泛抗肿瘤活性的p-榄香烯和莪术醇。2整体实验评价莪术油对特发性肺纤维化小鼠的效果及作用机制2.1方法①IR小鼠96只,随机分为4组：模型组(BLM)、莪术油中剂量组(OilM)、莪术油高剂量组(Oil H)及假手术组(Sham).模型组(BLM)、莪术油中剂量组(Oil M)、莪术油高剂量组(Oil H)经气管内注入博莱霉素(0.05 U/只)诱导肺纤维化,随即分别每日腹腔注射生理盐水和不同剂量莪术油(Oil M按100 mg·kg-1剂量给药,Oil H按200 mg·kg-1剂量给药)；假手术组(Sham)以生理盐水代替。各组动物均于气管内灌药后第7、14、28天分别处死8只,取肺组织。②HE染色,光镜下观察各组小鼠肺组织,进行肺泡炎、肺纤维化的分级。③用免疫组化方法和Western blotting检测各组小鼠肺内Ⅰ型胶原、Ⅲ型、TGF-β1、Smad3、Cat B、Cat D和Cat K的表达,并用图像分析软件检测各组的光密度。④实时荧光PCR法检测各组Cat B、Cat D、Cat K和Cat S的表达。⑤酶法检测肺组织匀浆中羟脯氨酸含量。2.2结果HE染色显示,假手术组肺泡结构完整、清晰,肺泡腔内无明显渗出物；模型组纤维化程度明显,在支气管周围有大片实变区,肺泡壁异常增厚,肺泡结构紊乱,腔内有渗出物,纤维化程度随时间的进展而加深加重；莪术油各剂量组肺泡结构较为清晰,在支气管周围有局部纤维化,肺泡间隔增厚,较模型组有所减轻。Mallory染色显示,假手术组小鼠肺肺切片中胶原层在支气管壁周围较薄,肺泡区散在；模型组肺间质、肺泡区、支气管、血管周围胶原纤维明显增多,并向间质沉积,增宽的肺泡间隔蓝色纤维增多尤为明显；两个治疗组的胶原沉积低于模型组。羟脯氨酸测定结果表明,模型组小鼠肺组织匀浆中的含量明显升高,莪术油高、中剂量组均降低其的含量,以第7天的作用最为明显。胶原免疫组化结果表明,Ⅰ型胶原主要表达于肺间质,随纤维化的进展加深加重,大量的Ⅰ型胶原在肺泡区沉积,破坏了肺泡的正常结构,两个治疗组肺切片中Ⅰ型胶原的沉积均较模型组的下降。Ⅲ型胶原主要表达于血管和支气管上皮,以第7天时的表达量最高,随时间延长肺组织中沉积的Ⅲ型胶原逐渐减少,到第28天时,Ⅲ型胶原的表达量接近正常水平。与假手术组相比,模型组Ⅲ型胶原的含量均升高,以第7天的表达量最高,14天次之,28天时的含量最低。两个治疗组在一定程度上降低Ⅲ型胶原在肺组织中的沉积。上皮细胞转分化相关蛋白及组织蛋白酶活性相关检测结果表明,特发性肺纤维化小鼠肺上皮细胞经历了上皮细胞间质转化,TGF-β1显著活化,促使α-SMA在肺泡区表达增强,下游信号分子Smad3表达增强,活化的Cat K、CatB和Cat S促使胶原降解,活化的Cat D加重了肺纤维化。两个治疗组较模型组中信号分子表达减弱,Cat K、Cat B和Cat S信号表达增强,Cat D的活化被抑制。3莪术油对A549细胞增殖抑制机理3.1方法本实验通过体外培养A549细胞,莪术油干预后,测定其对细胞增殖、细胞周期、细胞凋亡、胶原沉积及组织蛋白酶活性等方面的影响。3.2结果莪术油剂量为80 mg·mL-1时,细胞阻滞于S期,并伴随凋亡和坏死事件的发生；Cat D、Cat K的表达均上调。结论1使用GC-MS分析技术,对莪术油进行了化学成分研究,分离了23种化学结构,其中包含广泛报道的具有肿瘤抑制活性的p-榄香烯、莪术醇。2莪术油能改善特发性肺纤维化小鼠炎性细胞浸润,维持肺泡结构正常,以利于肺内气体交换,提高生存率；细胞外基质以Ⅰ型胶原的沉积为主,伴有Ⅲ型胶原的增生。3博来霉素诱导的肺纤维化模型中,上皮细胞经历了间质转化,莪术油能抑制纤维形成期上皮细胞的间质转化,从而有效的拮抗肺组织纤维化。4半胱氨酸组织蛋白酶K、S、B参与纤维化小鼠肺组织细胞外基质的重构,但其作用时间受到活化的TGF-β1的抑制而延后；莪术油能部分抑制TGF-β1免疫活性,提高纤维化肺组织中半胱氨酸组织蛋白酶K、S和B mRNA水平,降低大量沉积的细胞外基质而抑制纤维化的发展。组织蛋白酶D参与了肺纤维化的作用途径与半胱氨酸组织蛋白酶K、S和B不同,肺纤维化加速了Cat D蛋白肽链的断裂,在特发性肺纤维化中主要以成熟的重链形式(-33KDa)参与；莪术油能减少Cat D(～48KDa)中间体向成熟形式发展而抗纤维化。5莪术油能抑制A549细胞增殖,主要通过阻滞细胞于S期,减少进入分裂期的细胞比例；80 mg.mL-1的莪术油主要通过加速细胞衰老而上调Cat D的表达。
【Abstract】 Pulmonary fibrosis(PF) is a kind of diffuse interstitial lung disease which caused by many causes and has similar pathogenesis, Its pathogenic characterize is which alveolar structure destruction and diffuse alveolitise in the early, then cause over aggradation of extracellular matrix(ECM) and gaudily leading to fibrosis, consequently cause respiratory function destruction. Most of the patients died from respiratory failure from progressive pulmonary fibrosis. In clinic idiopathic pulmonary fibrosis(IPF) is the most common. IPF is a kind of pulmonary fibrosis, however, the mechanisma are still unknown. An emerging concept is that the alveolar typeⅡepithelial cells transition into mesenchymal cells via a process termed epithelial-mesenchymal transition(EMT). As pollution, environmental degradation, and so the number of seizures have increased trend every year. The laboratory studies of chinese medicine in the control organs fibrosis found that Zedoary Oil has the effect of remedying pulmonary fibrosis