GSK3β促肾小管间质纤维化的分子机制及抗肾纤维化措施探讨

发布时间：2018-04-30 22:01

本文选题：糖原合成酶激酶3β(GSK3β) + 法尼醇X受体(FXR)　；参考：《第三军医大学》2015年博士论文

【摘要】：肾脏纤维化是指在复杂的多种致病因子,包括氧化应激、药物、炎症、损伤等的作用下,肾脏间质细胞增多,基质蛋白合成增加,细胞外基质(extracellular matrix,ECM)大量堆积,由此导致的肾小球硬化和小管间质的纤维化,以及肾脏小血管的透明变性和硬化,最终导致肾实质梗阻和肾功能衰竭。肾脏纤维化不仅是绝大多数慢性肾脏疾病进展至终末期肾衰竭的最终共同通路,其发生及进展,尤其是肾小管间质纤维化程度,也是许多肾脏疾病进展和判断肾功能及预后的主要标志。肾脏纤维化可以在多种肾脏损伤因素如高血压、炎症、高糖高脂、药物损害后发生,但是研究发现,即使有效控制住这些原发病因,包括效控制血压、血糖、去除致损药物等,肾脏纤维化的进程有时仍难以遏止,因此,解析纤维化发生、发展的分子机制、寻找直接抑制纤维化的治疗靶点成为近年来研究的重要方向。肾脏纤维化的形成和发展是复杂的动态过程,包括炎性细胞浸润、成纤维细胞活化、ECM生成和堆积、肾小管萎缩及微血管退行性变等。在这一过程中,许多分子在其中发挥重要的作用。其中TGF-β1被认为是最重要的促纤维化因子,启动了纤维化的发生,促进了纤维化的进展。此外,炎性介质通过炎症反应诱导纤维化发生;PDGF、FGF2、CTGF和血管紧张素II等多种细胞因子促使纤维蛋白原的沉积和重修饰。然而,尽管对肾纤维化形成、发展的机制已有大量的研究,但目前临床上尚未找到确切有效的治疗手段,因此,进一步解析肾脏纤维化的发生机制、寻找新的潜在治疗靶点具有重要价值。近年来有研究提示糖原合成酶激酶3β(glycogen syntheses kinase 3β,GSK3β)具有促纤维化的作用,但是其机制以及在肾脏纤维化中的作用尚不清楚,因此本研究的第一部分探讨了GSK3β在肾小管间质纤维化中的作用及其机制。GSK3β是在1980年做为糖原合成酶抑制分子被发现,随后发现其广泛参与了细胞增殖、干细胞更新、凋亡和及发育的过程,通过胰岛素、Wnt/β-catenin和Hedgehog等信号通路发挥作用,与神经系统紊乱、糖尿病和炎症等疾病和病理过程有着密切关系。在肾脏疾病,有报道发现GSK3β参与了肾细胞凋亡和炎症相关通路。本论文第一部分研究发现:1、GSK3β在纤维化组织高表达;2、在体外肾小管上皮细胞纤维化模型中,以特异抑制剂抑制GSK3β活性可以抑制纤维化蛋白的表达;而过表达GSK3β可加重肾小管上皮细胞纤维化,由此证明GSK3β是促肾小管上皮细胞纤维化的重要因素。3、进一步的机制探讨发现,GSK3β促进了TGFβ1诱导的Smad3磷酸化,进而促进Smad3的入核;抑制GSK3β则可下调Smad3的活性。GSK3β的这种调节作用未见于包括Smad2在内的其它Smad分子。这一结果解析了GSK3β通过TGFβ1/Smad3发挥促肾纤维化(profibrotic)的机制和GSK3β与TGFβ1-Smad3信号通路的cross-talk,为防治肾脏小管间质纤维化新靶点的筛选提供新的依据。同时,我们还发现一个新的Smad3被GSK3β磷酸化位点。在第一部分新机制研究的基础上,本论文第二部分探讨了基于抑制GSK3β和Smad3的抗肾脏纤维化新策略。法尼酯X受体(farnesoid X receptor,FXR)是重要的抗炎核受体,近年来有报道其具有抗纤维化作用,但具体的分子机制尚不明了。我们证实,FXR是调节GSK3β和Smad3的重要转录因子,具有抗肾脏纤维化的作用。具体表现为:1、在肾纤维化组织中,FXR与GSK3β、Smad3的表达成负相关。2、FXR的活化可以从转录水平下调GSK3β、Smad3;荧光素酶报告基因实验发现FXR可以抑制GSK3β、Smad3启动子区活性。3、FXR激动剂可抑制小鼠肾脏纤维化模型(UUO模型)中肾脏的纤维化水平。本部分研究为FXR做为新的抗肾纤维化用药候选靶点提供了依据。在论文的第三部分,我们探讨了其它可能抑制肾纤维化的策略。目前抗肾纤维化的研究集中在以下几个方面:改善微循环;抗炎治疗;发育重构及表观遗传学重编程等。其中,表观遗传学的调控是新兴和极具发展潜力的治疗方向。表观遗传学的调控除了包括FXR在内的多种核受体调控以外,另外一方面重要的内容是对于染色体和组蛋白修饰的调控,包括乙酰化-去乙酰化、甲基化-去甲基化等。组蛋白去乙酰化酶抑制剂(histone acetyltransferases and histone deacetylase inhibitor,HDACi))是一类可以抑制组蛋白去乙酰化酶的化合物,广泛参与了炎症、增殖和分化的过程,近来有研究提示HDACi可缓解肝纤维化,但是其对于肾脏纤维化的作用及其机制尚未阐明。我们选用已被美国食品与药品管理局(FDA)批准应用于T细胞淋巴瘤治疗的药物HDACi制剂SAHA(Suberoylanilide hydroxamic acid)为研究对象,探讨其对肾纤维化的作用及其可能的机制。本部分实验证实了:1、SAHA从mRNA水平和蛋白水平抑制了肾细胞纤维化蛋白的表达,在细胞水平和动物实验均表现出抗肾脏纤维化作用。2、SAHA抑制了STAT3的磷酸化水平;以siRNA沉默STAT3及以STAT3磷酸化抑制剂处理细胞,均可以抗肾小管上皮细胞纤维化。SAHA可能是通过抑制STAT3磷酸化,发挥抗肾纤维化作用。3、SAHA抑制了STAT3上游调节分子ERK的活性,同时还可能下调TGFRI的表达,以及增强STAT3去磷酸化酶PTP7的表达。SAHA的抗纤维化作用为其成为新的抗肾脏纤维化候选药物提供了可能。综上所述,本研究针对肾脏小管间质纤维化的发生机制及治疗策略进行了研究。研究发现GSK3β通过TGFβ-Smad3通路发挥促肾脏小管间质纤维化作用,抑制GSK3β可以抗肾小管间质纤维化;核受体FXR可以从转录水平抑制GSK3β和Smad3的表达,其活化具有抗肾脏纤维化作用;HDACi制剂SAHA可以通过多种途径抑制STAT3的磷酸化水平,减轻肾纤维化。以上研究结果为理解肾脏纤维化的发生、发展提供了新的参考,同时为深入研究拮抗和治疗肾脏纤维化提供了一定的理论依据。此外,肾脏和肝、肺、小肠及皮肤的纤维化有很多相同的病理表现和进程,因此它们可能具有类似的纤维化发生机制,因此深入研究肾小管间质纤维化的发生机制以及拓展治疗和减缓肾纤维化的措施不仅对于该疾病本身有临床意义,而且具有应用到其它纤维化疾病的前景。
[Abstract]:Renal fibrosis is a complex variety of pathogenic factors, including oxidative stress, drugs, inflammation, damage and so on, the increase of renal interstitial cells, the increase of matrix protein synthesis, the accumulation of extracellular matrix (extracellular matrix, ECM), resulting in renal small ball sclerosis and tubulointerstitial fibrosis, and the transparency of the small blood vessels of the kidney. Denaturation and hardening eventually lead to renal parenchyma obstruction and renal failure. Renal fibrosis is not only the ultimate common pathway for the overwhelming majority of chronic renal diseases to end-stage renal failure, and its occurrence and progress, especially the degree of renal tubulointerstitial fibrosis, are also the main signs of the progression of renal disease and the judgment of renal function and prognosis. Renal fibrosis can occur after a variety of renal damage factors such as hypertension, inflammation, high glucose, high fat, and drug damage. However, the study found that the process of renal fibrosis is