甘草酸抑制糖基化修饰调控EMT的机制研究

发布时间：2018-05-16 09:46

本文选题：甘草酸 + 肺间质纤维化　；参考：《大连医科大学》2016年博士论文

【摘要】：肺间质纤维化(Pulmonary fibrosis)是一种慢性疾病,病程逐渐发展、不可逆转,是间质性肺疾病的终末期,据统计其平均生存期短,不足5年。肺间质纤维化的患者呼吸困难明显,生活难以自理,且花费大,给社会、家庭、及个人带来沉重的精神及经济负担。因此延缓肺间质纤维化进展、降低死亡率是我国肺病防治所面临的巨大挑战和重大需求。虽然导致肺间质纤维化的病因不甚清楚,但肺间质纤维化的病理表现与信号通路主要表现为:肺间质中大量炎症细胞浸润,肌成纤维细胞显著增多从而产生过多的细胞外基质积聚,最终出现纤维化的病理改变。目前尚无有效手段阻止其进展。因此,阐明肺间质纤维化发生、发展机制,寻找有效的新的干预治疗策略,对阻止疾病发展、降低死亡率有着重大的意义。但是,目前我们尚不十分明确有关肺间纤维化这一疾病的发病机制。肺间质纤维化疾病病程中关键的病理改变包括肌成纤维细胞大量、异常增多。目前针对肌成纤维细胞的来源的研究较多,说法不一,争议较大。其研究结果显示多数观点倾向主要来源为肺泡上皮细胞,即EMT(epithelial to mesenchymaltransition)。肺泡上皮细胞活化后的一个重要效应是:转化为肌成纤维细胞,促进细胞外基质合成,同时活化多种参与固有免疫、炎症的基因等,促进炎症、氧化应激损伤,从而加重肺间质纤维化进展。由此推测:肺泡上皮细胞转分化(EMT)是肺间质中肌成纤维细胞的主要来源。深入研究EMT的机制及其活化后导致的重要分子生物学效应,是寻求干预靶点的重要防治策略。综上所述,肺间质纤维化是一种难治、复杂的疾病。目前传统激素及单通路受体阻滞剂(如吉非替尼)可能获得部分疗效,但不能阻止肺间质纤维化的进展。近年来有关我国中草药方剂及其单体活性成分在疾病治疗中的研究越来越多,其在肺间质纤维化的治疗中的作用也越来越引起大家的重视。本课题组前期实验证实活血化瘀方剂(黄芪、川芎、丹参、当归、红景天、浙贝母、甘草)对肺间质纤维化有治疗作用。其中甘草酸(Glycyrrhizic acid,GA)作为甘草中最主要的活性成分,大量研究表明甘草酸对脏器纤维化(如肝脏)有显著的治疗作用。但甘草酸对肺间质纤维化是否有改善作用我们尚不清楚。因此我们提出以下科学问题:多种炎症、多条信号通路的过度激活是肺间质纤维化的重要基础,EMT又是导致肺间质纤维化的重要途径,是什么调控了这些炎症因子和信号通路活性?甘草酸是否能够找到控制它们活性的共同靶点?近年,糖蛋白组学的飞速发展为我们解答以上问题提供了新的研究方向。核心岩藻糖基化(core fucosylation,CF)是一种重要的蛋白质糖修饰,是由α-1,6岩藻糖转移酶(Fucosyltransferase8,FUT8)特异性地催化转移岩藻糖,将其连接在靶蛋白的特定氨基酸位点的核心糖链上,从而完成靶蛋白质的折叠、空间构象。我们课题组研究表明脏器纤维化中其关键蛋白均为糖蛋白,所以,我们思考:甘草酸是否通过CF修饰调控这些炎症因子及信号通路的活性?要解决上述科学问题,本实验拟通过建立博来霉素(Bleomycin,BLM)诱导肺间质纤维化大鼠模型明确甘草酸对肺间质纤维化有无作用;在大鼠肺间质纤维化模型上观察甘草酸对EMT的影响;在此基础上,通过观察甘草酸对CF的影响进而深入解析甘草酸抑制EMT的具体机制。本实验首次发现甘草酸能减轻BLM诱导的肺间质纤维化及抑制EMT;其次从糖生物学全新的角度阐释了甘草酸治疗肺间质纤维化的机制,填补了糖生物学在肺间质纤维化领域的空白,为临床研发新药提供了全新思路。第一部分甘草酸减轻BLM致肺间质纤维化目的:探讨甘草酸对BLM致大鼠肺间质纤维化的病理影响,明确甘草酸对肺间质纤维化有无干预作用。方法:利用BLM致大鼠肺间质纤维化模型,成功获得肺脏大体标本,使用不同剂量甘草酸观察其对肺脏病理的影响及明确对肺间质纤维化是否有治疗作用。首先大体观察甘草酸湿/干重比的影响;使用HE、Masson染色方法观测甘草酸对肺脏病理的影响;利用免疫组化及免疫印迹技术检测胶原蛋白Ⅰ、Ⅲ的变化;其次利用ELISA法检测大鼠的肺组织中Hyp含量,明确甘草酸对肺间质纤维化是否有干预作用。结果:与正常组相比,BLM诱导的大鼠肺间质纤维化中肺湿/干重比明显升高;HE、Masson染色显示肺脏病理明显加重;免疫组化及免疫印迹显示胶原蛋白Ⅰ、Ⅲ表达明显增加;ELISA检测Hyp的含量明显升高。经甘草酸治疗后,肺湿/干重比明显降低;,肺脏病理明显减轻,胶原蛋白Ⅰ、Ⅲ及Hyp表达较纤维化组明显降低,并随甘草酸剂量的升高而明显减轻,其中高剂量甘草酸具有显著的抑制作用。结论:在BLM致大鼠肺间质纤维化的模型中,甘草酸对抑制及保护肺间质纤维化起到了一定作用,并呈剂量依赖性。第二部分甘草酸对EMT的影响目的:阐述甘草酸对肺上皮细胞转分化的影响,明确甘草酸阻抑EMT。方法:在用博来霉素制备的大鼠肺间质纤维化模型中,使用特有标记物E-cadherin定位肺泡上皮,观察不同剂量甘草酸在肺泡上皮细胞转分化成肌成纤维细胞中的影响,明确甘草酸阻抑EMT进而减轻肺间质纤维化。免疫荧光共染方法检测不同剂量甘草酸对各组肺组织中E-cadherin、α-SMA的表达变化,免疫印迹方法对各组肺标本中上述蛋白采用定量分析,明确不同剂量甘草酸对上皮细胞转分化现象的影响;免疫荧光检测各组大鼠肺组织中p-Smad2/3、β-catenin的表达变化,利用免疫印迹方法对上述蛋白进行定量分析,明确不同剂量甘草酸对肺脏纤维化关键蛋白的影响。结果:免疫荧光及免疫印迹结果显示正常肺组织E-cadherin中等量表达,α-SMA低量表达。与正常组相比较,BLM致大鼠肺间质纤维化模型中α-SMA表达显著升高,E-cadherin表达显著下降;经甘草酸治疗后,免疫荧光及免疫印迹显示上述两种蛋白呈逆向变化,对EMT现象有明显抑制作用。免疫荧光及免疫印迹结果显示正常肺组织中p-Smad2/3、β-catenin低量表达。与正常组相比,BLM诱导大鼠肺间质纤维化模型中p-Smad2/3、β-catenin蛋白的表达显著升高;经甘草酸治疗后,免疫荧光及免疫印迹显示甘草酸明显抑制上述两种蛋白的表达。上述指标均显示与甘草酸的剂量相关。结论:在BLM致大鼠肺间质纤维化中,EMT通路关键蛋白起到重要作用,甘草酸能够干预EMT及上述蛋白的活化。第三部分甘草酸通过CF修饰调控EMT的机制目的:探讨核心岩藻糖基化修饰对EMT关键信号通路的影响,深入解析甘草酸调控FUT8阻抑EMT。方法:利用BLM致大鼠肺间质纤维化模型,观察核心岩藻糖基化修饰水平变化及核心岩藻糖基化修饰对TGF-β/Smad2/3、Wnt/β-catenin信号通路的影响,深入解析甘草酸对核心岩藻糖基化修饰的水平变化。首先免疫荧光双染法观察大鼠肺脏中CF表达及甘草酸对大鼠肺脏CF修饰水平的变化;其次应用免疫荧光、免疫印迹检测TGF-βR、WNT核心岩藻糖基化水平及甘草酸对上述受体蛋白及CF修饰水平变化。结果:正常肺脏组织中存在中等量的CF修饰水平。与正常组比较,BLM致大鼠肺间质纤维化中,CF修饰水平明显增加;大鼠肺脏中TGF-βR、WNT及其核心岩藻糖基化水平显著升高(p0.001);甘草酸能明显抑制BLM诱导纤维化大鼠肺脏中TGF-βR、WNT受体蛋白的核心岩藻糖基化修饰水平(p0.01),但是并未影响受体本身的表达。结论:在BLM致大鼠肺间质纤维化模型中,关键受体蛋白TGF-βR、WNT翻译后CF修饰在EMT中起到关键作用,甘草酸调控TGF-βR、WNT核心岩藻糖基化修饰后能够干预EMT,进而减轻肺间质纤维化。
