【作者】 唐海英；

【导师】 吴泰华；

【作者基本信息】 大连医科大学 ， 内科学， 2015， 博士

【摘要】 特发性肺间质纤维化(Idiopathic Pulmonary Fibrosis,IPF)严重威胁人类健康和生命,是最难治的慢性纤维化性肺部疾病,其发病率呈逐渐上升趋势,死亡率高。但因发病机制至今仍不明确,目前尚无有效的治疗方法。2011指南指出肺移植是目前唯一有效的治疗办法,尚没发现哪种药物对IPF有效,研究提示某些药物对IPF患者可能有益。因此,继续寻找有效的治疗IPF的药物仍是目前研究之重。丹参是一种经典传统中药,对活血化淤有效,从丹参中提取的脂溶性活性成分之一丹参酮ⅡA(TanshinoneⅡA)是目前研究较多的一种单体成分,主要因为丹参酮ⅡA是众多丹参酮成分中最为稳定的有效活性成分,且其含量最高,分子结构明确,具有丹参的大部分药物效应,在临床上常用于心血管病的治疗。近来大量研究表明丹参酮ⅡA作用很多,包括清除氧自由基、抗炎,抗肿瘤等多种药学活性及多种脏器保护作用。肺泡巨噬细胞过度浸润与肺纤维化的异常炎症反应有关,先前的研究不仅发现丹参酮ⅡA抑制博来霉素诱导的大鼠肺组织炎症反应,而且可以减轻肺纤维化。因此,我们首先用博来霉素建立肺纤维化大鼠模型,进一步验证丹参酮ⅡA抑制纤维化肺组织炎症反应的作用,发现丹参酮ⅡA可使肺泡巨噬细胞标志物CD68表达下降。同时从形态学角度证实丹参酮ⅡA可以减轻肺纤维化,抑制胶原沉积。最新的研究认为,肺泡上皮损伤和病理性损伤修复在IPF发生发展中起重要作用,具体表现在以下几个方面:肺泡上皮损伤;上皮细胞间质转化(Epithlial to mesenchymal transition,EMT);纤维细胞灶形成;端粒受损变短等,其中有研究报道,EMT是成纤维细胞的主要来源之一,主要表现细胞黏附分子中的E-钙粘蛋白(E-cadherin)表达缺失,上皮细胞标志物下调,间质细胞标志物中的波形蛋白(Vimentin)、纤维连接蛋白(Fibronetin)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)表达上调,细胞极性消失,类似成纤维细胞形态。越来越多的证据表明EMT发生与肺纤维化进展密切相关,是目前研究热点内容。FU等发现丹参酮ⅡA阻断MCF-7和HCC1973乳腺癌细胞系EMT的发生。Wang等也报道丹参酮ⅡA抑制EMT及体内肝细胞癌转移。这两个实验证明丹参酮ⅡA在乳腺癌及肝癌中的EMT的发生有抑制作用。我们前期工作发现丹参酮ⅡA有抑制肺纤维化的作用,因此,猜测丹参酮ⅡA有可能是通过抑制EMT而减轻肺纤维化,然而,关于丹参酮ⅡA对纤维化肺疾病EMT作用的细胞及分子机制的研究很少。我们预实验结果表明博来霉素诱导的纤维化肺组织E-cadherin表达减少,而Fibronectin和Vimnetin表达增加,经过丹参酮ⅡA预处理组上述因子变化被逆转,证明丹参酮ⅡA可以抑制EMT发生。因此将对其进行深入研究。转化生长因子-β(Transforming growth factor-β,TGF-β)是一种细胞因子,功能较多,主要通过影响细胞的增生、分化、凋亡、细胞外基质生成来调节组织的形态发生及分化,被公认为最重要的促纤维化因子,是各种纤维化中EMT的主要触发因素,其中包括肾小管上皮细胞、角膜上皮细胞和肺泡上皮细胞EMT等。参与肺纤维化EMT的信号通路有很多,其中TGF-β/Smad是最经典的一种信号通路,Smad是TGF-β的下游因子,研究证明,TGF-β能诱导肺泡上皮细胞发生EMT,主要是通过活化了的Smad2/3,促进肺纤维化形成。有学者研究丹参酮IIA抑制肾间质纤维化的作用,发现在肾脏成纤维细胞中,丹参酮IIA主要通过抑制TGF-β1/Smads信号传导通路发挥作用;最近的研究展示丹参酮ⅡA可以显著减少博来霉素诱导的肺纤维化的炎症反应及减少大鼠肺组织TGF-β1的表达。但是,丹参酮ⅡA对TGF-β信号依赖的EMT发生及对肺纤维化疾病潜在机制的作用还不是很清楚,因此,我们的研究假设丹参酮ⅡA能调整IPF的发生过程中TGF-β相关的EMT过程,从体内体外两个方面研究其潜在的分子机制。小窝蛋白-1(Caveolin-1)具有维持小窝形态结构及功能的作用,是小窝的标志性蛋白。其中脚手架结构域(Caveolin scaffolding domain,CSD)是Caveolin-1与胞内其他信号分子相互作用的区域,为多种信号分子的负性调节因子。Caveolin-1在多数分化细胞中表达丰富,其中包括肺泡Ⅰ型上皮细胞、平滑肌细胞、成纤维细胞等。近几年,一些学者研究发现:IPF患者及博来霉素诱导大鼠肺组织中存在着Caveolin-1的表达下调;Caveolin-1基因敲除小鼠,Caveolin-1表达明显下降,肺部出现类似肺纤维化病变,Kasper等发现经TGF-β1处理后的肺组织,上皮细胞中Caveolin-1的表达明显下降,以上研究均提示Caveolin-1参与了肺纤维化的发生发展过程。又因Caveolin-1负性调节多种信号通路发挥作用,其中TGF-β/Smad信号通路研究较多。已有文献报道,Caveolin-1与TGF-β1Ⅰ型、TGF-βⅡ型受体及Smad2等共同定位在细胞质膜丰富的区域,Caveolin-1通过CSD与TGF-β1Ⅰ型受体及TGF-βⅡ型受体作用,负性调节TGF-β与受体结合,阻断TGF-β/Smad信号通路。我们通过对丹参酮ⅡA抑制TGF-β依赖的EMT过程的体内体外研究,发现丹参酮ⅡA抑制博来霉素诱导的肺巨噬细胞增加、胶原沉积、EMT过程及TGF-β/Smad信号通路的激活。体外实验也证实丹参酮ⅡA抑制A549细胞中TGF-β信号通路以及EMT,但是丹参酮ⅡA是通过哪些途径起到这样的作用尚不清楚,Caveolin-1是TGF-β信号通路的上游因子,在肺纤维化过程中,Caveolin-1的表达下降;增加Caveolin-1的表达可以抑制TGF-β信号通路,达到一定程度抑制肺纤维化的目的,因此,既然Caveolin-1和丹参酮ⅡA都有抑制TGF-β信号通路的作用,我们是否可以猜想丹参酮ⅡA抑制肺纤维化的作用可能与Caveolin-1有关呢?我们主要通过体外实验研究丹参酮ⅡA是否通过Caveolin-1调控下游的TGF-β/Smad信号通路介导的EMT过程,从而达到抑制肺纤维化目的?