上皮-间充质转变（EMT）在百草枯中毒大鼠肺纤维化中的作用及机制探讨
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摘要
百草枯(paraquat, PQ)一种在世界范围内广泛使用的高效除草剂。近20年来,随着其在我国国内的普及使用,PQ中毒的发病率急剧上升,2001～2010年全国文献报道的总例数是过去10年的15倍。由于PQ致死剂量极低(人经口致死量30-40mg/kg),又缺乏特效的解毒剂治疗,临床死亡率却一直居高不下,国内外报道高达60%～87.8%。PQ中毒一直是近年急诊医学领域备受关注的焦点之一,并已逐步上升为一个严峻的社会问题。
肺是PQ中毒的主要靶器官,由于肺泡I,II型细胞和细支气管细胞膜上多胺转运系统的大量表达,肺组织PQ的浓度要比血浆浓度高6-10倍,而PQ对肺泡细胞的氧化损伤和随后出现的肺纤维化导致的快速进展的呼吸衰竭是PQ中毒导致患者死亡的主要原因。快速进展的弥漫性肺纤维化,是PQ中毒的特征性病理改变。但是,目前关于PQ中毒肺纤维化的起源、形成机制仍不清楚,国内外相关的基础研究,十分匮乏。
肺纤维化特征性的病理生理过程是肌成纤维细胞(MFb)的活化与增生,大量的肌纤维母细胞堆积并过分产生细胞外基质是肺纤维化病理过程形成和发展的原因。但是关于MFb的起源和形成过程,传统的观点正在受到越来越多的挑战。传统的观点认为,MFb来源于肺内固有的间充质细胞,而近年的研究认为,MFb除来源于肺内固有的间充质细胞外,还可能来源于外周血(骨髓源性)。新近,支气管、肺泡上皮细胞可能通过“上皮一间充质转变(epithelial----mesenchymal transition,EMT)”而成为MFb的部分来源的观点,正得到越来越多的研究证明而备受关注。EMT是指上皮细胞在形态学上发生间充质细胞表型的转变,并获得转移能力的过程,很久以来一直被认为在胚胎发育和恶性肿瘤的细胞转分化中起主导作用。近10年的研究证实,EMT与纤维化的发生发展也有着密切的联系,EMT在肾间质、心肌、肝脏等脏器的纤维化中,起着十分重要,甚至是关键性的作用。EMT在肺纤维化中的作用,也正日益被证实：体外培养的肺泡II型上皮细胞可以通过EMT而成为MFb的部分来源；博莱霉素诱导的肺纤维化小鼠模型中,发现了肺泡、支气管上皮细胞能通过EMT向MFb转变；特发性肺纤维化患者的活检标本中,也观察到EMT参与了肺纤维化的形成。
那么,PQ中毒作为近20年才出现的一种新的疾病,作为肺纤维化疾病中的一个特殊病种,其肺纤维化的形成是否也有EMT参与呢?如果有,在分子水平上,参与EMT调节的细胞内信号转导途径又有哪些?
EMT时,会发生下列特征性的变化：上皮细胞失去其上皮表型,上皮细胞标志物“上皮细胞钙粘素(E-Cadherin, E-Cad)"表达丢失,并获得间质细胞的表型特征,α平滑肌肌动蛋白(α-SMA).波形蛋白(Vimentin)等间质细胞标志物表达上调。基于此机理,我们设计了本实验研究。
首先,采用一次性腹腔注射PQ的方法,制作PQ中毒大鼠模型。然后,收集染毒后不同时间点中毒大鼠的肺组织,用苦味酸天狼星红染色(picrosirius red staining)观察肺纤维化的动态变化；用免疫组化、Real time RT-PCR、 Western blot等方法,观察上皮细胞和间质细胞标志物基因和蛋白表达的动态变化,探讨PQ中毒大鼠肺纤维化模型中肺上皮细胞是否通过EMT参与了肺纤维化的形成过程。最后,对肺泡上皮细胞发生EMT的分子生物学机制进行初步探索,观察TGF-131/Smads、 Wnt/β-catenin两条分子内信号转导途径在肺泡上皮细胞发生EMT时的可能作用。
目的：
探讨肺组织上皮细胞是否通过“上皮—间充质转变(EMT)”参与了PQ中毒大鼠的肺纤维化形成过程,并初步探讨其可能的分子生物学机制。
方法：
第一章PQ中毒大鼠的模型建立。用一次性腹腔注射PQ(15mg/Kg)的方法,建立PQ中毒大鼠模型。72只大鼠随机分成实验组(中毒组,n=48)和对照组(n=24),实验组腹腔注射20%PQ(剂量15mg/Kg),对照组腹腔注射等体积的生理盐水,分别于染毒后第1、3、7、10、14、21d处死大鼠,实验组每次8只,对照组每次4只,留取肺组织标本。两组标本行苏木精-伊红(H-E)染色,光镜下观察肺组织病理变化；行苦味酸天狼星红(Picrosirius red)胶原特异染色,观察肺组织纤维化程度变化,计算胶原容积积分(collagen volume fraction, CVF)。
第二章EMT在PQ中毒大鼠肺纤维化中的作用。用免疫组化(En Vision两步法)、Real time RT-PCR、 Western blot方法,观察：(1)上皮细胞标志物E-Cad的基因和蛋白表达的动态变化。(2)间质细胞标志物αa-SMA、 Vimentin和成纤维细胞特异蛋白(FSP-1)的基因和/或蛋白表达的动态变化。(3)I型胶原蛋白(collagen Ⅰ,Co1-Ⅰ)和Ⅲ型胶原蛋白(ollagen Ⅲ, Col-Ⅲ)基因和/或蛋白表达的动态变化。(4)转录因子Slug (Snail2)和Twist的基因表达的动态变化。