pharmacology. Zedoary Oil is an effective part in Zedoary. Studies show that Zedoary Oil can inhibit the donor cell proliferation, promote the donor cell apoptosis. But the control mechanisms of cellular and molecular about Zedoary Oil are not clear yet. To evaluate the effect of the pathogenesis and role of pulmonary fibrosis induced by Zedoary Oil. Mice model induced by Bleomycin(BLM) and alveolar epithelial cellⅡ(A549)as a model, and describes the effect of Zedoary Oil proliferation, and to discuss the potential mechanism of the inhibiting effect of Zedoary Oil pulmonary fibrosis.Methods & Results1 The chemical constituents and yield of the Zedoary Oil1.1 Method The chemical componentswere of Zedoary Oil are separated and identified by GC/MS. Taking Nist98 standard mass spectrometer database and contrast as control, the structure of themass spectrogram was identified, and relatively reserved time and peak area percentage occupy the peak total area percentage.1.2 Results 23 compositions were identified from Zedoary Oil. Three single peaked area are larger than ten percentage of the peak total area. Three single peaked area are one out of five or ten of the peak total area,β-Elemene and Curcumol can inhibit the tumor cells growth in the 23 compositions.2 To study medicine of the Zedoary Oil2.1 Methods①96 IR mice was divided into four groups:modle group (BLM), medium dose group(Oil M), the high dose group(Oil H) and sham operation group (Sham). A single dose of 0.05 U bleomycin in 100μL of saline’solution was administered by transtracheal puncture. Oil M and Oil H reeeived the same amount of bleomycin as BLM, followed by different amouts Zedoary oil. Sham received normal saline’solution by tratracheally. These mice were killed on 7,14,28 day post bleomycin.The lung tissues were obtained.②Apply the HE staining to investigate the lung inflammation and lung fibrosis under the routine mieroseope.③Use the immunohistochemistry staining and western blotting to study the expression of collagenⅠ、collagenⅢ、TGF-β1、Smad3、Cat B、Cat D and Cat K. Use image analysis software to measure the density.④Use the real time PCR to measure the expression of Cat B. Cat D、Cat K and Cat S.⑤The lung hydroxyproline content was analyzed using a enzymes response method in minced lung.2.2 Results HE staining display that the alveolar structure of sham is complete and clear. There were no inflammatory cells in alveolus. pulmonary fibrosis were obviously in sham. Consolidation area appeared around bronchus:abnormal thickening of alveolar walls, architectural distortion of alveolar, exudation in alveolus. As time went on, pulmonary fibrosis become serious. thickening and Exudation were seen in sham group that didn’t exist in rest group.Mallory staining results:the collagen layer of tissue section of lung of mice were thin around bronchus. There were the change visible in alveolus.Collagen fibers and deposition of BLM group increased considerably in interstitial lung, alveolar, bronchus, vessels.Blue fabric of BLM group increased obviously in alveolus interval.The changes of two treatment groups is lower than BLM group.Immunohistochemistry(IHC) staining results:normal structure of alveolar was destroyed by the deposition of I type collagen as fibrosis went on. The changes of two treatment groups is lower than BLM group.