發(fā)布時(shí)間：2018-04-30 22:01

  本文選題：糖原合成酶激酶3β(GSK3β) + 法尼醇X受體(FXR)��； 參考：《第三軍醫(yī)大學(xué)》2015年博士論文

【摘要】：腎臟纖維化是指在復(fù)雜的多種致病因子,包括氧化應(yīng)激、藥物、炎癥、損傷等的作用下,腎臟間質(zhì)細(xì)胞增多,基質(zhì)蛋白合成增加,細(xì)胞外基質(zhì)(extracellular matrix,ECM)大量堆積,由此導(dǎo)致的腎小球硬化和小管間質(zhì)的纖維化,以及腎臟小血管的透明變性和硬化,最終導(dǎo)致腎實(shí)質(zhì)梗阻和腎功能衰竭。腎臟纖維化不僅是絕大多數(shù)慢性腎臟疾病進(jìn)展至終末期腎衰竭的最終共同通路,其發(fā)生及進(jìn)展,尤其是腎小管間質(zhì)纖維化程度,也是許多腎臟疾病進(jìn)展和判斷腎功能及預(yù)后的主要標(biāo)志。腎臟纖維化可以在多種腎臟損傷因素如高血壓、炎癥、高糖高脂、藥物損害后發(fā)生,但是研究發(fā)現(xiàn),即使有效控制住這些原發(fā)病因,包括效控制血壓、血糖、去除致?lián)p藥物等,腎臟纖維化的進(jìn)程有時(shí)仍難以遏止,因此,解析纖維化發(fā)生、發(fā)展的分子機(jī)制、尋找直接抑制纖維化的治療靶點(diǎn)成為近年來(lái)研究的重要方向。腎臟纖維化的形成和發(fā)展是復(fù)雜的動(dòng)態(tài)過(guò)程,包括炎性細(xì)胞浸潤(rùn)、成纖維細(xì)胞活化、ECM生成和堆積、腎小管萎縮及微血管退行性變等。在這一過(guò)程中,許多分子在其中發(fā)揮重要的作用。其中TGF-β1被認(rèn)為是最重要的促纖維化因子,啟動(dòng)了纖維化的發(fā)生,促進(jìn)了纖維化的進(jìn)展。此外,炎性介質(zhì)通過(guò)炎癥反應(yīng)誘導(dǎo)纖維化發(fā)生;PDGF、FGF2、CTGF和血管緊張素II等多種細(xì)胞因子促使纖維蛋白原的沉積和重修飾。然而,盡管對(duì)腎纖維化形成、發(fā)展的機(jī)制已有大量的研究,但目前臨床上尚未找到確切有效的治療手段,因此,進(jìn)一步解析腎臟纖維化的發(fā)生機(jī)制、尋找新的潛在治療靶點(diǎn)具有重要價(jià)值。近年來(lái)有研究提示糖原合成酶激酶3β(glycogen syntheses kinase 3β,GSK3β)具有促纖維化的作用,但是其機(jī)制以及在腎臟纖維化中的作用尚不清楚,因此本研究的第一部分探討了GSK3β在腎小管間質(zhì)纖維化中的作用及其機(jī)制。GSK3β是在1980年做為糖原合成酶抑制分子被發(fā)現(xiàn),隨后發(fā)現(xiàn)其廣泛參與了細(xì)胞增殖、干細(xì)胞更新、凋亡和及發(fā)育的過(guò)程,通過(guò)胰島素、Wnt/β-catenin和Hedgehog等信號(hào)通路發(fā)揮作用,與神經(jīng)系統(tǒng)紊亂、糖尿病和炎癥等疾病和病理過(guò)程有著密切關(guān)系。在腎臟疾病,有報(bào)道發(fā)現(xiàn)GSK3β參與了腎細(xì)胞凋亡和炎癥相關(guān)通路。本論文第一部分研究發(fā)現(xiàn):1、GSK3β在纖維化組織高表達(dá);2、在體外腎小管上皮細(xì)胞纖維化模型中,以特異抑制劑抑制GSK3β活性可以抑制纖維化蛋白的表達(dá);而過(guò)表達(dá)GSK3β可加重腎小管上皮細(xì)胞纖維化,由此證明GSK3β是促腎小管上皮細(xì)胞纖維化的重要因素。3、進(jìn)一步的機(jī)制探討發(fā)現(xiàn),GSK3β促進(jìn)了TGFβ1誘導(dǎo)的Smad3磷酸化,進(jìn)而促進(jìn)Smad3的入核;抑制GSK3β則可下調(diào)Smad3的活性。GSK3β的這種調(diào)節(jié)作用未見(jiàn)于包括Smad2在內(nèi)的其它Smad分子。這一結(jié)果解析了GSK3β通過(guò)TGFβ1/Smad3發(fā)揮促腎纖維化(profibrotic)的機(jī)制和GSK3β與TGFβ1-Smad3信號(hào)通路的cross-talk,為防治腎臟小管間質(zhì)纖維化新靶點(diǎn)的篩選提供新的依據(jù)。同時(shí),我們還發(fā)現(xiàn)一個(gè)新的Smad3被GSK3β磷酸化位點(diǎn)。在第一部分新機(jī)制研究的基礎(chǔ)上,本論文第二部分探討了基于抑制GSK3β和Smad3的抗腎臟纖維化新策略。