第三章TGF-β1/Smads信号转导途径在PQ中毒肺纤维化EMT中的作用。用Real time RT-PCR方法,检测染毒后不同时间点大鼠肺组织TGF-β1、Smad2和Smad7mRNA的动态变化。
第四章Wnt/β-catenin信号转导途径在PQ中毒肺纤维化EMT中的作用。用Real time RT-PCR方法,检测染毒后不同时间点大鼠肺组织TGF-β1、Wnt2和β-catenin mRNA的动态变化。
结果：
第一章急性肺泡炎是PQ染毒后第1w的主要病理表现,第14d见肺泡间隔内大量胶原纤维沉积,第21d有所减轻。在实验组,各不同时间点肺组织CVF变化,多组间比较差异有统计学意义(P<0.05),对照组无统计学意义(P>0.05)。实验组,CVF从染毒后第3d开始升高,第14d达高峰,第21d有所回落,与同期对照组比较,差异均有统计学意义(P<0.05或0.01))。模型建立成功。
第二章在实验组,各不同时间点肺组织E-Cad、 α-SMA、 Vimentin、 FSP-1、 Col-Ⅰ、 Col-Ⅲ、 Slug和Twist的基因和/或蛋白表达变化,多组间比较差异有统计学意义(P<0.05)；对照组无统计学意义(P>0.05)。实验组,PQ染毒后第3d, E-Cad表达开始逐渐下调,第14d降至谷底,第21d有所回升；PQ染毒后第3～7d,α-SMA、 Vimentin、 FSP-1、 Col-Ⅰ、 Col-Ⅲ、 Slug、 Twist的基因(和蛋白)表达开始逐渐上调,第14d达峰值,第21d有所回落。
第三章在实验组,各不同时间点肺组织TGF-β1和Smad2的基因表达变化,多组间比较差异有统计学意义(P<0.05)；对照组无统计学意义(P>0.05)。在实验组,PQ染毒后第7d, TGF-β1mRNA表达开始逐渐上调,第10d, Smad2mRNA表达开始逐渐上调,两者均在第14d达峰值,第21d有所回落。在实验组和对照组,不同时间点的Smad7mRNA表达均无明显变化。
第四章在实验组,各不同时间点肺组织Wnt2和β-catenin mRNA表达变化,多组间比较差异有统计学意义(P<0.05)；对照组无统计学意义(P>0.05)。在实验组,PQ染毒后第10d, Wnt2和β-catenin mRNA表达突然大幅度上调,第21d达峰值,观察期内未见回落。
结论和展望：
PQ中毒肺纤维化模型大鼠肺组织上皮细胞发生了较为典型的EMT现象,肺泡上皮细胞通过EMT转变为间质细胞,可能是MFb的一个重要来源,在PQ中毒肺纤维化进程中发挥着重要作用。TGF-β1是PQ中毒肺纤维化发生发展过程中重要的细胞因子,其持续高表达可能通过Smad2依赖的TGF-β1/Smads信号转导途径和非Smad依赖的Wnt/β-catenin信号转导途径诱导肺上皮细胞发生了EMT。
PQ中毒肺组织EMT现象的新发现,是PQ中毒肺纤维化形成机制的重要突破,将为PQ中毒的临床治疗带来新思路、新策略、新前景,为PQ中毒特效解毒剂的寻找、靶基因治疗、新药物的开发等提供重要的理论依据。但PQ中毒肺上皮细胞发生EMT的最终确认、EMT来源的MFb占肺组织全部MFb的比例、EMT发生的确切机制,尚需使用转基因动物模型、流式细胞分析技术、共聚焦显微技术、基因芯片技术等进行更深入的研究；而且,本研究只观察到大鼠PQ中毒后第3w便终止了实验,PQ中毒后更长时间段的肺纤维化规律和发生机制也值得进一步的观察和探讨。
Paraquat dichloride (methyl viologen; PQ) is an effective and widely used herbicide all over the world. Over the last two decades, with the growing widespread application of PQ in China, the morbidity of PQ poisoning rapidly elevated and the number of reported cases in recent10years is15times that of the past10years from China Journal Full-text Database. PQ poisoning is an extremely frustrating condition to manage clinically, and the mortality was as high as60%～87.8%according to the domestic and abroad reports, due to the very lower fatal dose (ingestion of30～40mg/kg) and the lack of magic antidote. PQ poisoning has been becoming one of the focal questions in emergency medicine and even a severe social problem.
