探討巴拉刈造成小鼠腎膈細胞細胞自噬之訊息傳遞路徑

The Study on Paraquat-Induced Signaling Pathway of Autophagy in Mouse Mesangial Cells

陳又嘉(Yu-Chia Chen)

指導教授 ： 彭福佐

國立臺灣大學/醫學院/毒理學研究所/碩士(2015年)

https://doi.org/10.6342/NTU201600273

摘要

巴拉刈 (Paraquat,PQ) 又稱百草枯，其化學名為1,1’-dimethyl-4,4’-bipyridylium dichloride，分子式為 C12H14Cl2N2，屬於聯嘧啶類 (bypyridiniums) 的化合物，在農藥分類上被歸類為四集胺類 (quaternary ammonium) 除草劑，已經在全球超過一百二十個國家正式登記並使用，為全球使用最廣泛的除草劑之一。巴拉刈中毒大多由口服途徑引起，進入體內後，最早達到最高組織濃度的器官為腎臟，同時它也是最主要的排泄器官。在腎臟功能正常下，進入體內後24小時內巴拉刈以原型型態近於百分之百由腎臟排出。若腎功能不正常，無法及時代謝巴拉刈則會造成組織中的濃度增高，進而加劇巴拉刈對其他器官的毒性。當巴拉刈進到腎臟細胞內後，隨即進行氧化還原循環 (redox cycling)，消耗 NADPH，產生大量的超氧化物 (superoxide anion，O2•̄) 等自由基，導致腎臟的傷害。
根據2001到2002年的統計資料指出，在台灣所有農藥中毒事件中，巴拉刈中毒佔了18.9%，造成的死亡率為72.1%。過去文獻顯示，高劑量巴拉刈中毒後，會引發多重器官衰竭，其中包括急性腎損傷 (acute kidney injury, AKI)，在24至96小時內出現的蛋白尿 (proteinuria)、膿尿 (pyuria)、氮血症 (azotemia)、血尿 (hematuria) 等較輕微症狀會出現，一旦出現寡尿或無尿，則代表更嚴重的急性腎小管壞死及腎絲球腎炎的情況。研究已證實暴露巴拉刈與腎絲球腎炎 (glomerulonephritis，GN) 的發生具相關性，且在腎臟組織可觀察到腎絲球腎膈細胞 (mesangial cells，MC) 的病變。然而造成腎絲球腎膈細胞傷害之機制的探討目前仍未釐清。因而啟發本研究以細胞膜式來探討巴拉刈對腎臟腎膈細胞細胞自噬之訊息傳遞路徑。
本研究以巴拉刈處理小鼠腎膈細胞株 (mouse mesangial cells, MMCs)，探討巴拉刈誘發細胞自噬作用之訊息傳遞路徑。實驗結果得知巴拉刈造成MMCs存活率的下降，其24小時的 IC50為145μM 。使用西方墨點法分析 (western blotting)，發現 MMCs 在暴露巴拉刈3小時時 LC3-II 表現量達到最大量，此時形成細胞自噬體的相關蛋白 Beclin 1 及 Atg5-Atg12-Atg16L1複合體的蛋白質表現量相對於對照組具有顯著性的增加，而 p62 蛋白質表現量為顯著性下降。實驗藉由螢光免疫染色法，觀察到巴拉刈確實誘導 MMCs 形成細胞自噬體 (autophagosome)。進一步利用西方墨點法了解參與細胞自噬的分子訊息途徑，結果顯示 GSK-3β，ERK，AKT，mTOR 及 p70S6K 參與 ROS 介導的自噬途徑; 而 p-eIF2α 及 ERK 參與內質網壓力 (ER stress) 介導的自噬途徑的分子皆有表現量增加的趨勢。此外，在探討細胞自噬作用扮演的角色的研究中發現，透過分別給予 MMCs 前處理細胞自噬誘導劑 Rapamycin 及細胞自噬抑制劑 3-MA (3-Methyladenine)，巴拉刈24小時處理下的細胞存活率分別為16%的提升及25%的下降，由此可知巴拉刈在 MMCs 上所誘導產生的細胞自噬具有保護細胞之角色。綜合以上實驗結果，我們發現巴拉刈暴露於 MMCs 中，在初期時所產生的 ROS 以及誘發內質網壓力而啟動細胞自噬作用，以保護細胞避免巴拉刈的毒性對腎絲球腎膈細胞的傷害。此篇結果提供對於巴拉刈腎毒性的細胞自噬所扮演的角色與其分子調控機制，作為早期治療參考之依據。