[Abstract]:Pulmonary interstitial fibrosis (Pulmonary fibrosis) is a chronic disease. The course of the disease is gradually developing and irreversible. It is the end stage of interstitial lung disease. It is estimated that the average life period is short and less than 5 years. The patients with pulmonary fibrosis are difficult to breathe, and the life is difficult to take care of themselves, and it is costly to bring to society, families, and individuals. It is a great challenge and great demand for the prevention and control of pulmonary diseases in China. Although the cause of pulmonary fibrosis is not clear, the pathological manifestations and signal pathways of pulmonary fibrosis are mainly manifested in the infiltration of inflammatory cells and myofibroblast in the interstitial lung. There has been a significant increase in the accumulation of extra extracellular matrix and the eventual pathological changes of fibrosis. There is no effective means to prevent its progress. Therefore, it is of great significance to elucidate the development of interstitial lung fibrosis, the development mechanism, and the search for effective new intervention strategies for preventing the development of the disease and reducing the mortality. We are not very clear about the pathogenesis of the disease of pulmonary fibrosis. The key pathological changes in the course of pulmonary fibrosis include myofibroblast and abnormal increase. There are many studies on the origin of myofibroblast. The source is alveolar epithelial cells, that is, EMT (epithelial to mesenchymaltransition). An important effect of the activation of alveolar epithelial cells is: transforming into myofibroblast, promoting the synthesis of extracellular matrix, activating a variety of genes involved in inherent immunity and inflammation, promoting inflammation, oxidative stress injury, and aggravating pulmonary fibrosis. It is speculated that pulmonary alveolar epithelial cell transdifferentiation (EMT) is the main source of myofibroblast in the interstitial lung. The in-depth study of the mechanism of EMT and the important molecular biological effects caused by activation is an important strategy to seek intervention targets. To sum up, pulmonary interstitial fibrosis is a refractory, complex disease. And single pathway receptor blockers (such as gefitinib) may have some effect, but it does not prevent the progress of pulmonary fibrosis. In recent years, more and more studies have been made about Chinese herbal prescription and its monomer active components in the treatment of disease. The role of the Chinese herbal medicine in the treatment of pulmonary fibrosis has also attracted more and more attention. The previous experiments confirmed that activating blood and removing stasis prescription (Huang Qi, chuanxiong, Salvia miltiorrhiza, angelica, Rhodiola, Fritillaria thunbergii, licorice) has a therapeutic effect on pulmonary fibrosis. Glycyrrhizic acid (GA) is the most important active ingredient in Glycyrrhiza. A large number of studies have shown that glycyrrhizic acid has a significant therapeutic effect on visceral fibrosis (such as liver). It is not clear whether oxalic acid has an improved effect on pulmonary fibrosis. Therefore, we propose the following scientific questions: multiple inflammation, excessive activation of multiple signal pathways is an important basis for pulmonary fibrosis, and EMT is an important way of inducing interstitial fibrosis. What regulates the activity of these inflammatory factors and signaling pathways? Can oxalic acid find common targets to control their activity? In recent years, the rapid development of glycoproteins provides us with new research directions for solving the above problems. Core fucosylation (CF) is an important protein sugar modification, which is specific to alpha -1,6 fucose transferase (Fucosyltransferase8, FUT8). We catalyze the transfer of fucose and connect it to the core