CHOP蛋白、氧化壓力及發炎反應在腎臟缺血再灌流引起細胞凋亡上之角色探討

Roles of CCAAT/enhancer-binding Protein Homologous Protein (CHOP), Oxidative Stress and Inflammation in Renal Ischemia/Reperfusion-induced Cell Apoptosis

陳柏霖(Bo-Lin Chen)

指導教授 ： 劉興華

國立臺灣大學/醫學院/毒理學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.11234

摘要

腎臟缺血再灌流為最主要造成急性腎衰竭的原因之一，然而截至目前為止對其引起的損傷並無良好的治療方式且對其之機制仍未完全明瞭。目前已知氧化壓力為其重要的損傷機制之一，且最近的研究也發現腎臟缺血再灌流所引起之氧化壓力可以進一步引起內質網壓力最終造成腎臟的損傷及功能的喪失。CCAAT/enhancer結合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein, CHOP)是一個重要的內質網壓力蛋白，其為一轉錄因子負責引發內質網壓力所造成之細胞凋亡。目前研究認為內質網壓力在腎臟缺血再灌流所引起之損傷中至為重要，但對於CHOP在其中所扮演的角色仍然不甚清楚，因此本研究希望同時利用動物及細胞模式來研究CHOP在腎臟缺血再灌流中所扮演的角色，並測試抗氧化物質槲皮素是否可以調控缺血再灌流所引起的CHOP表現及其他可能的保護機制。結果顯示，在處理缺血在灌流後，CHOP基因剔除小鼠之肌酐酸及尿素氮數值相較於野生基因型小鼠來的低，代表CHOP基因剔除小鼠其腎功能損傷較少。病理組織染色也觀察到類似的情形，CHOP基因剔除小鼠在腎臟缺血再灌流後其組織受損及免疫細胞浸潤的情況較輕微。進一步發現CHOP基因缺失可減低其缺血再灌流後的caspase-3, caspase-8的表現，也可減低脂質過氧化程度並增加內生性抗氧化物質的活性，顯示CHOP基因缺失的確可減低腎臟缺血再灌流所造成的氧化壓力。在缺血再灌流之細胞模式中，以微RNA抑制CHOP表現後可發現過氧化氫產生相較於對照組有顯著的下降，同時細胞凋亡及發炎反應分子表現也減少許多。內生性抗氧化物在CHOP抑制的情況下則有更強的活性。利用過氧化氫及內生性氧化壓力產生物menadione也可發現CHOP抑制減低了氧化壓力引起之細胞凋亡及發炎反應分子表現。抗氧化劑槲皮素過去已知可保護缺血再灌流造成的傷害。本研究發現，抑制槲皮素所誘發的細胞自噬反應會降低槲皮素的保護作用，由此可知除了抗氧化能力之外，槲皮素也能透過誘發細胞自噬作用減低缺血再灌流所造成的細胞傷害。近一步利用動物模式來觀察可以發現，槲皮素有效的降低腎臟缺血再灌流所造成的腎臟功能損失及組織的傷害。利用西方點墨法發現，槲皮素有效的降低了腎臟缺血再灌流所引起的CHOP表現。另外槲皮素也增加了adenosine monophosphate-activated protein kinase (AMPK)的磷酸化及誘發了細胞自噬作用，同時也降低mammalian target of rapamycin的磷酸化。進一步以螢光免疫染色發現，槲皮素的確增加細胞自噬小體的形成。由結果可知，在腎臟缺血再灌流的情況下，槲皮素的確顯著的加強了AMPK-自噬作用這條訊息傳遞路徑。總結來說，我們發現CHOP的表現不僅僅造成了細胞凋亡的產生，同時也會增加氧化壓力及發炎反應，抗氧化劑槲皮素有效的降低了缺血再灌流所造成的CHOP表現及活化了AMPK-自噬作用的路徑。在未來，調控內質網壓力的藥物及抗氧化劑槲皮素也許可成為治療腎臟缺血再灌流的可能策略。

關鍵字

缺血再灌流 ； 內質網壓力 ； CHOP蛋白 ； 細胞凋亡 ； 槲皮素 ； 發炎反應

並列摘要

Renal ischemia-reperfusion (I/R) is a major cause of acute renal failure. Generation of reactive oxygen species was one of the most important mechanisms in renal I/R injury. Recent studies showed that ROS could induced renal cell death through ER stress activation. CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is involved in the ER stress signaling pathways. CHOP is a transcription factor and a major mediator of ER stress-induced apoptosis. However, the role of CHOP in renal I/R injury is still undefined. Here, we investigated whether CHOP could regulate I/R-induced renal injury using CHOP-knockout mice and cultured renal tubular cells as models. In CHOP-knockout mice, loss of renal function induced by I/R was prevented. Renal proximal tubule damage was induced by I/R in wild-type mice; however, the degree of alteration was significantly less in CHOP-knockout mice. CHOP deficiency also decreased the I/R-induced activation of caspase-3 and -8, apoptosis, and lipid peroxidation, whereas the activity of endogenous antioxidants increased. In an in vitro I/R model, small interfering RNA targeting CHOP significantly reversed increases in H2O2 formation, inflammatory signals, and apoptotic signals, while enhancing the activity of endogenous antioxidants in renal tubular cells. To the best of our knowledge, this is the first study which demonstrates that CHOP deficiency attenuates oxidative stress and I/R-induced acute renal injury both in vitro and in vivo. Antioxidants such as quercetin are considered to be a potential therapeutic strategy for I/R in recent years. Quercetin could protect renal cell against injury induced by in vitro I/R model through adenosine monophosphate-activated protein kinase (AMPK)-autophagy pathway. In this studies, we found that quercetin attenuated I/R-induced renal damage in mice. Furthermore, quercetin treatment increased the phosphorylation of AMPK and the cleavage of microtubule-associated protein light chain 3 (LC3). We also found that quercetin could enhance the autophagasome formation by LC3 Immunofluorescence staining. Those results suggest AMPK-autophagy pathway may participated in the protective effects of quercetin in renal I/R. Taken together, our study indicate CHOP regulates not only apoptosis-related signaling but also ROS formation and inflammation in renal tubular cells during I/R. Antioxidant quercetin treatment could inhibit ER stress related signal expression and activate AMPK-autophagy pathway. In future, ER stress modulating compounds and quercetin may be potential therapeutic strategies for renal I/R.

並列關鍵字

ischemia/reperfusion ； ER stress ； CHOP ； quercetin ； AMPK ； autophagy

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