sometimes difficult to stop even if effective control of these primary causes, including effective control of blood pressure, blood sugar, and removal of drugs, therefore, the development of fibrosis is resolved. Molecular mechanisms, looking for therapeutic targets for direct inhibition of fibrosis, have become an important research direction in recent years. The formation and development of renal fibrosis are complex dynamic processes, including inflammatory cell infiltration, fibroblast activation, ECM formation and accumulation, renal tubule atrophy and microvascular degeneration. In this process, many molecules are in it TGF- beta 1 is considered to be the most important fibrotic factor that activates the development of fibrosis and promotes fibrosis. In addition, inflammatory mediators induce fibrosis through inflammatory reactions; PDGF, FGF2, CTGF, and angiotensin II and other cytokines promote fibrinogen deposition and reconditioning. Although there has been a lot of research on the pathogenesis of renal fibrosis, the exact and effective treatment methods have not been found in clinic. Therefore, it is of great value to further analyze the mechanism of renal fibrosis and to find new potential therapeutic targets. In recent years, a study of glycogen synthase kinase 3 beta (glycogen syntheses kinase) has been suggested. 3 beta, GSK3 beta, has a role in promoting fibrosis, but its mechanism and role in renal fibrosis is not clear. Therefore, the first part of this study explored the role of GSK3 beta in renal tubulointerstitial fibrosis and its mechanism,.GSK3 beta was found in 1980 as a glycogen synthase inhibitor, and then found that it was widely involved in the cells. Proliferation, stem cell renewal, apoptosis, and development processes through insulin, Wnt/ beta -catenin and Hedgehog signaling pathways are closely related to neurological disorders, diabetes and inflammation and other diseases and pathological processes. In renal diseases, it is reported that GSK3 beta is involved in renal cell apoptosis and inflammation related pathways. Some studies have found that: 1, GSK3 beta is highly expressed in fibrotic tissue; 2, in the model of renal tubular epithelial fibrosis in vitro, inhibition of GSK3 beta activity by specific inhibitors can inhibit the expression of fibrin protein, and overexpression of GSK3 beta can aggravate the fibrosis of renal tubular epithelial cells, thus proving that GSK3 beta is the fibrosis of renal tubular epithelial cells. Important factor.3, further mechanism study found that GSK3 beta promoted Smad3 phosphorylation induced by TGF beta 1 and promoted the nucleation of Smad3, and the inhibitory effect of GSK3 beta on the regulation of Smad3 active.GSK3 beta was not found in other Smad molecules including Smad2. This result shows that GSK3 beta is used to promote renal fibrosis through TGF beta. The mechanism of ofibrotic) and the cross-talk of the GSK3 beta and TGF beta 1-Smad3 signaling pathway provide a new basis for the screening of new targets for renal tubulointerstitial fibrosis. At the same time, we also found a new Smad3 by GSK3 beta phosphorylation site. On the basis of the study of the first part of the new mechanism, the second part of this paper is based on the inhibition of GSK3 beta. Smad3's new anti fibrosis strategy. The farnesoid X receptor (FXR) receptor (farnesoid X receptor, FXR) is an important anti-inflammatory nuclear receptor. In recent years it has been reported to have anti fibrosis effect, but the specific molecular mechanism is still unknown. We have confirmed that FXR is an important transcription factor for regulating GSK3 beta and Smad3, and has the effect of anti fibrosis. 