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標題: | 細胞自噬現象與內質網壓力對腎臟傷害之探討:以馬兜鈴酸與4-苯基丁酸為例 Detection of Autophagy and Endoplasmic Reticulum Stress in Renal injury:Analyze with Aristolochic Acid and 4-phenylbutyrate |
作者: | Ching-Chin Yang 楊靜欽 |
指導教授: | 劉興華 |
關鍵字: | 馬兜鈴酸,細胞自噬現象,中草藥腎病變,自噬小體,3-甲基腺嘌呤,腎小管間質纖維化,內質網壓力,4-苯基丁酸,單側輸尿管阻塞,轉化生長因子, Aristolochic Acid,autophagy,Chinese Herb Nephropathy,autophagosome,3-Methyladenine,Renal tubulointerstitial fibrosis,Endplasmic Reticulum Stress,4-phenylbutyrate,unilateral ureteral obstruction,transforming growth factor-β, |
出版年 : | 2014 |
學位: | 博士 |
摘要: | 第一部份 馬兜鈴酸誘發細胞自噬現象促進腎臟細胞凋亡
細胞自噬(autophagy),為自我吞噬 (self-eating)的過程,屬於真核細胞中第二型的計畫性細胞死亡(Type II programmed cell death,簡稱PCD);細胞自噬的產生乃為了分解受損的胞器,或是細胞質中未正確折疊的蛋白(unfolded protein)。細胞自噬有一特定家族基因與蛋白稱作自噬相關基因(autophagy-related gene;Atg),這些自噬相關蛋白與自噬體(autophagesomes)發生、形成及成熟過程相關,自噬體成熟後再與溶酶體(lysosomes)融合並分解包圍在其中的的物質。自噬相關基因首先於酵母菌中被發現,但現今發現其同源基因,在哺乳動物及其他動物體內的細胞自噬現象中也扮演重要角色。在哺乳動物體內的Beclin-1為酵母菌內Atg6的同源基因, 促成小泡結核(vesicle nucleation)與早期自噬體的成形。哺乳動物體內的LC3為Atg8的同源,會被Atg4蛋白進行切割形成LC3-II並結合膜上的磷脂醯乙醇胺( phosphatidyl ethanolamine),並完成自噬小體的延伸及擴張。傳統而言,細胞自噬被認為是細胞因應養分耗盡或飢餓狀態的反應模式,細胞將會消化部分細胞質蛋白轉化成養分以利細胞存活。然而,近期的研究指出細胞自噬作用可能是細胞面對壓力的常態反應。依照實驗型態的設計,細胞自噬作用可能直接導致細胞死亡或是扮演細胞保護的角色。儘管近來對細胞自噬相關的實驗進展迅速,但對於腎臟細胞、組織或系統方面的所知仍極為有限。中草藥導致腎毒性在全球的發生率已屢見不鮮,不慎的誤用中草藥包括馬兜鈴酸的使用,乃是造成中草藥腎臟病變的主因之一。馬兜鈴酸腎病變的主要病徵包含大規模腎小管萎縮,發炎細胞的浸潤及腎間質纖維化,但腎小球相較之下較不受傷害。經由缺氧、壞死或是腎臟毒物造成的腎臟傷害,像是馬兜鈴酸所導致的腎病變是典型的腎臟傷害模式,會造成細胞死亡、組織傷害、以及腎臟功能缺失或是腎臟衰竭。細胞自噬是否會在馬兜鈴酸腎病變下產生?在此病理狀態下細胞自噬是否對腎臟細胞損傷及凋亡或是保護細胞存活有所貢獻?本實驗乃利用馬兜鈴酸腎病變模式來探討並解釋上述相關問題,在此我們假設馬兜鈴酸的攝取將會導致腎臟自噬小泡與自噬小體的形成。在近曲小管細胞培養模式下,馬兜鈴酸伴隨著細胞凋亡,在劑量效應與時間效應下造成自噬作用的形成。馬兜鈴酸的攝取會顯著的造成細胞自噬作用的產生。我們的初步發現分別前處理自噬作用抑制劑3-MA(3-methyladenine)與Atg5-shRNA,馬兜鈴酸處理下的腎小管細胞產生自噬作用的現象減少,更可抑制部分經由馬兜鈴酸造成的細胞凋亡。馬兜鈴酸腎毒性會導致自噬作用的產生,並在細胞存活或致死機制上扮演重要的角色。 第二部份 化學伴護蛋白-4-苯基丁酸減輕內質網壓力造成之腎臟纖維化 腎小管間質纖維化是腎臟終末期腎臟疾病的常見和後期的病理變化。內質網壓力(endoplasmic reticulum stress,簡稱ER stress)為刺激腎臟纖維化發展的病理生理機制。根據本實驗是過去研究,我們發現化學分子伴侶4-苯基丁酸對腎臟纖維化的生體和細胞模式下的保護效果。在大鼠單側輸尿管阻塞(unilateral ureteral obstruction,簡稱 UUO )模型, 4-苯基丁酸可模仿內生性內質網伴侶蛋白以避免無法抵抗的內質網壓力傷害。 4-苯基丁酸顯著減弱單側輸尿管阻塞模式下所增加的葡萄糖調節蛋白(Glucose Regulated Protein,簡稱GRP78) , C/EBP-homologous protein (簡稱CHOP) , activating transcription factor (簡稱ATF4)蛋白表現和c-Jun N-terminal kinases (簡稱JNK)的蛋白磷酸化,並可回復因單側輸尿管阻塞而減少表現的拼接型X-box binding protein 1 (簡稱spliced XBP1)。 4-苯基丁酸也可緩解單側輸尿管阻塞大鼠腎臟組織中α-平滑肌肌動蛋白(α-smooth muscle actin)和結締組織生長因子(Connected Tissue Growth Factor,簡稱CTGF)蛋白表現的增加。 此外,單側輸尿管阻塞造成的腎小管間質損傷,纖維化和細胞凋亡也可通過給予4-苯基丁酸而得到緩解。另一方面,因轉化生長因子(Transforming Growth Factor-β,簡稱TGF-β)刺激而顯著增加正常大鼠腎臟上皮細胞(Normal Rat kidney-52E,簡稱NRK-52E)的內質網壓力相關分子蛋白、促纖維化因子蛋白以及細胞凋亡相關標記蛋白表現,也會受到4-苯基丁酸的抑制。 4-苯基丁酸也能緩減正常大鼠腎臟上皮細胞因轉化生長因子-β刺激而增加的結締組織生長因子活性螢光表現量及其信息RNA的表達。綜合來看,4-苯基丁酸可模擬內質網伴護蛋白來改善過度的內質網壓力和防止腎小管細胞凋亡與腎臟纖維化。 Part 1:Autophagy induction promotes aristolochic acid-I-induced renal injury in vivo and in vitro. Autophagy as cell self-eating process , which belongs to eukaryotic cells type II programmed cell death (PCD); autophagy has been organized to produce the decomposition damaged organelles , or misfolded proteins in cytoplasm. Specific autophagy gene and protein family known as autophagy-related genes (Atg) regulate the phenomenon of autophagosome occurance, formation and maturation. Matured autophagosome then fuse with lysosome to form autolysome and lysis inner materials. Autophagy-related genes were first discovered in yeast , and the homologous genes in mammals and other animals were also found plays an important role in autophagy regulation. Beclin-1 gene in mammals is homologous to Atg6 in the yeast , contributed to vesicle nucleation and forming of the preautophagic structure phagophore. Mammal gene LC3 is homologous to Atg8, will be cut by Atg4 protein to form LC3-II and binding to the membrane of phosphatidyl ethanolamine to complete the extending and expansion of autophagic vesicles. Traditionally, autophagy is considered to be a cell response