Lung is the main target organ of PQ poisoning, and the pulmonary concentrations of PQ can be6to10times higher than those in the plasma, which can be explained by the participation of the polyamine transport system abundantly expressed in the membrane of alveolar cells type Ⅰ, Ⅱ, and Clara cells. The major cause of death in paraquat poisoning is a rapidly progressive respiratory failure due to an oxidative insult to the alveolar epithelium with subsequent fulminant obliterating fibrosis. Progressive and extensive pulmonary fibrosis is the distinctive pathological changes in PQ poisoning, but its source and molecular biological mechanism are still unknown so far and the correlative research is scanty.
The activation and proliferation of myofibroblast (MFb) are the distinctive pathophysiologic changes of pulmonary fibrosis, and the accumulation of a large number of MFb is responsible for exaggerated and uncontrolled production of extracellular matrix during the development and progression of pathological fibrosis. But the traditionary opinion about the origin of myofibroblast which deemed that the myofibroblast comed from the resident mesenchymal cells in the lung is beening challenged by more and more new experiments. Epperly et al. discovered that there is a significant contribution of bone marrow-derived cells in the pathophysiology of murine lung irradiation fibrosis in2003, and the resent opinion concerning the contribution of alveolar epithelial cell (AEC) to the development of pulmonary fibrosis by Epithelial-Mesenchymal Transition (EMT) is been certificating. EMT is a graded response whereby epithelial cells reversibly acquire mesenchymal features and enhanced capacity of invasion, which has been considered a leading role in the cell transdifferentiation of embryonic development and malignant tumor for a long time. In recent10years, a large number of evidences have highlighted a link between EMT and the fibrosis. EMT plays a significant and even a key pole in fibrosis of renal interstitiumcancer, myocardium and liver. The importance of EMT as a factor of pulmonary fibrosis is also increasingly recognized. Kasai H et al. reported TGF-betal induces human alveoiar epithelial in vitro culture to mesenchymal cell transition in2005, and EMT was detected in airways of a bleomycin induced pulmonary fibrosis model derived from an a-smooth muscle actin-Cre Transgenic mouse in2009. Furthermore, EMT was observed even in patients with idiopathic pulmonary fibrosis (IPF).
Well, PQ poisoning, a newly emerged disease20years ago, a specific one of pulmonary fibrosis diseases, is involved in EMT? Whether EMT participates in the pulmonary fibrosis of PQ poisoning or not? If it does, by which intra-cellular signal transduction pathway might EMT be regulated at molecular level?
When EMT happened, epithelial cells would lose their features of epithelial phenotype, lose the gene and protein expressions of E-cadherin which is the marker of epithelial cells, and acquire the features of mesenchymal phenotype:the protein of expression of α-SMA in cytoplasm significantly enhances, the cell skeleton fracture changes, and the gene and protein expressions of markers of mesenchymal cells increase including Vimentin, Fibronectin and N-cadherin. Based on this theory, we designed this study to investigate the role of EMT in pulmonary fibrosis caused by PQ poisoning in rat and the possible mechanisms of EMT.
At first, rat models of pulmonary fibrosis caused by PQ poisoning were established by intraperitoneal injection at the dose of15mg/Kg and the lung speciments at different time points were collected. And then, collagen specific picrosirius red staining was performed to observe the degree of pulmonary fibrosis. Subsequently, Gene and protein expression of the epithelial and mesenchymal phenotype markers were determined by real time reversed transcriptional polymerase chain reaction (Real time RT-PCR), immunohistochemistry and western blot analysis to investigate whether the alveolar epithelial cells participate the pulmonary fibrosis by EMT. At last, the roles of TGF-β1/Smads and Wnt/β-catenin signal transduction pathways in AECs EMT were explored to investigate the possible mechanism of AECs EMT primitively.
Objective:
To investigate the role of EMT and its possible mechanism in pulmonary fibrosis caused by paraquat poisoning in rats.
