甲基乙二醛和碘酸鈉對視網膜色素上皮細胞之細胞死亡模式

Abstract

在已發展國家中，糖尿病視網膜病變(DR)和老年性黃斑部病變(AMD)是後天性失明的兩個主要原因。晚期糖化終產物（Advanced glycation end products, AGEs）在視網膜色素上皮細胞的堆積，在糖尿病視網膜病變及AMD中佔有重要角色。其中，晚期糖化終產物中的甲基乙二醛 (Methylglyoxal, MGO) 對蛋白質結構和功能，有著不可逆的影響。另外，氧化壓力是視網膜色素上皮細胞損傷的主要因素，是導致老年性黃斑部病變的主要原因。碘酸鈉(Sodium iodate, NaIO3)是一種氧化毒性劑，其選擇性破壞視網膜色素上皮細胞，使其成為老年性黃斑部病變的可重複模型。雖然碘酸鈉是一種氧化壓力誘導劑，但活性氧化物（ROS）在碘酸鈉誘導的訊息路徑和細胞活性中的角色尚未釐清，而碘酸鈉對視網膜色素上皮細胞自噬作用仍然不清楚。 在我們的研究中，我們研究了兩種化學甲基乙二醛和碘酸鈉對視網膜色素上皮細胞死亡的機轉。我們利用ARPE-19細胞，了解甲基乙二醛誘導的視網膜色素上皮細胞死亡的機制。研究結果顯示甲基乙二醛通過非依賴性凋亡蛋白酶(caspase-independent)的方式，誘導視網膜色素上皮細胞死亡，會造成活性氧化物形成，粒線體膜電位（MMP）喪失，細胞內鈣離子上升和內質網壓力反應。抑制活性氧化物的產生可以逆轉甲基乙二醛的活性氧化物產生，粒線體膜電位損失，細胞內鈣離子增加和細胞死亡。此外，存儲運作的鈣離子通道抑制劑(store-operated calcium channel inhibitors) MRS1845和YM-58483，而不是肌醇1,4,5-三磷酸 (inositol 1,4,5‐trisphosphate, IP3）受體抑制劑xestospongin C，可以阻止甲基乙二醛對ARPE-19細胞誘導的活性氧化物產生，粒線體膜電位損失和持續的細胞內鈣離子增加。最後，內質網壓力的抑制劑的salubrinal和4-PBA，可以減少甲基乙二醛誘導的細胞內變化和細胞死亡。 此外，我們研究了碘酸鈉誘導的細胞死亡的機制。在人類ARPE-19細胞中，我們使用annexin V/PI染色來確定細胞活性，使用免疫墨點法去確定蛋白質表達和訊息級聯，共軛焦顯微鏡以確定粒線體動力學和粒線體自噬，以及海馬分析以確定粒線體氧化磷酸化。我們發現碘酸鈉可以顯著誘導細胞質，而不是粒線體活性氧化物產生。碘酸鈉還可以活化ERK、p38、JNK和Akt，增加LC3II表達，誘導Drp-1磷酸化和粒線體分裂，但抑制粒線體呼吸。共軛焦顯微鏡數據顯示碘酸鈉和巴弗洛黴素(bafilomycin )A1對LC3點狀形成的協同作用，顯示會誘發自噬作用。使用細胞質抗氧化劑N-乙醯半胱氨酸(NAC)，我們發現p38和JNK是活性氧化物的下游信息，並且涉及碘酸鈉誘導的細胞毒性，但不參與粒線體動力學，而活性氧化物也參與LC3II表達。出乎意料的是，碘酸鈉的刺激及同時使用NAC，會導致粒線體片段化和加強細胞死亡。此外，抑制自噬作用和Akt，會進一步加強細胞對碘酸鈉的易感性。 總括來說，我們的結果顯示甲基乙二醛可以降低視網膜色素上皮細胞活性，是透過內質網壓力導致細胞內活性氧化物產生，粒線體膜電位損失和細胞內鈣離子增加所致。由於甲基乙二醛是老年性黃斑部病變中玻璃疣(druen)的成分之一，並且是糖尿病視網膜病變中的晚期糖化終產物加成物，因此本研究可以作為老年性黃斑部病變和糖尿病視網膜病變的發病機制和相關治療提供有價值的研究。另外，碘酸鈉誘導的氧化壓力和細胞質活性氧化物產生，會誘發多種訊息路徑，去協調控制視網膜色素上皮細胞的死亡。雖然活性氧化物依賴性p38和JNK的活化會參與細胞毒性，但ROS誘導的自噬作用以及粒線體動態平衡，可以減少由碘酸鈉誘導的視網膜色素上皮細胞死亡。

