- 學位論文
以動物模式來探討巴拉刈對小鼠腎臟腎絲球的傷害及造成小鼠腎膈細胞細胞凋亡之訊息傳遞路徑
The Studies on Paraquat-Induced Glomerulonephritis in Mouse Model and Signaling Pathway of Apoptosis in Mouse Mesangial Cells
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摘要
巴拉刈（paraquat，PQ）為全球使用量第二大的除草劑，其化學名稱為1,1’-dimethyl-4,4’-bipyridylium dichloride，分子式為C12H14Cl2N2，屬於聯嘧啶類（bypyridiniums）的化合物。由於其毒性對人體的健康造成危害，巴拉刈已被許多國家限制及禁止使用。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%。巴拉刈中毒主要由口服誤食或自殺而進入體內，腎臟是其最早達到組織濃度最高的器官，也為最主要的排泄器官。在腎臟功能正常情況下，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將造成組織中的濃度將增高，進而加劇巴拉刈對其他器官的毒性。當巴拉刈進入腎臟細胞後，隨即進行氧化還原循環（redox cycling），消耗NADPH，產生的氧化性自由基（reactive oxygen species，ROS）將直接破壞細胞膜構造和胞器，並引發嚴重的發炎反應，最終導致細胞死亡。過去文獻指出，在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並在24至96小時內出現蛋白尿及血尿等症狀；若出現寡尿或無尿的現象，則代表已造成更嚴重的急性腎小管壞死及腎絲球腎炎。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，因而啟發本研究以動物及細胞模式來探討巴拉刈對腎臟的傷害及其造成腎絲球細胞死亡的分子機制。 本研究首先以腹腔注射高劑量的55 mg/kg BW 巴拉刈於八週齡的雌性C57BL/6鼷鼠，觀察腎功能及其腎絲球的病理組織變化。動物實驗結果顯示暴 露巴拉刈後，小鼠腎功能指數與對照組相比有異常顯著增加的現象，且隨暴露天數的增加，腎功能指數也有上升的趨勢。由組織切片中觀察到腎小管刷狀緣減少、細胞排列不規則及腎絲球結構受損之現象。進一步以免疫組織化學染色和TUNEL等方法證實腎膈細胞受到傷害及走向細胞凋亡（apoptosis）途徑。 另以巴拉刈處理小鼠腎膈細胞株 （mouse mesangial cells，MMC），研究造成其死亡之訊息傳導路徑。實驗結果得知巴拉刈造成MMC存活率的下降，其24小時的IC50為145 μM。後續進行其ROS產生量的檢測，發現巴拉刈處理細胞12小時後，ROS變化量相較於對照組皆有顯著增加的趨勢，且有時間及劑量效應關係。而以Annexin V/PI進行細胞凋亡實驗的結果顯示，巴拉刈誘發MMC的凋亡細胞量顯著高於對照組。以與超氧自由基反應的Dihydroethidine （DHE）和DNA的特異性染劑Hoechst 33342進行細胞螢光染色，藉由影像分析證實了巴拉刈處理的MMC其氧化壓力和凋亡細胞皆有增加的現象。進一步利用西方點墨法（western blotting）了解參與細胞凋亡的分子訊息途徑，結果顯示caspase-12，CHOP，p-JNK，p-p38等參與內質網壓力（ER stress）介導的凋亡途徑的分子皆有表現量增加的趨勢;而p-p53，Bax，caspase-9和caspase-3的增加也暗示著粒線體內源性凋亡途徑（mitochondrial intrinsic apoptosis pathway）的啟動。此外，在探討轉錄因子Nrf2在細胞凋亡途徑扮演的角色的研究中發現，Nrf2參與了巴拉刈誘導ROS產生進而造成細胞凋亡的過程。透過Nrf2的抑制實驗也發現其調控內質網壓力和粒線體失調造成的訊息傳遞途徑，最後導致MMC的死亡。 綜合以上實驗結果，巴拉刈進入鼷鼠後造成腎臟中腎絲球的傷害，而其細胞的傷害是藉由ROS的產生引發內質網壓力和粒線體失調並引起下游相關的訊息傳遞，進而誘導細胞凋亡。本實驗為第一篇探討巴拉刈對MMC造成傷害之 機制的研究，為以後類似的研究提供了參考的依據。
並列摘要
Paraquat (PQ) is one of the most widely used herbicides in the world and is well known for its nephrotoxicity. The molecular formula of PQ is C12H14Cl2N2 and it has been classified as bipyridylium quaternary ammonium herbicides. PQ poisoning is the most common cause of fulminated and fatal herbicide intoxication. PQ intoxication contributed to 18.9 % of all pesticide poisonings in Taiwan from 2001 to 2002. Previous studies showed that high doses of PQ lead to multiple organ failure such as lung, kidney, liver and so on. Kidney has the highest concentration at any detection time when compare to the other organs. PQ mainly excreted by kidney and undergoes a process of redox-cycling which ultimately leads to reactive oxygen species (ROS) production at the expense of NADPH. Excessive ROS elevation in cells may cause cellular dysfunction and lead to nephrotoxicity. PQ-induced acute kidney injury (AKI) caused proteinuria, pyuria, azotemia, uremia and other minor symptoms within 24-96 hours. Despite