sugar chain of the specific amino acid site of the target protein to complete the folding and space conformation of the target protein. Our research group studies show that the key proteins in the visceral fibrosis are glycoproteins. Therefore, we think whether glycyrrhizic acid can regulate these inflammatory factors and signal through CF modification. To solve these scientific problems, the experiment is to establish a rat model of pulmonary fibrosis induced by Bleomycin (BLM) to determine whether glycyrrhizic acid has an effect on pulmonary fibrosis, and to observe the effect of glycyrrhizic acid on EMT in rat pulmonary fibrosis model, and on this basis, the effect of glycyrrhizic acid on CF is observed. It is the first time that glycyrrhizic acid can reduce BLM induced pulmonary fibrosis and inhibit EMT. Secondly, the mechanism of glycyrrhizin in the treatment of interstitial fibrosis of the lung is explained from a new angle of sugar biology, which fills the gap in the field of pulmonary fibrosis in sugar biology and develops new drugs for the clinical development of EMT. In the first part, glycyrrhizic acid alleviates BLM induced pulmonary fibrosis: the pathological effects of glycyrrhizic acid on pulmonary fibrosis in rats induced by BLM and the effect of glycyrrhizic acid on interstitial fibrosis of the lungs. Methods: the lung interstitial fibrosis model of rats was made by BLM, and the gross specimens of lung were successfully obtained and the different doses were used. The effects of glycyrrhizic acid on lung pathology and the treatment of pulmonary fibrosis were observed. First, the effects of glycyrrhizin wet / dry weight ratio were observed. The effects of glycyrrhizic acid on lung pathology were observed by HE and Masson staining, and the changes of collagen I and III were detected by immunohistochemistry and immunoblotting techniques, and then EL was used. The ISA assay was used to detect the Hyp content in the lung tissue of rats and the effect of glycyrrhizin on pulmonary fibrosis. Results: compared with the normal group, the lung wet / dry weight ratio in pulmonary fibrosis induced by BLM was significantly higher than that in the normal group; HE, Masson staining showed that the lung pathology was obviously aggravated; immunohistochemistry and immunoblotting showed the expression of collagen I and III The content of Hyp was significantly increased by ELISA. After glycyrrhizic acid treatment, the lung wet / dry weight ratio decreased obviously, the lung pathology obviously decreased, the expression of collagen I, III and Hyp decreased significantly compared with the fibrosis group, and obviously decreased with the increase of glycyrrhizic acid, and the high dose glycyrrhizic acid had significant inhibitory effect. Conclusion: in BLM In the model of pulmonary fibrosis in rats, glycyrrhizic acid plays a role in inhibiting and protecting pulmonary fibrosis, and is dose-dependent. Second the effect of glycyrrhizic acid on EMT: the effect of glycyrrhizic acid on the transdifferentiation of lung epithelial cells, and the method of glycyrrhizin inhibition EMT.: the pulmonary interstitial fiber in rats prepared with bleomycin The effects of different doses of glycyrrhizic acid on the transformation of the alveolar epithelial cells into myofibroblasts were observed in the alveolar epithelium using a specific marker E-cadherin, and the inhibition of glycyrrhizic acid to inhibit EMT and then reduce pulmonary fibrosis. The immunofluorescence CO staining method was used to detect E-cadherin, alpha -S in different doses of glycyrrhizic acid