1, in renal fibrosis, the expression of FXR and GSK3 beta, Smad3 expression is negatively correlated with.2. The activation of FXR can reduce GSK3 beta from the transcription level, Smad3, and the luciferase reporter gene experiment found that FXR can inhibit GSK3 beta, Smad3 promoter region activity.3, FXR agonist can inhibit the level of renal fibrosis in the mouse kidney fibrosis model. Part of the study provides a basis for FXR as a candidate target for anti renal fibrosis. In the third part of the paper, we explored other strategies that may inhibit renal fibrosis. The present study of anti renal fibrosis is focused on the following aspects: improving microcirculation; anti-inflammatory therapy; development reconfiguration and epigenetic reprogramming. The regulation of epigenetics is a new and potential therapeutic direction. The regulation of epigenetics is in addition to the regulation of various nuclear receptors, including FXR. On the other hand, the regulation of the modification of chromosomes and histone, including acetylation - deacetylation, methylation - demethylation, and so on. Histone deacetylase Inhibitor (histone acetyltransferases and histone deacetylase inhibitor, HDACi)) is a class of compounds that can inhibit histone deacetylase. It has been widely involved in the process of inflammation, proliferation and differentiation. Recent studies have suggested that HDACi can alleviate liver fibrosis, but its role in renal fibrosis and its mechanism have not been elucidated. We selected the drug HDACi agent SAHA (Suberoylanilide hydroxamic acid), which has been approved by the US Food and Drug Administration (FDA) for the treatment of T cell lymphoma (Suberoylanilide hydroxamic acid) as the research object, to explore its role in renal fibrosis and its possible mechanisms. This part of the experiment confirmed that: 1, SAHA inhibits the renal cell fiber from the level of mRNA and protein. The expression of protein, both in cell level and in animal experiments, showed anti fibrosis effect.2, and SAHA inhibited the phosphorylation level of STAT3. SiRNA silenced STAT3 and STAT3 phosphorylation inhibitor treated cells, all of which could resist renal tubular epithelial fibrotic.SAHA may be through inhibiting STAT3 phosphorylation and exerting anti renal fibrosis effect.3, SAHA inhibits the activity of ERK in the upstream regulator of STAT3, and may also reduce the expression of TGFRI, as well as the anti fibrosis effect of.SAHA on the expression of STAT3 de phosphorylase PTP7, which provides the possibility of becoming a new candidate for anti renal fibrosis. In summary, this study aims at the mechanism and treatment of renal tubulointerstitial fibrosis. The strategy has been studied. The study found that GSK3 beta plays a role in promoting renal tubulointerstitial fibrosis through the TGF beta -Smad3 pathway and inhibits GSK3 beta to resist renal tubulointerstitial fibrosis; nuclear receptor FXR can inhibit the expression of GSK3 beta and Smad3 from the transcriptional level, and its activation has the role of anti renal fibrosis; HDACi preparation SAHA can be inhibited by a variety of ways. The phosphorylation level of STAT3 reduces renal fibrosis. These results provide a new reference for understanding the occurrence and development of renal fibrosis, and provide a theoretical basis for the in-depth study of antagonism and treatment of renal fibrosis. In addition, there are many same pathological manifestations and processes in the renal and liver, lung, small intestine and skin fibrosis. Therefore, they may have a similar mechanism of fibrosis, so the in-depth study of the mechanism of renal tubulointerstitial fibrosis and the measures to expand the treatment and slow down the renal fibrosis are not only of clinical significance to the disease itself, but also in the foreground of other fibrotic diseases.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R692

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