苯芘透過活性氧化物誘發視網膜色素上皮細胞 上皮間質轉換機制之探討

Abstract

苯芘 ( Benzo[a]pyrene, B[a]P ) 為香菸中廣泛存在的多環芳香烴之一，可由多環芳香烴受體 ( Aryl hydrocarbon receptor, AhR ) 活化生物代謝途徑，進而促進細胞色素 P450 ( Cytochromes P450, CYP450 ) 將其水解代謝而產生活性氧化物( Reactive oxygen species, ROS )。近年流病研究統計，抽菸會增加眼部病變之機率，其中與 B[a]P 代謝過程所產生之 ROS 攻擊視網膜之視網膜色素上皮細胞 ( Retinal pigment epithelium, RPE ) 有關。RPE 可維持眼部代謝功能之平衡，其功能失常可引發視網膜病變，如增生性玻璃體視網膜病變（proliferativevitreoretinopathy, PVR）即為 RPE 的上皮細胞間質化 ( epithelial-mesenchymal transition, EMT ) 現象所造成，此疾病是視網膜手術後視網膜剝離的主要原因，且文獻指吸菸的人會因此疾病導致視網膜再次剝離的風險高於一般人，但病變之機制仍待進一步釐清。文獻指出 AhR 活化或者 ROS 皆會促進細胞 EMT，而香菸中的 B[a]P 同時刺激此兩個促進因素，因此本研究主要探討香菸中物質 B[a]P 是否造成 RPE 引起 EMT 現象，並探討其機制以釐清是否為導致 PVR 的原因之一。實驗使用 ARPE-19 細胞株，由細胞爬行試驗發現 B[a]P 會促進細胞爬行，並以 westernblot 發現 B[a]P 會促進 Vimentin 蛋白表現量增加，而當 AhR 受到抑制之下， Vimentin 蛋白表現量即不受 B[a]P 所誘導，證明 AhR 的確會調控 Vimentin 表現量。且從 ROS 螢光圖中顯示 B[a]P 會誘導 RPE 中 ROS 含量上升，而使用 ROS 抑制劑 ( N-Acetyl-cysteine, NAC ) 結合western bolt實驗後，發現抑制B[a]P產生之ROS後，Vimentin 表現量即不受B[a]P所影響，證明 AhR 透過代謝 B[a]P 產生 ROS 促使 Vimentin 蛋白表現量上升，進一步探討 B[a]P 透過 ROS 引起 EMT 的路徑，發現使用ROS抑制劑後，B[a]P 誘導 β-Catenin 的表現量上升即，GSK3β 表現量下降及不受 B[a]P 影響。綜合上述結果，本研究發現 B[a]P 的暴露會導致 RPE 細胞中產生 ROS，進而抑制 β-Catenin 的降解，導致細胞EMT；除此之外本實驗室過去研究亦發現， RPE 中之 AhR 與 抗氧化轉錄因子 -NF-E2 相關因子2（ nuclear factor erythroid-2-related factor, Nrf2 ）蛋白表現量顯著高於其它人類細胞株，Nrf2 可活化下游抗氧化基因，藉此保護細胞不受 ROS 傷害，但詳細機轉仍屬未知。因此本實驗最後也探討 B[a]P 暴露 RPE中 AhR 及 Nrf2 之間的交互作用，發現 AhR 除了利用轉譯後修飾調節Nrf2，從 RT-PCR 實驗結果也得知加入 B[a]P 後 Nrf2 mRNA表現量上升。進一步利用 CHIP 實驗證實 B[a]P 促使 AhR 入核，結合在 Nrf2 的 promoter region 使得 Nrf2 轉錄量上升，顯示 AhR 於眼部代謝扮演重要角色，而 AhR-Nrf2 之調控亦影響眼部疾病的發生。

Benzo[a]pyrene, one of the polycyclic aromatic hydrocarbons widely present in cigarettes and it would produce reactive oxygen species (ROS) after AhR metabolic processes. In recent years, studies have shown that smoking increases the risk of eye disease may be related to ROS produced by B[a]P metabolism attacking the retinal pigment epithelium cells (RPE). RPE can maintain the balance of ocular metabolic function, and its dysfunction can cause retinopathy, such as proliferative vitreoretinopathy (PVR), which is caused by epithelial-mesenchymal transition (EMT) of RPE. This disease is the main cause of retinal detachment after retinal surgery, and the literature indicates that people who smoke will have a higher risk of re-detachment of the retina than the average person, but the mechanism of the PVR remains to be further clarified. The literature indicates that both AhR activation and ROS promote cell EMT, while B[a]P in cigarettes stimulates these two stimulating factors. Therefore, the aim of this research is to study whether B[a]P in cigarettes causes RPE EMT, and clarify its mechanism. The ARPE-19 cell line was used in the experiment. The result shows that B[a]P promoted cell migration and western blot result show that B[a]P promote Vimentin protein expression, while when AhR was inhibited, Vimentin protein expression was not induced by B[a]P, demonstrating that AhR regulates Vimentin. It is shown from the ROS fluorescence that B[a]P induces ROS level in RPE, whereas after ROS inhibitors (N-Acetyl-cysteine, NAC) inhibition the expression of Vimentin was not affected by B[a]P, which proved that B[a]P produced ROS through AhR metabolism promoted the expression of Vimentin protein. Further, confirming the mechanism of ROS inducing EMT. We found that B[a]P induced the expression of β-Catenin and decreased GSK3β. After ROS inhibition, β-Catenin and GSK3β protein expression were not affected by B[a]P. Based on the above results, this study found that exposure to B[a]P leads to the production of ROS in RPE cells, which in turn inhibits the degradation of β-Catenin, leading to cellular EMT. In addition, Studies in our laboratory have also found that The protein expression of AhR and nuclear factor erythroid-2-related factor (Nrf2) in RPE is significantly higher than in other human cell lines. Nrf2 activates antioxidant genes, thereby protecting cells from ROS damage, Further previous studies showed that Nrf2 may be regulated by AhR. Therefore, the interaction between AhR and Nrf2 by B[a]P exposed in RPE was also discussed at the end of the experiment. It was found that AhR not only uses post-translational modification to regulate Nrf2, from RT-PCR results, it is also known that after B[a]P treatment, The amount of Nrf2 mRNA expression increased. Further used CHIP experiments to confirm that B[a]P promoted AhR nuclear import and binding to the promoter region of Nrf2 results in an increase of Nrf2 transcription level.