为临床新药开发提供更多的理论基础。第一部分丹参酮ⅡA抑制博来霉素诱导的肺纤维化目的:探讨丹参酮ⅡA对肺纤维化大鼠的肺组织形态学变化及Ⅰ型胶原、羟脯氨酸含量、CD68表达的影响,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,体重约250g,采用随机数字表法分为对照组(Control),丹参酮ⅡA组(TanⅡA),纤维化组(BLM),丹参酮ⅡA+纤维化(BLM+TanⅡA),每组6只。各组干预28天,处死所有大鼠,取肺组织,计算肺组织的湿/干重比,对肺组织进行石蜡包埋后行组织病理学检测及ELISA检测各组大鼠肺组织中的Ⅰ型胶原及羟脯氨酸含量,通过免疫组织化学方法检测肺巨噬细胞标志物CD68的表达。结果:丹参酮ⅡA+纤维化组的大鼠肺湿/干重比明显低于纤维化组;HE染色可见丹参酮ⅡA+纤维化组与纤维化组比较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,Masson染色提示胶原纤维减少;纤维化组Ⅰ型胶原及羟脯氨酸含量、CD68表达与对照组比较明显升高,丹参酮ⅡA+纤维化组与纤维化组比较Ⅰ型胶原及羟脯氨酸含量、CD68表达下降。结论:丹参酮ⅡA抑制博来霉素诱导的大鼠肺组织肺泡巨噬细胞标志物CD68表达,减轻炎症反应;丹参酮ⅡA抑制Ⅰ型胶原及羟脯氨酸含量,减少细胞外基质沉积,减轻肺纤维化。第二部分丹参酮ⅡA抑制TGF-β依赖的EMT机制研究目的:研究丹参酮ⅡA对博来霉素诱导肺纤维化大鼠肺组织及TGF-β1刺激的A549细胞EMT生成及TGF-β/Smad通路的影响,进一步探讨丹参酮ⅡA改善肺间质纤维化的作用机制。方法:体内实验分为对照组(Control),丹参酮ⅡA组(TanⅡA),纤维化组(BLM),丹参酮ⅡA+纤维化组(BLM+TanⅡA),每组6只。各组干预28天,处死所有大鼠,取肺组织;体外实验分为4组:对照组(Control):10μM的DMSO;丹参酮ⅡA组(TanⅡA):10μM的丹参酮ⅡA;TGF-β1组(TGF-β1):10μM的DMSO+10ng/ml的TGF-β1;丹参酮ⅡA+TGF-β1(TGF-β1+TanⅡA):10μM的丹参酮ⅡA+10ng/ml的TGF-β1。通过免疫荧光方法检测各组大鼠肺组织及各组细胞E-cadherin的表达;Real-time PCR检测各组大鼠肺组织TGF-β1RNA水平的表达;Western blotting检测各组大鼠肺组织及各组细胞TGF-β1、Smad2、p-Smad2、Smad3、p-Smad3、E-cadherin、α-SMA、Fibronectin和Vimentin蛋白水平的表达。结果:免疫荧光染色检测结果提示纤维化组与对照组及丹参酮ⅡA组比较,E-cadherin的阳性染色减少,丹参酮ⅡA预处理后E-cadherin的表达被部分恢复;Western blotting及Real-time PCR结果发现纤维化组、TGF-β1组分别与对照组比较,E-cadherin表达明显下降,α-SMA、Fibronectin和Vimentin蛋白表达明显升高,TGF-β1RNA及蛋白表达、p-Smad2、p-Smad3表达均升高,加入丹参酮ⅡA干预性治疗组上述因子表达均被逆转,差异有显著性。而且丹参酮ⅡA对Smad2、Smad3的表达无影响,仅能影响Smad2、Smad3的磷酸化水平。结论:丹参酮ⅡA通过抑制博来霉素诱导的肺组织EMT及TGF-β/Smad信号通路的激活,减轻肺纤维化;丹参酮ⅡA抑制TGF-β1刺激的A549细胞TGF-β信号依赖的EMT形成。第三部分丹参酮ⅡA通过上调Caveolin-1抑制TGF-β1刺激的A549细胞EMT目的:进一步体外实验探讨丹参酮ⅡA是否通过Caveolin-1调控下游TGF-β/Smad信号通路及EMT过程,为丹参酮ⅡA应用于临床,开发新药提供更多的理论基础。方法:我们首先用Western blotting检测第二部分四组细胞Caveolin-1蛋白水平表达。然后构建并筛选了转染效率高的Caveolin-1特异性sh RNA表达载体用于细胞转染,共分为7组:对照组(A);丹参酮ⅡA组(B):10μM的丹参酮ⅡA处理2h;Caveolin-1 sh RNA+丹参酮ⅡA(C):转染Caveolin-1 sh RNA 24h,然后用10μM的丹参酮ⅡA处理2h;TGF-β1(D):10ng/ml的TGF-β1处理48h;Caveolin-1sh RNA+TGF-β1(E):转染Caveolin-1 sh RNA 24h,然后用10ng/ml的TGF-β1处理48h;丹参酮ⅡA+TGF-β1(F):10μM的丹参酮ⅡA处理2h,然后用10ng/ml的TGF-β1处理48h;Caveolin-1 sh RNA+丹参酮ⅡA+TGF-β1(G):转染Caveolin-1sh RNA 24h,然后用10μM的丹参酮ⅡA处理2h,再用10ng/ml的TGF-β1处理48h。RT-PCR及Western blotting检测各组细胞TGF-β1、E-cadherin、Fibronectin和Vimentin RNA及蛋白水平表达。Western blotting检测各组细胞p-Smad2、p-Smad3表达。结果:TGF-β1组与对照组及丹参酮ⅡA组比较Caveolin-1的表达水平明显下降,经过丹参酮ⅡA预处理后Caveolin-1的表达明显升高,说明丹参酮ⅡA在纤维化过程中影响了Caveolin-1的表达;RT-PCR及Western blotting均提示丹参酮ⅡA(B组)与对照组(A组)比较TGF-β1、p-Smad2、p-Smad3、E-cadherin、Fibronectin、Vimentin的表达变化不明显,但转染Caveolin-1sh RNA(C组)后,TGF-β1、p-Smad2、p-Smad3表达升高,上皮细胞标志物E-cadherin表达下降,而间质细胞标志物Fibronectin与Vimentin表达升高;A549细胞转染Caveolin-1 sh RNA 24 h后,10 ng/ml TGF-β1处理细胞48 h(E组)与仅接受TGF-β1处理48 h(D组)比较,Caveolin-1 sh RNA+丹参酮ⅡA+TGF-β1(G组)与丹参酮ⅡA+TGF-β1(F组)比较,TGF-β1、p-Smad2、p-Smad3表达升高,E-cadherin的表达明显下降,Fibronectin与Vimentin的表达升高,而经过丹参酮ⅡA治疗后上述因子的表达均被逆转。结论:丹参酮ⅡA增加了TGF-β1刺激的A549细胞Caveolin-1的表达;丹参酮ⅡA通过上调Caveolin-1抑制TGF-β1刺激的A549细胞TGF-β信号依赖的EMT。更多还原