while cell undergo nutrient depletion or starvation model, part of misfolded proteins and broken organelles would be digested to generate nutrients to facilitate cell survival. However, recent studies indicate that autophagy may directly resulted to cell death or played a cyto protective role. Despite of rapid progress in recent experiments on autophagy -related study, there were still extremely limited knowledge about autophagy for kidney cells, tissues or systems. Chinese herb products induce renal toxicity occurred in the global more and more frequently, inadvertent misuse of Chinese herbs including aristolochic acid was one of the main causes of kidney disease. The main symptoms of aristolochic acid nephropathy contains massive tubular atrophy, inflammatory cell infiltration and renal interstitial fibrosis, but in contrast less susceptible in glomerular damage. Kidney poison damage induced kidney hypoxia, renal failure or cell death, was mimic to the typical renal injury model that induced by aristolochic acid nephropathy, would lead to cell death , tissue damage, and loss of kidney function or renal failure. Whether autophagy occurred in aristolochic acid nephropathy? Whether autophagy contribute to renal cell damage apoptosis or cytoprotective to cell survival in this pathological state? In this experiment we use aristolochic acid nephropathy model to explore and explain these issues ,and assume that aristolochic acid will result in the formation of autophagy phenomenon and autophagosome in kidney. In tubular cell culture model, aristolochic acid induced the formation of autophagy along with apoptosis, in the dose-depend response and time effects. Intake of aristolochic acid would significant produce autophagy . Our preliminary findings identified that alternatively pre- treated with autophagy inhibitor 3-MA (3-methyladenine) and Atg5-shRNA would not only reduce autophagy phenomenon but also inhibited partial apoptosis triggered by aristolochic acid. Aristolochic acid renal toxicity could cause autophagy generation, and the phenomenon of autophagy would play important role in the mechanism of cell survival or apoptosis. Part 2:Chemical chaperon 4-phenylbutyrate protects against the endoplasmic reticulum stress-mediated renal fibrosis in vivo and in vitro. Renal tubulointerstitial fibrosis is the common and final pathologic change of kidney in end-stage renal disease. Endoplasmic reticulum (ER) stress contributes to the pathophysiological mechanisms during the development of renal fibrosis. Here, we investigated the protective effect of chemical chaperon 4-phenylbutyrate (4-PBA) on renal fibrosis in vivo and in vitro. In a rat unilateral ureteral obstruction (UUO) model, 4-PBA mimicked endogenous ER chaperon in the kidneys. 4-PBA significantly attenuated UUO-increased renal GRP78, CHOP, ATF4, and phosphorylated JNK protein expressions and UUO-decreased renal splicing XBP1 protein expression. 4-PBA also attenuated the increases of α-smooth muscle actin and connective tissue growth factor (CTGF) protein expressions in the kidneys of UUO rats. The tubulointerstitial injury, fibrosis, and apoptosis in the kidneys of UUO rats could also be attenuated by the administration of 4-PBA. Moreover, transforming growth factor (TGF)-β markedly increased the protein expressions of ER stress-related molecules, profibrotic factors, and apoptosis-related markers in the normal rat kidney epithelial cells (NRK-52E), which could be significantly attenuated by 4-PBA treatment. 4-PBA treatment also attenuated TGF-β-increased CTGF-luciferase activity and CTGF mRNA expression in NRK-52E cells. Taken together, 4-PBA acts as an ER chaperone to ameliorate ER stress and protect against renal tubular cell apoptosis and renal fibrosis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58598 |
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