法尼酯X受體(farnesoid X receptor,FXR)是重要的抗炎核受體,近年來(lái)有報(bào)道其具有抗纖維化作用,但具體的分子機(jī)制尚不明了。我們證實(shí),FXR是調(diào)節(jié)GSK3β和Smad3的重要轉(zhuǎn)錄因子,具有抗腎臟纖維化的作用。具體表現(xiàn)為:1、在腎纖維化組織中,FXR與GSK3β、Smad3的表達(dá)成負(fù)相關(guān)。2、FXR的活化可以從轉(zhuǎn)錄水平下調(diào)GSK3β、Smad3;熒光素酶報(bào)告基因?qū)嶒?yàn)發(fā)現(xiàn)FXR可以抑制GSK3β、Smad3啟動(dòng)子區(qū)活性。3、FXR激動(dòng)劑可抑制小鼠腎臟纖維化模型(UUO模型)中腎臟的纖維化水平。本部分研究為FXR做為新的抗腎纖維化用藥候選靶點(diǎn)提供了依據(jù)。在論文的第三部分,我們探討了其它可能抑制腎纖維化的策略。目前抗腎纖維化的研究集中在以下幾個(gè)方面:改善微循環(huán);抗炎治療;發(fā)育重構(gòu)及表觀遺傳學(xué)重編程等。其中,表觀遺傳學(xué)的調(diào)控是新興和極具發(fā)展?jié)摿Φ闹委煼较�。表觀遺傳學(xué)的調(diào)控除了包括FXR在內(nèi)的多種核受體調(diào)控以外,另外一方面重要的內(nèi)容是對(duì)于染色體和組蛋白修飾的調(diào)控,包括乙�；�-去乙酰化、甲基化-去甲基化等。組蛋白去乙�；敢种苿�(histone acetyltransferases and histone deacetylase inhibitor,HDACi))是一類可以抑制組蛋白去乙酰化酶的化合物,廣泛參與了炎癥、增殖和分化的過(guò)程,近來(lái)有研究提示HDACi可緩解肝纖維化,但是其對(duì)于腎臟纖維化的作用及其機(jī)制尚未闡明。我們選用已被美國(guó)食品與藥品管理局(FDA)批準(zhǔn)應(yīng)用于T細(xì)胞淋巴瘤治療的藥物HDACi制劑SAHA(Suberoylanilide hydroxamic acid)為研究對(duì)象,探討其對(duì)腎纖維化的作用及其可能的機(jī)制。本部分實(shí)驗(yàn)證實(shí)了:1、SAHA從mRNA水平和蛋白水平抑制了腎細(xì)胞纖維化蛋白的表達(dá),在細(xì)胞水平和動(dòng)物實(shí)驗(yàn)均表現(xiàn)出抗腎臟纖維化作用。2、SAHA抑制了STAT3的磷酸化水平;以siRNA沉默STAT3及以STAT3磷酸化抑制劑處理細(xì)胞,均可以抗腎小管上皮細(xì)胞纖維化。SAHA可能是通過(guò)抑制STAT3磷酸化,發(fā)揮抗腎纖維化作用。3、SAHA抑制了STAT3上游調(diào)節(jié)分子ERK的活性,同時(shí)還可能下調(diào)TGFRI的表達(dá),以及增強(qiáng)STAT3去磷酸化酶PTP7的表達(dá)。SAHA的抗纖維化作用為其成為新的抗腎臟纖維化候選藥物提供了可能。綜上所述,本研究針對(duì)腎臟小管間質(zhì)纖維化的發(fā)生機(jī)制及治療策略進(jìn)行了研究。研究發(fā)現(xiàn)GSK3β通過(guò)TGFβ-Smad3通路發(fā)揮促腎臟小管間質(zhì)纖維化作用,抑制GSK3β可以抗腎小管間質(zhì)纖維化;核受體FXR可以從轉(zhuǎn)錄水平抑制GSK3β和Smad3的表達(dá),其活化具有抗腎臟纖維化作用;HDACi制劑SAHA可以通過(guò)多種途徑抑制STAT3的磷酸化水平,減輕腎纖維化。以上研究結(jié)果為理解腎臟纖維化的發(fā)生、發(fā)展提供了新的參考,同時(shí)為深入研究拮抗和治療腎臟纖維化提供了一定的理論依據(jù)。此外,腎臟和肝、肺、小腸及皮膚的纖維化有很多相同的病理表現(xiàn)和進(jìn)程,因此它們可能具有類似的纖維化發(fā)生機(jī)制,因此深入研究腎小管間質(zhì)纖維化的發(fā)生機(jī)制以及拓展治療和減緩腎纖維化的措施不僅對(duì)于該疾病本身有臨床意義,而且具有應(yīng)用到其它纖維化疾病的前景。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R692

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 熊浩君;趙凱;陳姍;何鳳田;陳丙波;;法尼酯衍生物X受體(FXR)在糖尿病腎病中的研究進(jìn)展[J];中國(guó)比較醫(yī)學(xué)雜志;2012年07期

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本文編號(hào)：1826396