in each group of lung tissues. The expression of MA and Western blot were quantified to determine the effects of glycyrrhizin on the transdifferentiation of epithelial cells, and the changes in the expression of p-Smad2/3 and beta -catenin in lung tissues of each group were detected by immunofluorescence, and the above protein was quantified by immunoblotting. The effect of different doses of glycyrrhizin on the key protein of lung fibrosis. Results: the results of immunofluorescence and immunoblotting showed that the expression of E-cadherin in normal lung tissue was equal and the expression of alpha -SMA was low. Compared with the normal group, the expression of alpha -SMA in the pulmonary interstitial fibrosis model of BLM rats increased significantly, the expression of E-cadherin decreased significantly, and the treatment was treated with glycyrrhizic acid. After the treatment, immunofluorescence and immunoblotting showed that the above two proteins were reversed and had obvious inhibitory effect on the EMT phenomenon. The results of immunofluorescence and immunoblotting showed the low expression of p-Smad2/3 and beta -catenin in normal lung tissue. Compared with the normal group, BLM induced the expression of p-Smad2/3 and beta -catenin protein in the pulmonary interstitial fibrosis model of rats. After the treatment of glycyrrhizic acid, immunofluorescence and immunoblotting showed that glycyrrhizic acid obviously inhibited the expression of the above two proteins. All of these indexes were related to the dose of glycyrrhizin. Conclusion: the key protein of EMT pathway plays an important role in BLM induced pulmonary interstitial fibrosis in rats, and glycyrrhizic acid can interfere with the activation of EMT and the above proteins. The three part of glycyrrhizic acid modified the mechanism of regulating EMT through CF modification: To explore the effect of core fucoidylation on the key signaling pathway of EMT, and to deeply analyze the FUT8 inhibition of EMT. by glycyrrhizin: using BLM to induce rat pulmonary fibrosis model, and observe the changes in the level of the core fucose trimming and the core fucose modification of the core fucose to TGF- beta /Smad2/3, the effect of Wnt/ beta -catenin signaling pathway, the level of glycyrrhizinate modification was deeply analyzed. First, the expression of CF in the lungs of rats was observed by immunofluorescence and the changes of glycyrrhizic acid on the level of CF modification in the lungs of rats. Secondly, immunofluorescence and immunoblotting were used to detect TGF- beta R and WNT core fucose base water. The changes in the levels of the receptor protein and CF modification were observed. Results: the level of CF modification in normal lung tissues was equal. Compared with the normal group, the level of CF modification in BLM induced pulmonary fibrosis in rats was significantly increased; the TGF- beta R, WNT and its core fucose levels in the lungs of rats increased significantly (p0.001), and glycyrrhizic acid could be obvious. Inhibition of BLM induced TGF- beta R, WNT receptor protein core fucose modification level (P0.01), but did not affect the expression of the receptor itself. Conclusion: the key receptor protein TGF- beta R in rat pulmonary fibrosis model induced by BLM and the key role of CF modification after WNT translation, glycyrrhizic acid regulates TGF- beta. Heart fucose modification can interfere with EMT, thereby reducing pulmonary interstitial fibrosis.

【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R563

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