【Abstract】 Idiopathic Pulmonary Fibrosis(IPF) is a serious disease which threatens human health and life; its morbidity and mortality increased in recent yeas. The pathogenesis of IPF remains unclear; and it is lack of effective treatment. Evidences showed there had no effective drugs which could heal IPF except lung transplantation according to 2011 Guide. But studies suggested that some drugs might be beneficial to IPF, which implies it could be important to look for effective drugs to treat and control the progress of pulmonary fibrosis.Tanshinone II A(Tan II A) is a bioactive constituent extracted from the root of salvia miltiorrhiza Bunge, a Chinese herbal medicine. Tan II A is used in the treatment of cardiovascular disease in clinic. It is not only a specific molecular structure, but also the highest levels of many tanshinone compositions, the most stable and effective active ingredient, with most of the drug effect of salvia miltiorrhiza. A removal of oxygen free radicals, anti-inflammatory, anti-tumor activities, cardioprotective and neuroprotective effects of Tan II A have been reported in many earlier studies. Studies have shown that Tan II A can relieve the bleomycin-induced pulmonary fibrosis in rats. The excessive infiltrations of alveolar macrophages contribute to inflammation of lung fibrosis. Our study showed that lung expression of CD68, a specific mark of alveolar macrophage, was decreased after treating with Tan II A in bleomycin-induced pulmonary fibrosis which suggested Tan II A can inhibit inflammatory response. Rats model of pulmonary fibrosis reduced by bleomycin were used to confirm Tan II A can inhibit lung inflammation and alleviate fibrosis of lung by inhibiting collagen deposition.The pathological character of IPF was the accumulation and proliferation of undetermined mesenchymal cell including fibroblast and myofibroblast. The latest studies suggested IPF was induced by alveolar epithelial injury, EMT and formation of fibroblast foci. EMT was considered the main source of fibroblasts.EMT manifested as cells obtained characters of fibroblast as decreased of E-cadherin and increased of mesenchymal cells markers such as vimentin, fibronection and alpha smooth muscle actin. EMT was closely associated with pulmonary fibrosis, which had become the focous in the pathogenesis of pulmonary fibrosis.Studies showed that EMT of breast cancer cell lines as MCF-7 and HCC1973, and liver cancer cell were blocked and by Tan II A. Our