Methods:
Chapter one Establishment of rat models of pulmonary fibrosis caused by paraquat poisoning. Seventy-two adult male Sprague-Dawley (SD) rats were randomized to two groups:PQ poisoning group (n=48) and control group(n=24). PQ was administrated by intraperitoneal injection at a dose of15mg/Kg to the rats in PQ poisoning group to establish rat models of the pulmonary fibrosis, and equivalent volume of normal sodium to the rats in contral group by intraperitoneal injection, too. Rats were sacrificed at days1,3,7,10,14and21after the PQ treatment, each time8rats in PQ poisoning group and4rats in control group. Lungs were excised from rat bodies and lung speciments were processed and reserved for later histological evaluation, immunohistochemical analyses and the determination of gene and protein expression respectively by real-time RT-PCR assay and western blot analysis.
In this chapter, H-E staining was performed to observe the pathological changes and collagen specific picrosirius red staining was performed to observe the degree of pulmonary fibrosis and to calculate the collagen volume fraction (CVF).
Chapter two The role of EMT in pulmonary fibrosis caused by paraquat poisoning in rats. Gene expressions of the epithelial phenotype marker (E-cad) mesenchymal phenotype markers (α-SMA, FSP-1and Vimentin), fibrogenesis markers (collagens type Ⅰ and Ⅲ) and transcription factor (Slug and Twist) was determined by real time reversed transcriptional polymerase chain reaction (Real time RT-PCR). In addition, protein expression of E-cad, α-SMA and collagens type Ⅰ was determined by immunohistochemistry and western blot analysis.
Chapter three The role of TGF-β1/Smads signal transduction pathway in AECs EMT in pulmonary fibrosis induced by paraquat poisoning in rats. Gene expression of TGF-β1,Smad2and Smad7in pulmonary tissues obtained at different time points was determined by Real time RT-PCR.
Chapter four The role of Wnt/β3-catenin signal transduction pathway in AECs EMT in pulmonary fibrosis induced by paraquat poisoning in rats. Gene expression of TGF-β1, Wnt2and β-catenin in pulmonary tissues obtained at different time points was determined by Real time RT-PCR.
Results:
Chapter one Acute pulmonary alveolitis was the chief pathological change of paraquat poisoning in the first week, and collogen deposition was observed in alveolar septum in the second week which arrived peaking on day14but decreased on day21. Obvious pulmonary fibrosis marked the successful modeling of pulmonary fibrosis by paraquat poisoning in rat. In PQ poisoning group, the pulmonary tissue CVF began to rise on the3rd day and arrived peaking on the14st day, but down-regulated on the21st day, with significant difference (P<0.01). On day3,7,10,14and21, the values of CVF in PQ poisoning group were significantly higher than coinstantaneous that in control group (P<0.05or0.01).
Chapter two There were significant difference among the gene and/or protein expressions of E-Cad, α-SMA, Vimentin, FSP-1, Col-Ⅰ, Col-Ⅲ, Slug and Twist at different time points in PQ poisoning group(P<0.05), but not in control group (P>0.05). In PQ poisoning group, the gene and protein expression of E-Cad down regulated on the3rd day and arrived valley bottom on the14st day, but up-regulated on the21st day. Also in PQ poisoning group, the expressions of α-SMA, Vimentin, FSP-1, Col-Ⅰ, Col-Ⅲ, Slug and Twist up-regulated during the first week and arrived peaking on the14st day, but showed up-regulation on21st day.
Chapter three There was significant difference among the gene expressions of TGF-β1and Smad2in PQ poisoning group at different time points in PQ poisoning group(P<0.05), but not in control group (P>0.05). The expression of TGF-β1mRNA enhanced on the7th day and Smad2mRNA did on the10th day. Both of them arrived peaking on the14st day and down-regulated on the21st day. There was not significant difference among the gene expressions of Smad7at different time points either in PQ poisoning group or in control group (P>0.05).
Chapter four There was significant difference among the gene expressions of Wnt2and β-catenin at different time points in PQ poisoning group(P<0.05), but not in control group (P>0.05). In PQ poisoning group, the expressions of Wnt2and β-catenin mRNA suddenly enhanced in a large argument on the day10, and arrived peaking on the21st day.
Conclusions:
Our study shows that some alveolar epithelial cells in pulmonary fibrosis model rat caused by paraquat poisoning underwent EMT. EMT may be an imporportant one of the sources where the myofibroblasts in lung tissue derived from and play a key role in pulmonary fibrosis caused by paraquat poisoning. TGF-betal, the critical cytokine in pulmonary fibrosis, showed a persistent homo-expression in our study and might induce AECs to occur EMT by TGF-betal/Smads pathway depending on Smad2and by Wnt/β-catenin pathway which not depending on Smads. If it is really like it, our observation of AECs EMT would brings a new breakthrough in the mechanisms of pulmonary fibrosis caused by paraquat poisoning and might provide base theoretic support for the search of magic antidote, exploitation of new drug and target gene treatment in clinical medcine of PQ poisoning. But as to the confirmation of AECs EMT, the percentage of MFb from AECs EMT and the indeed mechanism of EMT, much more studies should be done by transgenic animal model, flow cytometry, confocal microtechnique, gene chip and so on. In addition, our experiment lasted just for3weeks and the characteristics of pulmonary fibrosis by PQ poisoning over3weeks deserves our further observation and exploration.
