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Title: | 萘誘導NF-κB與SP1交互作用影響視網膜色素上皮細胞發炎機制之探討 The mechanism of Naphthalene- induced inflammation through NF-κB and SP1 interaction in ARPE-19 cells |
Authors: | Yi-Shiou Wei 魏伊秀 |
Advisor: | 康照洲(Jaw-Jou Kang) |
Keyword: | ?,1,4-??,視網膜色素上皮細胞,發炎反應,ICAM-1,NF-κB,SP1,CYP2F1, Naphthalene,1,4-Naphthoquinone,retinal pigment epithelium cells,inflammation,ICAM-1,NF-κB,SP1,CYP2F1, |
Publication Year : | 2019 |
Degree: | 碩士 |
Abstract: | 萘(Naphthalene, NAP)為廣泛性存在於環境的汙染物,屬二苯環之多環芳香烴(Polycyclic Aromatic Hydrocarbons, PAHs),主要經有機物不完全燃燒而生成,來源如汽機車廢氣、生質燃料及香菸煙霧等,隨著近代工業發展,近年流行病學研究調查發現 PAHs 暴露會增加罹患眼部疾病風險,如眼睛乾澀、發炎甚至影響視網膜血流量。近年研究發現,急性及慢性職業暴露NAP可造成工作者產生白內障和視網膜損傷;另從環境監測資料得知,NAP無論在日常生活或工作場所占所有PAHs暴露中最大比率可高達60%到70%,且 NAP 已於動物實驗證實具有眼部病變之特異性,易導致眼部白內障(Cataract)及視網膜退化(Retina degeneration),然而造成此特異性傷害之機制仍不清楚。目前文獻普遍認為 NAP 造成眼部病變可能源於 P450 代謝,並懷疑其代謝物萘醌(Naphthoquinone)為主要產生毒性之物質,透過造成細胞毒性與細胞間修復機制不斷循環所引起;另發炎反應已知為細胞修復前驅反應之一,本研究透過NAP暴露探討典型發炎調控因子 NF-κB 與 CYP2F1 間產生對視網膜傷害之關聯性,期以瞭解眼部發炎特異性反應與眼部疾病成因。實驗利用 ARPE-19 人類視網膜色素上皮細胞株,分別處理不同濃度及暴露時間之 NAP 及其代謝物 1,4-naphthoquinone (1,4-NQ),發現 10 μM 1,4-NQ 對 RPE 細胞有顯著毒性,而 NAP 與 1,4-NQ 皆會使 p65之磷酸化增加,且NAP 會顯著增加 RPE 下游細胞黏附因子(Intercellular Adhesion Molecule 1, ICAM-1)蛋白表現量;然而1,4-NQ 卻使 RPE 之 ICAM 表現量下降,顯示 1,4-NQ 可能透過非 NF-κB訊息傳遞路徑調控ICAM。此外,我們證實NAP會誘發CYP2F1蛋白及mRNA表現量增加,但不影響CYP1A1和CYP1B1;相反地,1,4-NQ不會改變CYP2F1表現量。進一步透過NF-κB活化抑制劑BAY11-7082不僅抑制NF-κB訊息傳遞路徑,也減少SP1、CYP2F1蛋白表現量,且使SP1活性下降。實驗結果也顯示,抑制NF-κB活化會減少NAP所促進的SP1-p65蛋白間交互作用,並減少SP1入核,而此機制與MAPK調控訊息傳遞有關。綜合上述,我們發現低劑量的NAP暴露可透過活化NF-κB 並調控SP1方式影響CYP2F1表現量增加,進而導致NAP誘導視網膜損傷,未來研究可進一步釐清 NF-κB 與 CYP2F1 間是否有相互調控關係,以提出了在預防眼部退化早期階段,NAP所誘導的發炎效應與其調節機制。 Naphthalene (NAP) is the most widespread environmental contaminant of polycyclic Aromatic Hydrocarbons (PAHs), which was generated from fuel combustion, transportation, and cigarette smoking. In recent years, studies indicated that PAHs might associate with the increase risk of eye diseases such as dry eyes, ocular tissue inflammation, even affecting blood flow rate of retina. The clinical cases of Cataract and retina injury had been reported following acute and chronic occupational exposure to NAP. Due to NAP is the largest proportion of PAH exposure, which highest about 60-70% both in daily life and in workplace. Moreover, NAP had the specificity of ocular lesions in animal studies, which can lead to cataract and retina degeneration. Mostly, the past studies were focused on the causation of cataract but less on the mechanism of retina degeneration by NAP, elucidating the effect of changing retinal physiology is still needed. Generally, previous studies revealed that NAP caused ocular lesions may be derived from P450 metabolism, and suspected its metabolite naphthoquinone (NQ) is mainly induced cytotoxicity and cellular repair process which lead to ocular damage, but the underlying mechanism is still unclear. The pro-inflammatory response is known as one of the cell repair preliminary reaction; therefore, this study focused on NAP-induced pro-inflammatory factor on affecting CYP2F1 expression, the axis may involve in retinal lesion within NAP exposure. In this study, the retinal pigment epithelium (RPE) cells treated with dose- and time- dependent of NAP and its metabolite 1,4-naphthoquinone (1,4-NQ). Our results showed that 1,4-NQ decreased the retinal pigment epithelium (RPE) cell viability within 10 μM, but not NAP. Both NAP and 1,4-NQ up-regulated the phosphorylation of p65 which lead to increase nuclear translocation and NF-κB activation in RPE cells. NAP also increased such pro-inflammatory factor ICAM-1 dependently. Except for 1,4-NQ the down-regulation of ICAM expression, the similar effect was found by 1,4-NQ-treated RPE cells. Furthermore, we confirmed that NAP up- regulated CYP2F1 but unaffected both CYP1A1 and CYP1B1 on mRNA and protein expression; in contrast to NAP, 1,4-NQ has no change on CYP2F1 expression. Interestingly, the NF-κB activation inhibitor, BAY11-7082 not only inhibited NF-κB, but decreased both SP1 and CYP2F1 protein expression, and the activity of SP1. Moreover, the inhibition of NF-κB decreased NAP-induced SP1-p65 protein interaction, either nuclear translocation of SP1, these were regulated through MAPK signaling regulation. Taken together, we revealed that low level of NAP exposure could up-regulate CYP2F1 via activation of NF-κB/SP1 axis, which may contribute to NAP-induced retina lesion. As further studies are needed, this study propose a possible regulation of NAP-induced inflammatory effect during earlier stages for preventing macular degeneration. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74308 |
DOI: | 10.6342/NTU201902837 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 毒理學研究所 |
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