previous work demonstrated that Tan II A could inhibit lung fibrosis, which suggested Tan II A might inhibit EMT to alleviate fibrosis. However, few studies have been carried about the molecular mechanism of Tan II A inhibiting EMT in pulmonary fibrosis. Lung expression of E-cadherin decreased and expression of Fibronection and Vimnetin increased in rats with pulmonary fibrosis; but these factors expressions could be reversed after treated by Tan II A, which confirmed the inhibitory effect of Tan II A on EMT. So, further study should be continued to explore the mechanism of Tan II A inhibitory effect on EMT.Transforming growth factor-β(TGF-β) is a kind of multifunctional cytokine by affecting cell proliferation, differentiation, apoptosis, extracellular matrix(ECM) formation to adjust the morphogenesis and differentiation of the organization. TGF-β was considered as recognized fibrosis factor and induced transformation of EMT, including renal tubular epithelial cells, corneal epithelium cells and alveolar epithelial cells. TGF-β/Smad is an important signaling pathway, participating in the EMT of pulmonary fibrosis. Smad is downstream factor of TGF-β. Studies have shown that TGF-β can be induced EMT through activating Smad-2/3 and promote the formation of pulmonary fibrosis. Renal interstitial fibrosis could be relieved through inhibiting the TGF-β1/Smads signaling pathway in renal interstitial fibroblasts after dealing with Tan II A. Besides, inflammations in pulmonary fibrosis and lung expression of TGF-β1 decreased by Tan II A management. But mechanism about the effect of Tan II A on TGF-β depended EMT need to be further study. Therefore, in the present study, we hypothesized that Tan ⅡA could mitigate TGF-β-related EMT process during IPF progress and investigated the potential molecular mechanisms in vivo and in vitro.Caveolin-1 is a iconic protein of caveola, a small uncoated pit in the cell membrane, which plays important role in maintaining the structure and function of caveola. Caveolin scaffolding domain(CSD), interacting with other signaling factors in Caveolin-1, can down-regulate activity of many signaling factors. Caveolin-1 were expressed in many differentiated cells, especially in alveolar type I epithelial cells,smooth muscle cell, fibroblasts, adipose cells, endothelial cells and other terminally differentiated cells, which implied Caveolin-1 might be a kind of negative regulate factor. In recent years, studies showed that expression of Caveolin-1 decreased in lung of pulmonary fibrosis; meanwhile, mice of Caveolin-1 gene being knock-out showed lung changes as pulmonary fibrosis; besides, Caveolin-1 expression in epithelial