肺是PQ中毒的主要靶器官,由于肺泡I,II型细胞和细支气管细胞膜上多胺转运系统的大量表达,肺组织PQ的浓度要比血浆浓度高6-10倍,而PQ对肺泡细胞的氧化损伤和随后出现的肺纤维化导致的快速进展的呼吸衰竭是PQ中毒导致患者死亡的主要原因。快速进展的弥漫性肺纤维化,是PQ中毒的特征性病理改变。但是,目前关于PQ中毒肺纤维化的起源、形成机制仍不清楚,国内外相关的基础研究,十分匮乏。
肺纤维化特征性的病理生理过程是肌成纤维细胞(MFb)的活化与增生,大量的肌纤维母细胞堆积并过分产生细胞外基质是肺纤维化病理过程形成和发展的原因。但是关于MFb的起源和形成过程,传统的观点正在受到越来越多的挑战。传统的观点认为,MFb来源于肺内固有的间充质细胞,而近年的研究认为,MFb除来源于肺内固有的间充质细胞外,还可能来源于外周血(骨髓源性)。新近,支气管、肺泡上皮细胞可能通过“上皮一间充质转变(epithelial----mesenchymal transition,EMT)”而成为MFb的部分来源的观点,正得到越来越多的研究证明而备受关注。EMT是指上皮细胞在形态学上发生间充质细胞表型的转变,并获得转移能力的过程,很久以来一直被认为在胚胎发育和恶性肿瘤的细胞转分化中起主导作用。近10年的研究证实,EMT与纤维化的发生发展也有着密切的联系,EMT在肾间质、心肌、肝脏等脏器的纤维化中,起着十分重要,甚至是关键性的作用。EMT在肺纤维化中的作用,也正日益被证实：体外培养的肺泡II型上皮细胞可以通过EMT而成为MFb的部分来源；博莱霉素诱导的肺纤维化小鼠模型中,发现了肺泡、支气管上皮细胞能通过EMT向MFb转变；特发性肺纤维化患者的活检标本中,也观察到EMT参与了肺纤维化的形成。
那么,PQ中毒作为近20年才出现的一种新的疾病,作为肺纤维化疾病中的一个特殊病种,其肺纤维化的形成是否也有EMT参与呢?如果有,在分子水平上,参与EMT调节的细胞内信号转导途径又有哪些?
EMT时,会发生下列特征性的变化：上皮细胞失去其上皮表型,上皮细胞标志物“上皮细胞钙粘素(E-Cadherin, E-Cad)"表达丢失,并获得间质细胞的表型特征,α平滑肌肌动蛋白(α-SMA).波形蛋白(Vimentin)等间质细胞标志物表达上调。基于此机理,我们设计了本实验研究。
首先,采用一次性腹腔注射PQ的方法,制作PQ中毒大鼠模型。然后,收集染毒后不同时间点中毒大鼠的肺组织,用苦味酸天狼星红染色(picrosirius red staining)观察肺纤维化的动态变化；用免疫组化、Real time RT-PCR、 Western blot等方法,观察上皮细胞和间质细胞标志物基因和蛋白表达的动态变化,探讨PQ中毒大鼠肺纤维化模型中肺上皮细胞是否通过EMT参与了肺纤维化的形成过程。最后,对肺泡上皮细胞发生EMT的分子生物学机制进行初步探索,观察TGF-131/Smads、 Wnt/β-catenin两条分子内信号转导途径在肺泡上皮细胞发生EMT时的可能作用。
目的：
探讨肺组织上皮细胞是否通过“上皮—间充质转变(EMT)”参与了PQ中毒大鼠的肺纤维化形成过程,并初步探讨其可能的分子生物学机制。
方法：
第一章PQ中毒大鼠的模型建立。用一次性腹腔注射PQ(15mg/Kg)的方法,建立PQ中毒大鼠模型。72只大鼠随机分成实验组(中毒组,n=48)和对照组(n=24),实验组腹腔注射20%PQ(剂量15mg/Kg),对照组腹腔注射等体积的生理盐水,分别于染毒后第1、3、7、10、14、21d处死大鼠,实验组每次8只,对照组每次4只,留取肺组织标本。两组标本行苏木精-伊红(H-E)染色,光镜下观察肺组织病理变化；行苦味酸天狼星红(Picrosirius red)胶原特异染色,观察肺组织纤维化程度变化,计算胶原容积积分(collagen volume fraction, CVF)。
第二章EMT在PQ中毒大鼠肺纤维化中的作用。用免疫组化(En Vision两步法)、Real time RT-PCR、 Western blot方法,观察：(1)上皮细胞标志物E-Cad的基因和蛋白表达的动态变化。(2)间质细胞标志物αa-SMA、 Vimentin和成纤维细胞特异蛋白(FSP-1)的基因和/或蛋白表达的动态变化。(3)I型胶原蛋白(collagen Ⅰ,Co1-Ⅰ)和Ⅲ型胶原蛋白(ollagen Ⅲ, Col-Ⅲ)基因和/或蛋白表达的动态变化。(4)转录因子Slug (Snail2)和Twist的基因表达的动态变化。
第三章TGF-β1/Smads信号转导途径在PQ中毒肺纤维化EMT中的作用。用Real time RT-PCR方法,检测染毒后不同时间点大鼠肺组织TGF-β1、Smad2和Smad7mRNA的动态变化。
