苯芘對視網膜色素上皮細胞AhR / Stat3路徑所調控之Socs3角色探討

Abstract

近年來，許多研究發現PAHs可增加諸多眼部病變，流病資料統計亦發現抽菸與眼睛疾病發生有關。Benzo[a]pyrene (B[a]P)為多環芳香烴(Polycyclic Aromatic Hydrocarbons, PAHs)的其中之一員，由碳的不完全燃燒所產生，是一種廣泛存在環境的的致癌化學物質。B[a]P為Aryl Hydrocarbon Receptor (AhR) ligand，進入細胞後會和AhR蛋白結合進一步調控下游多項基因轉錄。過去研究顯示，B[a]P可能會透過AhR調控發炎反應，但具體機轉仍不清楚。Suppressor of cytokine signaling-3 (Socs3)作為一發炎調控因子，可藉由負回饋機轉抑制發炎反應，而發炎是導致視網膜病變的主要因素之一，因此本研究希望觀察眼部暴露B[a]P後之AhR路徑對Socs3的影響。我們利用ARPE-19人類視網膜色素上皮細胞株證實B[a]P暴露會透過活化AhR，近一步誘導Socs3表現；然而當AhR消失時，Socs3表現下降，但是隨B[a]P劑量暴露，依舊可以觀察到Socs3表現的誘導現象，伴隨Stat3的磷酸化增加。在炎症進展中，JAK / STAT途徑在信號轉導中起關鍵作用，後續實驗利用免疫熒光、免疫沈澱法以及Stat3磷酸化抑制劑證實在沒有AhR的情況下則會透過Stat3活化取代AhR增加Socs3表現。另外，過去文獻指出，B[a]P會導致發炎反應上升，然Socs3一般被認為扮演抑制發炎角色，所以接下來實驗想探討B[a]P對發炎的影響以及Socs3所扮演的角色。在以單一3µM B[a]P劑量24小時暴露後，測量發炎因子IL-6、TGF-beta1，並未發現以上發炎因子表現改變情況，相關B[a]P暴露對視網膜色素上皮細胞發炎的影響，仍待後續更全面劑量、暴露時間及不同cytokine的研究。

Recently, studies demonstrated that PAHs might increase a number of eye lesions. Statistics of epidemics suggested that smoking was a risk factor for oculardiseases. Benzo[a]pyrene (B[a]P), one of the polycyclic aromatic hydrocarbons (PAHs), a carcinogenic chemical substance that is widely present in the environment. It is produced by incomplete combustion of carbon. B[a]P is an aryl hydrocarbon receptor (AhR) ligand that binds to the AhR protein and further regulates downstream gene transcription. In addition, studies had also shown that B[a]P might affect the cellular inflammatory response through activation of the AhR pathway, but the relevant mechanisms remained unclear. Suppressor of cytokine signaling-3 (Socs3), an inflammatory regulator, act as an inhibitor of inflammation by negative feedback. Inflammation is one of the major causes of retinopathy. Therefore, this study aims to observe the effect of the AhR –regulated Socs3 pathway with B[a]P exposure. Here, we used ARPE-19 human retinal pigment epithelial cell line that B[a]P induced Socs3 expression through the activation of AhR. Knock out of AhR attenuated the B[a]P-dependent induction of Socs3 expression, accompanied with the increase of the phosphorylated Stat3. Subsequent experiments using immunofluorescence and Stat3 phosphorylation inhibitors demonstrated that in the absence of AhR, activation of Stat3 replaced AhR and increased the expression of Socs3. In addition, after exposure to 3 μM B[a]P for 24 hours, inflammatory cytokines IL-6 and TGF-beta1 were measured and no change in the protein expression was found. More studies should be done to further confirm the effect of B[a]P to ARPE-19 cells.