cell decreased in lung after dealing with TGF-β1. These results demonstrated that Caveolin-1 was related with pulmonary fibrosis. Caveolin-1 together with type I, ⅡTGF-β1 receptor and Smad2 located in cellular region with rich plasma membrane. Therefore, Caveolin-1 can inhibit TGF-β/Smad signaling pathway by preventing TGF-β to combine with its receptor. In the study regarding inhibitory effect of Tan II A on the TGF-β induced EMT, we discovered Tan II A could suppress BLM-induced activity of TGF-β/Smad signaling pathway, alveolar macrophages increased, collagen deposition and EMT; in vitro, Tan ⅡA was confirmed to inhibit the TGF-β/Smad pathway in A549 and TGF-β induced EMT. But the mechanism of Tan ⅡA inhibiting TGF-β/Smad and TGF-β induced EMT remains unclear. Caveolin-1, the upstream factor of TGF-β, decreased in pulmonary fibrosis and it inhibited TGF-β/Smad pathway to remit lung fibrosis. Since both caveolin-1 and Tan ⅡA had the effect of inhibiting TGF-β signaling pathways in pulmonary fibrosis, the effect of Tan ⅡA might be related with caveolin-1. Experiment in vitro was performed to test whether Tan ⅡA could regulate TGF-β/Smad induced EMT by caveolin-1. We aim to explore the effect of Tan ⅡA on treatment for pulmonary fibrosis and its mechanism to provide more theoretical basis for new drugs development.Part I Tanshinone IIA inhibited rat pulmonary fibrosis induced by bleomycinObjective: To study the effect of Tan ⅡA on lung morphology, type I collagen and hydroxyproline content to definite protective role of Tan ⅡA on pulmonary fibrosis.Methods: Sprague-Dawley(SD) rats, 8 weeks, weighing about 250 g, were divided into four groups: Control group(intratracheal instillation of normal sterile saline), BLM group(intratracheal instillation of saline containing BLM, 5 mg/kg), TanⅡA group(rats received daily injection of Tan ⅡA) and BLM + Tan ⅡA group(BLM-induced rats received daily injection of Tan ⅡA). Rats from each group were sacrificed on day 28 after the initial instillation. Lung tissues were obtained to examinewet/dry weight ratio, to observe the pathological changes by HE and Masson’s staining, to measure the concentration of type Ⅰcollagen, hydroxyproline by ELISA and to detect expression of CD68, the lung macrophage marker, by immunohistochemistry.Results: The wet/dry weight ratio of BLM + Tan ⅡA group was significantly lower than that in the BLM group. BLM instillation induced severe pulmonary lesions in rats, including thickened interstitium and excessive infiltration of inflammatory cells as compared with control group. As expected, these pathological alterations were significantly attenuated by intraperitoneal injection of