第四章Wnt/β-catenin信号转导途径在PQ中毒肺纤维化EMT中的作用。用Real time RT-PCR方法,检测染毒后不同时间点大鼠肺组织TGF-β1、Wnt2和β-catenin mRNA的动态变化。
结果：
第一章急性肺泡炎是PQ染毒后第1w的主要病理表现,第14d见肺泡间隔内大量胶原纤维沉积,第21d有所减轻。在实验组,各不同时间点肺组织CVF变化,多组间比较差异有统计学意义(P<0.05),对照组无统计学意义(P>0.05)。实验组,CVF从染毒后第3d开始升高,第14d达高峰,第21d有所回落,与同期对照组比较,差异均有统计学意义(P<0.05或0.01))。模型建立成功。
第二章在实验组,各不同时间点肺组织E-Cad、 α-SMA、 Vimentin、 FSP-1、 Col-Ⅰ、 Col-Ⅲ、 Slug和Twist的基因和/或蛋白表达变化,多组间比较差异有统计学意义(P<0.05)；对照组无统计学意义(P>0.05)。实验组,PQ染毒后第3d, E-Cad表达开始逐渐下调,第14d降至谷底,第21d有所回升；PQ染毒后第3～7d,α-SMA、 Vimentin、 FSP-1、 Col-Ⅰ、 Col-Ⅲ、 Slug、 Twist的基因(和蛋白)表达开始逐渐上调,第14d达峰值,第21d有所回落。
第三章在实验组,各不同时间点肺组织TGF-β1和Smad2的基因表达变化,多组间比较差异有统计学意义(P<0.05)；对照组无统计学意义(P>0.05)。在实验组,PQ染毒后第7d, TGF-β1mRNA表达开始逐渐上调,第10d, Smad2mRNA表达开始逐渐上调,两者均在第14d达峰值,第21d有所回落。在实验组和对照组,不同时间点的Smad7mRNA表达均无明显变化。
第四章在实验组,各不同时间点肺组织Wnt2和β-catenin mRNA表达变化,多组间比较差异有统计学意义(P<0.05)；对照组无统计学意义(P>0.05)。在实验组,PQ染毒后第10d, Wnt2和β-catenin mRNA表达突然大幅度上调,第21d达峰值,观察期内未见回落。
结论和展望：
PQ中毒肺纤维化模型大鼠肺组织上皮细胞发生了较为典型的EMT现象,肺泡上皮细胞通过EMT转变为间质细胞,可能是MFb的一个重要来源,在PQ中毒肺纤维化进程中发挥着重要作用。TGF-β1是PQ中毒肺纤维化发生发展过程中重要的细胞因子,其持续高表达可能通过Smad2依赖的TGF-β1/Smads信号转导途径和非Smad依赖的Wnt/β-catenin信号转导途径诱导肺上皮细胞发生了EMT。
PQ中毒肺组织EMT现象的新发现,是PQ中毒肺纤维化形成机制的重要突破,将为PQ中毒的临床治疗带来新思路、新策略、新前景,为PQ中毒特效解毒剂的寻找、靶基因治疗、新药物的开发等提供重要的理论依据。但PQ中毒肺上皮细胞发生EMT的最终确认、EMT来源的MFb占肺组织全部MFb的比例、EMT发生的确切机制,尚需使用转基因动物模型、流式细胞分析技术、共聚焦显微技术、基因芯片技术等进行更深入的研究；而且,本研究只观察到大鼠PQ中毒后第3w便终止了实验,PQ中毒后更长时间段的肺纤维化规律和发生机制也值得进一步的观察和探讨。
Paraquat dichloride (methyl viologen; PQ) is an effective and widely used herbicide all over the world. Over the last two decades, with the growing widespread application of PQ in China, the morbidity of PQ poisoning rapidly elevated and the number of reported cases in recent10years is15times that of the past10years from China Journal Full-text Database. PQ poisoning is an extremely frustrating condition to manage clinically, and the mortality was as high as60%～87.8%according to the domestic and abroad reports, due to the very lower fatal dose (ingestion of30～40mg/kg) and the lack of magic antidote. PQ poisoning has been becoming one of the focal questions in emergency medicine and even a severe social problem.