severe tubular necrosis, PQ may also cause glomerular damage and eventually lead to glomerulonephritis (GN). PQ exposure is associated with GN and the mesangial cells occupy a central position in the genesis of the cellular lesions seen in GN. However, the distinct mechanisms underlying PQ-induced GN and glomerular mesangial cells damage have not been clarified. Therefore, in this study we investigate PQ-induced GN in mice model and also investigate the mechanisms of PQ-induced cell death in glomerular mesangial cells. In animal model, 8 weeks female C57BL/6 mice were intraperitoneally administered with 55 mg/kg BW PQ. Kidney function evaluation revealed significant differences in serum BUN, serum Cr, urine total protein and urine Cr compared to the control group. Histopathologically, kidney showed tubular brush border loss, irregular cell arrangement and glomerulus damage. Further experiments through immunohistochemical stain and TUNEL assay demonstrated mesangial cells involved in glomerular lesions and went through apoptosis. MMC cell line provided an in vitro model to investigate the in detail mechanisms of PQ-induced apoptosis. We found that PQ caused a decrease in cell survival rate and a IC50 of 145 μM PQ was determined. ROS generation was observed in PQ-treated MMC and significant increased after the first 12 hours compared to the control group. ROS production after PQ exposure in MMC showed time and dose-dependent manner. Annexin V/PI apoptosis assay further demonstrated PQ-induced significant apoptosis in MMC. The signaling pathways were then investigated and we found that signal molecules involved in ER stress-mediated apoptosis pathway such as caspase-12, CHOP, p-JNK and p-p38 were significant elevated. p-p53, Bax, caspase-9 and caspase-3 which participate in mitochondrial intrinsic apoptosis pathway were also raised after PQ exposure. These results indicated that both ER-stress and mitochondrial signaling pathways involving in PQ-induced MMC cell death. In addition, we revealed the transcription factor, Nrf2 involved in apoptosis pathway. Nrf2 inhibition experiments also indicated that regulation of Nrf2 in ER stress and mitochondrial dysfunction was associated with the apoptosis of MMC. As a conclusion, the results of this study demonstrated that PQ caused kidney glomerular damage and induced GN in female C57BL/6 mice which involved the injury of mesangial cells. The production of ROS caused MMC apoptosis through activation of the ER stress-mediated and mitochondrial intrinsic apoptosis pathway. This experiment is the first to explore the exact mechanism of PQ-induced cell death in glomerular mesangial cells and provides useful information for other similar studies in future.
並列關鍵字
paraquat ； paraquat poisoning ； kidney ； glomerulonephritis ； mesangial cells ； cell apoptosis參考文獻
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