Tan ⅡA compared with BLM group. Similar inhibitory effect of Tan ⅡA on BLM-stimulated aberrant collagen deposition in the rat lung was also observed. Also, BLM-induced up-regulation of collagen Ⅰand hydroxyproline and CD68 was markedly suppressed by Tan ⅡA administration.Conclusion:(1) Tan ⅡA alleviated lung inflammatory response in BLM-induced pulmonary fibrosis by inhibiting the infiltration of lung alveolar macrophages.(2) Tan ⅡA reduced ECM deposition by inhibiting lung expression of type Ⅰcollagen and hydroxyproline to relieve pulmonary fibrosis.Part II Study on the mechanism of tanshinone ⅡA inhibiting TGF-β depended EMTObjective: To study the impact of Tan ⅡA on EMT and TGF-β/Smad signaling pathway in rats lung tissue with pulmonary fibrosis induced by BLM and A549 cells stimulated by TGF-β1 to discuss the mechanism of tanshinone ⅡA alleviating pulmonary fibrosis.Methods: SD rats, 8 weeks, weighing about 250 g, were divided into four groups: Control group, BLM group, TanⅡA group and BLM + Tan ⅡA group. Rats from each group were sacrificed to obtain lung tissues on day 28 after the initial instillation. Cells were divided into four groups: Control group(treated with 10 μM DMSO for 2 h), Tan ⅡA group(treated with 10 μM Tan ⅡA for 2 h), TGF-β1 group(stimulated with 10ng/ml recombinant TGF-β1for 48 h) and TGF-β1+ Tan ⅡA group(treated with 10μM Tan ⅡA for 2 h, followed by stimulation with 10 ng/m L recombinant TGF-β1 for 48 h). E-cadherin was examinated by immunofluorescence; TGF-β1 was detected by real-time PCR; TGF-β1、Smad2、p-Smad2、Smad3、p-Smad3、 E-cadherin、Fibronectin and vimentin were detected by western blot.Results: Immunofluorescence staining assay was used to detect the expression of epithelial marker, E-cadherin. Lung tissues from rats treated with BLM showed less positive staining cells of E-cadherin when compare with these from normal control or Tan ⅡA-treated control rats. This pulmonary reduction of E-cadherin could be partially reversed by Tan ⅡA injection. Western blot confirmed the immunofluorescence staining results. To the contrast, the expression levels of mesenchymal markers, fibronectin and vimentin, increased in BLM-treated rats and almost decreased to normal levels after Tan ⅡA treatment. Western blot method and real-time PCR showed BLM group, TGF-β1 groups compared with control group, respectively, E-cadherin expression decreased obviously, Fibronectin and vimentin protein expression significantly increased, TGF-β1 RNA and protein expression, p-Smad2 and p-Smad3 expression were higher. Tan ⅡA interventional therapy group, all the above factor expression reversed. Tan ⅡA have no effect on the expression of Smad2 and Smad3, but it affects Smad2 and Smad3 level of phosphorylation.Conclusion: Tan ⅡA ameliorated pulmonary fibrosis, probably through inhibition of TGF-β/Smad pathway-dependent EMT process. 