Lung is the main target organ of PQ poisoning, and the pulmonary concentrations of PQ can be6to10times higher than those in the plasma, which can be explained by the participation of the polyamine transport system abundantly expressed in the membrane of alveolar cells type Ⅰ, Ⅱ, and Clara cells. The major cause of death in paraquat poisoning is a rapidly progressive respiratory failure due to an oxidative insult to the alveolar epithelium with subsequent fulminant obliterating fibrosis. Progressive and extensive pulmonary fibrosis is the distinctive pathological changes in PQ poisoning, but its source and molecular biological mechanism are still unknown so far and the correlative research is scanty.
The activation and proliferation of myofibroblast (MFb) are the distinctive pathophysiologic changes of pulmonary fibrosis, and the accumulation of a large number of MFb is responsible for exaggerated and uncontrolled production of extracellular matrix during the development and progression of pathological fibrosis. But the traditionary opinion about the origin of myofibroblast which deemed that the myofibroblast comed from the resident mesenchymal cells in the lung is beening challenged by more and more new experiments. Epperly et al. discovered that there is a significant contribution of bone marrow-derived cells in the pathophysiology of murine lung irradiation fibrosis in2003, and the resent opinion concerning the contribution of alveolar epithelial cell (AEC) to the development of pulmonary fibrosis by Epithelial-Mesenchymal Transition (EMT) is been certificating. EMT is a graded response whereby epithelial cells reversibly acquire mesenchymal features and enhanced capacity of invasion, which has been considered a leading role in the cell transdifferentiation of embryonic development and malignant tumor for a long time. In recent10years, a large number of evidences have highlighted a link between EMT and the fibrosis. EMT plays a significant and even a key pole in fibrosis of renal interstitiumcancer, myocardium and liver. The importance of EMT as a factor of pulmonary fibrosis is also increasingly recognized. Kasai H et al. reported TGF-betal induces human alveoiar epithelial in vitro culture to mesenchymal cell transition in2005, and EMT was detected in airways of a bleomycin induced pulmonary fibrosis model derived from an a-smooth muscle actin-Cre Transgenic mouse in2009. Furthermore, EMT was observed even in patients with idiopathic pulmonary fibrosis (IPF).
Well, PQ poisoning, a newly emerged disease20years ago, a specific one of pulmonary fibrosis diseases, is involved in EMT? Whether EMT participates in the pulmonary fibrosis of PQ poisoning or not? If it does, by which intra-cellular signal transduction pathway might EMT be regulated at molecular level?
When EMT happened, epithelial cells would lose their features of epithelial phenotype, lose the gene and protein expressions of E-cadherin which is the marker of epithelial cells, and acquire the features of mesenchymal phenotype:the protein of expression of α-SMA in cytoplasm significantly enhances, the cell skeleton fracture changes, and the gene and protein expressions of markers of mesenchymal cells increase including Vimentin, Fibronectin and N-cadherin. Based on this theory, we designed this study to investigate the role of EMT in pulmonary fibrosis caused by PQ poisoning in rat and the possible mechanisms of EMT.
At first, rat models of pulmonary fibrosis caused by PQ poisoning were established by intraperitoneal injection at the dose of15mg/Kg and the lung speciments at different time points were collected. And then, collagen specific picrosirius red staining was performed to observe the degree of pulmonary fibrosis. Subsequently, Gene and protein expression of the epithelial and mesenchymal phenotype markers were determined by real time reversed transcriptional polymerase chain reaction (Real time RT-PCR), immunohistochemistry and western blot analysis to investigate whether the alveolar epithelial cells participate the pulmonary fibrosis by EMT. At last, the roles of TGF-β1/Smads and Wnt/β-catenin signal transduction pathways in AECs EMT were explored to investigate the possible mechanism of AECs EMT primitively.
Objective:
To investigate the role of EMT and its possible mechanism in pulmonary fibrosis caused by paraquat poisoning in rats.
Methods:
Chapter one Establishment of rat models of pulmonary fibrosis caused by paraquat poisoning. Seventy-two adult male Sprague-Dawley (SD) rats were randomized to two groups:PQ poisoning group (n=48) and control group(n=24). PQ was administrated by intraperitoneal injection at a dose of15mg/Kg to the rats in PQ poisoning group to establish rat models of the pulmonary fibrosis, and equivalent volume of normal sodium to the rats in contral group by intraperitoneal injection, too. Rats were sacrificed at days1,3,7,10,14and21after the PQ treatment, each time8rats in PQ poisoning group and4rats in control group. Lungs were excised from rat bodies and lung speciments were processed and reserved for later histological evaluation, immunohistochemical analyses and the determination of gene and protein expression respectively by real-time RT-PCR assay and western blot analysis.
