內質網壓力在赭麴毒素A誘發腎小管細胞傷害之角色

Abstract

黴菌毒素 (mycotoxins) 為真菌類所產生之低分子量的二次代謝物 (secondary metabolites)。赭麴毒素 (ochratoxin, OT) 主要是由麴黴菌屬 (Aspergillus) 和青黴菌屬 (Penicillium) 在黴變的飼料、穀類及咖啡豆中所產生的次級代謝物，其中以赭麴毒素A (ochratoxin A, 簡稱OTA) 的毒性最強，由於OTA廣泛地存在於食物及飼料中，因此，對於人類及動物的健康威脅很大。先前有許多研究證實OTA具有腎臟毒性、肝臟毒性、致畸胎毒性、基因毒性、免疫抑制性及致癌性，由於具有物種及性別特異性，在人類疾病中，OTA被認為可能是造成巴爾幹半島腎病變 (Balkan endemic nephropathy, BEN) 的原因之一，先前文獻指出OTA會經由抑制蛋白質合成、活化氧化壓力、促進細胞凋亡、影響鈣離子平衡等機制進而導致病變，然而對於OTA造成的腎臟傷害多以動物模式為主，在人類腎臟細胞中的詳細分子機制仍有待更多研究來釐清。當細胞受到壓力時，內質網會啟動未摺疊蛋白反應 (unfolded protein response, 簡稱UPR) 來緩解壓力，然而，鮮少有研究去探討OTA與UPR之間的關聯，因此，本實驗欲探討OTA在腎臟中是否會經由UPR去調控後續結果。本實驗以人類腎臟近曲小管上皮細胞 (human renal proximal tubular epithelial cells, 簡稱HK-2) 為實驗對象，首先進行細胞存活率試驗 (MTS assay)，觀察到OTA處理下細胞存活率明顯降低，進一步以流式細胞儀分析細胞週期，可以觀察到HK-2細胞在20 μM OTA處理下sub-G1 phase的細胞數明顯增加，再以西方墨點法分析細胞凋亡指標蛋白PARP的表現量，隨著OTA處理時間增加有上升的趨勢。綜合上述結果，推測OTA在HK-2細胞中會促進細胞凋亡。接著去分析在HK-2細胞中給予OTA是否會活化UPR途徑，以西方墨點法分析UPR路徑之蛋白 (Bip、ATF6、p-eIF2α、p-IRE1α)，實驗結果顯示，在20 μM OTA處理下，上述UPR路徑之蛋白皆有被誘導表現。再以西方墨點法分析UPR相關細胞凋亡路徑 (CHOP、caspase-4及JNK)，實驗結果顯示，只有JNK途徑有被活化。已知活性氧物質 (reactive oxygen species, 簡稱ROS) 的增加會活化JNK途徑，因此，以DCFDA偵測法觀察給予OTA後細胞內ROS的變化，實驗結果顯示ROS隨著OTA濃度增加有上升的趨勢，進一步給予ROS抑制劑－N-acetyl cysteine (NAC) 並分析細胞中ROS含量與細胞凋亡蛋白之變化量，結果顯示，合併NAC處理後能有效降低細胞中因OTA所引起的ROS增加，並能降低PARP的表現，然而，JNK的表現則未受影響。綜合上述結果，本篇研究確認了OTA在HK-2細胞中造成ROS增加最終導致細胞凋亡，然而JNK在這其中所扮演的角色是傾向細胞凋亡或是細胞存活仍有待進一步實驗來釐清。

Mycotoxins are the secondary metabolites produced by certain fungi that are capable of causing disease and death in human and other animals. Ochratoxin (OT) is a mycotoxin produced by Aspergillus and Penicillium. Ochratoxin A (OTA) is the most prevalent and deleterious toxin of this group, it is known to occur in commodities such as animal feeds, cereals and coffee. The exposure of the animal and human population to OTA represents a worldwide problem. Previous studies show that OTA is nephrotoxic, hepatotoxic, teratogenic, genotoxic, immunotoxic and carcinogenic to several species , which characterize in species and sex specificity manner. Epidemiological evidences suggest that OTA may be the risk factor of Balkan endemic nephropathy (BEN). To date, the mechanism of OTA leads to nephropathy remains controversial. Several mechanisms have been reported such as inhibition of protein synthesis, induction of reactive oxygen species (ROS), activation of apoptosis and disruption of calcium homeostasis. When cells counter stress, they will activate unfolded protein response (UPR) to restore normal function by suspending protein synthesis, activation of chaperon expression and degradation of misfolded protein. Overwhelmed stress, will lead to the activation of apoptosis. However, the relation between OTA and UPR in human remains unclear. Thus, the aim of this study is to investigate whether OTA induces UPR in human kidney. We use HK-2 cell as in vitro model. After OTA treatment, cell viability was decreased in a dose-dependent manner. In cell cycle study, OTA increased sub-G1 phase of HK-2 cell. The protein level of PARP, a marker of apoptosis, increased in a time-dependent manner. These results indicated that OTA induce apoptosis in HK-2 cell. To identify the relation between OTA and UPR, we analyzed the protein involved in UPR such as Bip, ATF6, p-eIF2α and p-IRE1α. Expression level of the proteins was increased by OTA treatment. Furthermore, we investigated which apoptosis pathway were induced by OTA treatment. The results showed that OTA induced JNK phosphorylatuin but not CHOP and caspase-4 dependent manner. It is known that increased ROS cause activation of JNK. Thus, we used DCFDA assay to detect ROS level after OTA treatment. The result revealed that ROS level significantly increased after OTA treatment. Next, we used NAC, an inhibitor of ROS, to examine the ROS and protein level. ROS and PARP level were significantly decreased by NAC treatment. Notably, treatment with NAC did not inhibit p-JNK induction. Taken together, our results suggested that OTA induced apoptosis in HK-2 cell through increasing ROS level. However, we need more experiments to examine whether JNK pathway lead to apoptosis or cell survival.