α-次亞麻油酸抑制硫酸吲哚誘導SV40 MES 13小鼠腎絲球間質細胞上皮間質轉化相關蛋白表現之研究

α-Linolenic acid inhibits indoxyl sulfate-induced epithelial-mesenchymal-transition-related protein expression in SV40 MES 13 mouse glomerular mesangial cells

沈佩盈(Pei-Ying Shen)

指導教授 ： 李健群

中山醫學大學/健康管理學院/營養學系/碩士(2019年)

https://doi.org/10.6834/CSMU201900229

摘要

美國國家腎臟基金會(The National Kidney Foundation, NKF)依腎功能衰退程度所提出的腎臟疾病分期標準，慢性腎臟病可依腎絲球過濾率的下降區分成一至五期。隨著病程發展尿毒素逐漸無法由腎臟排出體外，最終發展成末期腎病。硫酸吲哚(indoxyl sulfate, IS)為主要尿毒素成分，是加速慢性腎臟病惡化危險因子之一，已有研究證實IS大量堆積於腎臟時會促使腎小管細胞上皮間質轉化(epithelial-to-mesenchymal transition, EMT)，進而導致腎功能衰退。本研究以SV40 MES13小鼠腎絲球間質細胞，探討次亞麻油酸(α-Linolenic acid, ALA)調控IS所誘導EMT相關蛋白表現及相關調控機制。實驗結果顯示給予0.1 mM IS可顯著增加fibronectin、N-cadherin、α-smooth muscle actin (α-SMA)蛋白質及Twist1、Snail2等促EMT之轉錄因子表現。另外研究結果顯示，以0.1 mM IS處理細胞3小時可促進Snail2轉位進入細胞核內，而IS處理18小時後促使缺氧誘導因子-1α (hypoxia-inducible factor-1α, HIF-1α)於核內累積。預處理100 μM ALA 4小時，再給予0.1 mM IS 24小時，可顯著減少IS所誘發EMT相關蛋白質表現，並顯著減少核內HIF-1α及snail2蛋白累積。藉由HIF-1α siRNA將HIF-1α knockdown後可顯著抑制IS所誘發fibronectin、α-SMA、Twist1及Snail2表現。綜合上述實驗結果，ALA可透過減少IS所誘發核內HIF-1α轉錄因子累積，抑制EMT相關蛋白表現。另外，ALA抑制IS誘發SV40 MES13小鼠腎絲球間質細胞EMT可能也與Snail2核內累積減少有關。

關鍵字

硫酸吲哚次亞麻油酸 ； 上皮間質轉化 ； HIF-1α ； SV40 MES13細胞

並列摘要

The National Kidney Foundation defines Chronic kidney disease (CKD) is a condition characterized by a gradual loss of kidney function over time. CKD is classified into stage 1 to 5. Each stage of chronic kidney disease is related to the estimated glomerular filtration rate (eGFR) and kidney damage. On the progression of CKD, uremic toxics are difficult to excrete by the kidney, finally leading to end-stage renal disease. Indoxyl sulfate (IS), a uremic toxin, is one of the risk factors to accelerate CKD progression. The epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell-cell junction, and gain migratory and invasive properties to become mesenchymal cells. A previous study showed that IS promotes EMT in human proximal tubular cells, resulting in renal failure. The aim of this study is to investigate the effect of α-Linolenic acid (ALA) on IS-induced EMT-related proteins and underlying mechanism in SV40 MES13 mouse glomerular mesangial cells. Treatment with 0.1 mM IS significantly increased fibronectin, N-cadherin, α-smooth muscle actin (α-SMA) protein expression, and the level of EMT-related transcription factors, such as Twist1 and Snail2 were increased as well. Treatment with 0.1 mM IS for 3 hours promotes the accumulation of Snail2 in nucleus, and treatment with 0.1 mM IS for 18 hours transported HIF-1α (hypoxia-inducible factor-1α) into nucleus. Furthermore, pretreatment with 100 μM ALA for 4 hours significantly decreased IS-induced EMT-related protein levels, and the nuclear accumulation of HIF-1α and Snail2. Knockdown of HIF-1α by HIF-1α siRNA decreased IS-induced fibronectin, α-SMA, Twist1 and Snail2 protein levels. In conclusion, ALA decreased IS-induced nuclear accumulation of HIF-1α and inhibited EMT-related proteins expression. In addition, ALA inhibited IS-induced EMT-related proteins may connect with decreasing Snail2 translocated into nucleus.

並列關鍵字

indoxyl sulfate ； α-linolenic acid ； epithelial-to-mesenchymal transition ； HIF-1α ； SV40 MES13 cell

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