關鍵字

巴拉刈 ； 巴拉刈中毒 ； 腎臟 ； 腎絲球炎 ； 腎膈細胞 ； 細胞自噬

並列摘要

Paraquat (PQ) is the trade name for 1,1′-dimethyl-4,4′-bipyridinium dichloride and has a molecular formula of C12H14Cl2N2. The name paraquat is derived from the para positions of quaternary ammonium. PQ is registered and used in more than 130 countries, both developing and developed, and is considered as one of the most widely-used herbicides. PQ intoxication is caused by ingestion (swallowing) or through damaged skin, but may also be inhaled. It can cause multiple-organ failure including the lungs, heart, kidneys, adrenal glands, central nervous system, liver, muscles, as well as damaging the skin and eyes. Among which, kidney is the most sensitive via the ingestion route and has the highest concentration of PQ accumulation compared with the other organs at any detection time. Under normal homeostatic conditions, almost 100% of unlethal amount of PQ can be excreted in the urine within 24 hours of PQ ingestion. Since PQ is mainly eliminated by the kidney, damage of this organ is of particular concern to the outcome of PQ intoxication. After PQ reaches the kidney cells, they undergo a process of redox-cycling which, at the expense of NAPDH, in turn leads to the production of reactive oxygen species (ROS) such as superoxide anion, O2•̄.
A study at the emergency departments of two medical centers in southern Taiwan between January 2001 and December 2002 showed that PQ intoxication contributed 18.9% of all herbicide intoxication, which had caused a 72.1% fatality. Previous findings suggest that high dosage of PQ can cause serious kidney damage, for example, acute kidney injury (AKI). This is characterized by the symptoms of proteinuria, pyuria, azotemia and hematuria between 24 and 96 hours post-intoxication. Oliguria has also been reported after serious PQ intoxication, and this led to damages to the glomerular capillary wall and eventually glomerulonephritis (GN). Studies have shown that GN is caused by an elevated level of ROS which damages glomerular cells such as the mesangial cells. The glomerular mesangial cells constitute approximately 30 – 40% of the total glomerular cell populations, which act as contractile smooth muscle cells to modulate glomerular blood flow and filtration, as well as the uptake and clearance of macromolecules through their phagocytic / autophagic properties. Due to these reasons, the mechanisms on how PQ induces autophagy signalling in mesangial cells remains a critical research topic that needs to be unravelled.
This study used mouse mesangial cells (MMCs) as a model of examining PQ-induced autophagic signalling. Our results showed that 145μM of PQ (IC50) decreases cell survival rate. Western blot analyses demonstrated that PQ-induced LC3-II expression that peaked at 3 hours post-treatment, accompanied with the high expression of Beclin-1 and Atg5-Atg12-Atg16L1 complex, but reduction in p62 expression. Our immunofluorescence data showed the formation of autophagosomes in MMCs after PQ treatment. Further, PQ triggered autophagic signalling events mediated by GSK-3β, ERK, AKT, mTOR and p70S6K due to ROS production. Interestingly, p-eIF2α and ERK signalling was activated by endoplasmic reticulum (ER) stress. The addition of rapamycin or 3-Methyladenine (3-MA) increased cell survival by 16% or decreased by 25%, repectively. Overall, our data suggest that autophagy serves a protective role in PQ treatment of MMCs, and this is activated by an acute ROS production and ER stress. These results provide fundamental molecular mechanisms in the role of autophagy signalling in MMCs, which can be considered as an early reference in the clinical setting of PQ-induced kidney injuries.

並列關鍵字

paraquat ； paraquat poisoning ； kidney ； glomerulonephritis ； mesangial cells ； autophagy

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