。Conclusion:(1) Tan ⅡA alleviated BLM-induced pulmonary fibrosis by inhibiting lung EMT and activity of TGF-β/Smad signaling pathway.(2) Tan ⅡA relieved fibrosis by inhibiting TGF-β depended EMT in A549 cells stimulated by TGF-β1.Part III Tanshinone ⅡA suppressed TGF-β1 induced EMT in A549 cells by up-regulating caveolin-1Objective: To test whether Tan ⅡA could regulate TGF-β/Smad induced EMT by caveolin-1 to to provide more theoretical basis for new drugs development.Methods: High transfection efficiency of caveolin- 1 sh RNA expression vector was built for cell transfection. Cells are divided into 7 groups: Control group(A), Tanshinone ⅡA group(B)(10μM Tan ⅡA, 2h), Caveolin-1si RNA+ Tan ⅡA group(C)(transfection caveolin-1si RNA, 24h; 10μM tanshinone ⅡA, 2h), TGF-β1 group(D)(10ng/ml TGF-β1, 48h), TGF-β1+ caveolin-1si RNA group(E)(transfection caveolin-1si RNA, 24 h, then 10ng/ml TGF-β1, 48h), Tan ⅡA + TGF-β1 group(F)(10μM Tan ⅡA, 2h, and then 10ng/ml TGF-β1, 48h) and caveolin-1si RNA+ Tan ⅡA + TGF-β1 group(G)(transfection caveolin-1si RNA, 24 h, then 10μM Tan ⅡA, 2h, then with 10ng/ml TGF-β1, 48h). Caveolin-1, TGF-β1, E-cadherin, Fibronectin and vimentin expressions were detected by RT-PCR and western blot. p-Smad2 and p-Smad3 were detected by western blot.Results: TGF-β1 significantly reduced caveolin-1 expression compared with control group and Tan ⅡA treated control cells. Tan ⅡA pre-treatment restored caveolin-1 expression in A549 cells. Expression of Fibronectin and vimentin increased in TGF-β1-stimilated cells and decreased by pretreatment with Tan ⅡA. Factors changes as caveolin-1, TGF-β1, p-Smad2, p-Smad3, E-cadherin, Fibronection, Vimenin have no difference in A549 with or without Tan ⅡA. In transfection Caveolin-1sh RNA group, the expressions of TGF-β1, p-Smad2 and p-Smad3 increased; protein expression of E-cadherin decreased; fibronection and vimenin increased. In TGF-β1+caveolin-1sh RNA group, expression of TGF-β1, p-Smad2, p-Smad3, fibronection and vimentin increased significantly and E-cadherin decreased significantly, compared with TGF-β1 group. In caveolin-1sh RNA+ Tan ⅡA + TGF-β1 group, expression of TGF-β1, p-Smad2, p-Smad3, fibronection and vimentin increased significantly and E-cadherin decreased significantly, compared with Tan ⅡA +TGF-β1 group.Conclusion:(1) The expression of Caveolin-1 is increased by Tan ⅡA in A549 cells stimulated by TGF-β1.(2)Tanshinone ⅡA suppressed TGF-β1 induced EMT in A549 cells by up-regulating caveolin-1.更多还原