Ⅲtype collagen primarily on the vessels and bronchi. The collagen increased day by day and reached the highest at 7th day. deposition ofⅢtype collagen on the lung break down along with times.The expression ofⅢtype collagen approximate the normal level at 28th day.Compare to sham grounp, the content ofⅢtype collagen increassd day by day and reached the highest at 7th day, second at 14th day.The content get less at 28th day. two treatment groups can reduce the deposition of III type collagen on the lung.EMT of signal elements and Cathepisins of IHC staining results:EMT were urging TGF-β1 activated. The expression ofα-SMA in alveolar increased, and so the expression of Smad3. activated Cat K、Cat B and Cat S can promote degradation of collagen. Activated Cat D is correlated with pulmonary fibrosis. Compare to sham grounp, the expression of singnal in EMT decreased. the expression of Cat K、Cat B and Cat S increased. Cat D was inhibited.3 The pharmacologic mechanism of proliferation of A549 cell by the Zedoary Oil3.1 Methods The experiment measures that the effect on the regeneration of cell, the regulation of cell, the apoptosis of cell, the deposition of collagen and up cathepsins expression when the Zedoary Oil act on open educational A549 cell.3.2 Results when the concentration of the Zedoary Oil is 80 mg·mL-1, cell cycle was arrested at S stage. At the same time, some cell was apoptosis or necrosis. the expression of Cat D and Cat K increased.Conclusion1 The chemical componentswere separated and identified by GC/MS.23 compositions were identified from Zedoary Oil,β-Elemene and Curcumol which can inhibit the tumor cells growth.2 The Zedoary Oil can promote the gas eschange and the survival rate through maintaining the normal structure of alveolar. The exhibition of basis of cell give priority to the deposition of I type collagen.at the same time, the deposition of III type collagen increased.3 The Zedoary Oil can prevent the formation of Pulmonary fibrosis through inhibiting EMT in the modle of Pulmonary fibrosis which induced by BLM.4 The Zedoary Oil can prevent the formation of Pulmonary fibrosis throug hinhibiting some immunocompetence of TGF-β1, increasing the expression of Cat K、Cat S and Cat B mRNA and reducing the deposition of EMT. Cat D different from Cat K、Cat S and Cat B,5 The Zedoary Oil can inhibit the regeneration of cell through arresetting cell cycle at S stage and reducing the rate of cell division.80 mg·mL-1 can increase the expression of Cat D though accelerating cell senescence.