In this chapter, H-E staining was performed to observe the pathological changes and collagen specific picrosirius red staining was performed to observe the degree of pulmonary fibrosis and to calculate the collagen volume fraction (CVF).
Chapter two The role of EMT in pulmonary fibrosis caused by paraquat poisoning in rats. Gene expressions of the epithelial phenotype marker (E-cad) mesenchymal phenotype markers (α-SMA, FSP-1and Vimentin), fibrogenesis markers (collagens type Ⅰ and Ⅲ) and transcription factor (Slug and Twist) was determined by real time reversed transcriptional polymerase chain reaction (Real time RT-PCR). In addition, protein expression of E-cad, α-SMA and collagens type Ⅰ was determined by immunohistochemistry and western blot analysis.
Chapter three The role of TGF-β1/Smads signal transduction pathway in AECs EMT in pulmonary fibrosis induced by paraquat poisoning in rats. Gene expression of TGF-β1,Smad2and Smad7in pulmonary tissues obtained at different time points was determined by Real time RT-PCR.
Chapter four The role of Wnt/β3-catenin signal transduction pathway in AECs EMT in pulmonary fibrosis induced by paraquat poisoning in rats. Gene expression of TGF-β1, Wnt2and β-catenin in pulmonary tissues obtained at different time points was determined by Real time RT-PCR.
Results:
Chapter one Acute pulmonary alveolitis was the chief pathological change of paraquat poisoning in the first week, and collogen deposition was observed in alveolar septum in the second week which arrived peaking on day14but decreased on day21. Obvious pulmonary fibrosis marked the successful modeling of pulmonary fibrosis by paraquat poisoning in rat. In PQ poisoning group, the pulmonary tissue CVF began to rise on the3rd day and arrived peaking on the14st day, but down-regulated on the21st day, with significant difference (P<0.01). On day3,7,10,14and21, the values of CVF in PQ poisoning group were significantly higher than coinstantaneous that in control group (P<0.05or0.01).
Chapter two There were significant difference among the gene and/or protein expressions of E-Cad, α-SMA, Vimentin, FSP-1, Col-Ⅰ, Col-Ⅲ, Slug and Twist at different time points in PQ poisoning group(P<0.05), but not in control group (P>0.05). In PQ poisoning group, the gene and protein expression of E-Cad down regulated on the3rd day and arrived valley bottom on the14st day, but up-regulated on the21st day. Also in PQ poisoning group, the expressions of α-SMA, Vimentin, FSP-1, Col-Ⅰ, Col-Ⅲ, Slug and Twist up-regulated during the first week and arrived peaking on the14st day, but showed up-regulation on21st day.
Chapter three There was significant difference among the gene expressions of TGF-β1and Smad2in PQ poisoning group at different time points in PQ poisoning group(P<0.05), but not in control group (P>0.05). The expression of TGF-β1mRNA enhanced on the7th day and Smad2mRNA did on the10th day. Both of them arrived peaking on the14st day and down-regulated on the21st day. There was not significant difference among the gene expressions of Smad7at different time points either in PQ poisoning group or in control group (P>0.05).
Chapter four There was significant difference among the gene expressions of Wnt2and β-catenin at different time points in PQ poisoning group(P<0.05), but not in control group (P>0.05). In PQ poisoning group, the expressions of Wnt2and β-catenin mRNA suddenly enhanced in a large argument on the day10, and arrived peaking on the21st day.
Conclusions:
Our study shows that some alveolar epithelial cells in pulmonary fibrosis model rat caused by paraquat poisoning underwent EMT. EMT may be an imporportant one of the sources where the myofibroblasts in lung tissue derived from and play a key role in pulmonary fibrosis caused by paraquat poisoning. TGF-betal, the critical cytokine in pulmonary fibrosis, showed a persistent homo-expression in our study and might induce AECs to occur EMT by TGF-betal/Smads pathway depending on Smad2and by Wnt/β-catenin pathway which not depending on Smads. If it is really like it, our observation of AECs EMT would brings a new breakthrough in the mechanisms of pulmonary fibrosis caused by paraquat poisoning and might provide base theoretic support for the search of magic antidote, exploitation of new drug and target gene treatment in clinical medcine of PQ poisoning. But as to the confirmation of AECs EMT, the percentage of MFb from AECs EMT and the indeed mechanism of EMT, much more studies should be done by transgenic animal model, flow cytometry, confocal microtechnique, gene chip and so on. In addition, our experiment lasted just for3weeks and the characteristics of pulmonary fibrosis by PQ poisoning over3weeks deserves our further observation and exploration.
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Shimada H, Hirai K, Simamura E, Hatta T, et al.,2009. Paraquat toxicity induced by voltage-dependent anion channel 1 acts as an NADH-dependent oxidoreductase. J Biol Chem,284(42):28642-28649.
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