Diabetic retinopathy (DR) and age-related macular degeneration (AMD) are two important leading causes of acquired blindness in developed countries. As accumulation of advanced glycation end products (AGEs) in retinal pigment epithelial (RPE) cells plays an important role in both DR and AMD, and the methylglyoxal (MGO) within the AGEs exerts irreversible effects on protein structure and function, it is crucial to understand the underlying mechanism of MGO-induced RPE cell death. Moreover, oxidative stress is a major factor in RPE cells injury that contributes to AMD. Sodium iodate (NaIO3) is an oxidative toxic agent and its selective RPE cell damage makes it as a reproducible model of AMD. Although NaIO3 is an oxidative stress inducer, the roles of ROS in NaIO3-elicited signaling pathways and cell viability have not been elucidated, and the effect of NaIO3 on autophagy in RPE cells remains elusive. In our research, we investigated the mechanism of two chemicals MGO and NaIO3 on the RPE cell death. Using ARPE-19 as the cell model, our study revealed that MGO induces RPE cell death through a caspase‐independent manner, which relying on reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP) loss, intracellular calcium elevation and endoplasmic reticulum (ER) stress response. Suppression of ROS generation can reverse the MGO-induced ROS production, MMP loss, intracellular calcium increase and cell death. Moreover, store-operated calcium channel inhibitors MRS1845 and YM-58483, but not the inositol 1,4,5-trisphosphate (IP3) receptor inhibitor xestospongin C, can block MGO‐induced ROS production, MMP loss and sustained intracellular calcium increase in ARPE-19 cells. Lastly, inhibition of ER stress by salubrinal and 4-PBA can reduce the MGO-induced intracellular events and cell death. Moreover, we investigated the mechanism of NaIO3-induced cell death. In human ARPE-19 cells, we used Annexin V/PI staining to determine cell viability, immunoblotting to determine protein expression and signaling cascades, confocal microscopy to determine mitochondrial dynamics and mitophagy, and Seahorse analysis to determine mitochondrial oxidative phosphorylation. We found that NaIO3 can dramatically induce cytosolic but not mitochondrial ROS production. NaIO3 can also activate ERK, p38, JNK and Akt, increase LC3II expression, induce Drp-1 phosphorylation and mitochondrial fission, but inhibit mitochondrial respiration. Confocal microscopic data indicated a synergism of NaIO3 and bafilomycin A1 on LC3 punctate formation, indicating the induction of autophagy. Using cytosolic ROS antioxidant N-acetyl cysteine (NAC), we found that p38 and JNK are downstream signals of ROS and involve in NaIO3-induced cytotoxicity but not in mitochondrial dynamics, while ROS is also involved in LC3II expression. Unexpectedly NAC treatment upon NaIO3 stimulation leads to an enhancement of mitochondrial fragmentation and cell death. Moreover, inhibition of autophagy and Akt further enhances cell susceptibility to NaIO3. In summary, our data indicate that MGO can decrease RPE cell viability, resulting from the ER stress‐dependent intracellular ROS production, MMP loss and increased intracellular calcium increase. As MGO is one of the components of drusen in AMD and is the AGEs adduct in DR, this study could provide a valuable insight into the molecular pathogenesis and therapeutic intervention of AMD and DR. Moreover, NaIO3 induces oxidative stress and cytosolic ROS production exerts multiple signaling pathways that coordinate to control cell death in RPE cells. ROS-dependent p38 and JNK activation lead to cytotoxicity, while ROS-mediated autophagy and mitochondrial dynamic balance counteract the cell death mechanisms induced by NaIO3 in RPE cells.