探討 1-Undecanol 對小鼠巨噬細胞 RAW264.7 的毒性機制

The mechanism of 1-Undecanol induced toxicity in RAW264.7 macrophages

楊孟蓁(Meng-Chen Yang)

指導教授 ： 楊建洲

共同指導教授 ： 關宇翔(Yu-Hsiang Kuan)

中山醫學大學/醫學科技學院/生物醫學科學學系/碩士(2022年)

https://doi.org/10.6834/csmu202200026

摘要

1-Undecanol (1-U) 是一種脂肪酸為無色微溶於水液體，具柑橘味香氣可作為食品添加劑。1-U 會刺激眼睛、皮膚與肺，與乙醇具相近毒性作用，且 1-U 會與烹煮食物時的油煙一同產生，與 oleic acid 和 9-fluorenone 作為食用油烹飪時排放油煙之標誌物。目前對 1-U 毒性作用機制尚待闡明，本篇使用小鼠巨噬細胞 RAW264.7 細胞進行實驗，研究 1-U 對巨噬細胞的毒性作用機制。通過實驗結果可發現 1-U 誘導細胞 ROS 表現量上升，並且對抗氧化酶 catalase (CAT)、superoxide dismutase (SOD) 及 glutathione (GSH) 活性造成影響。此外透過 MTT、LDH 及 annexin V/PI 三項實驗結果，可知 1-U 會造成 RAW 264.7 細胞產生細胞毒性與細胞凋亡。通過 lyso green 染色結果可發現細胞中自噬囊泡體表現量增加。藉由 western blot 對蛋白表現進行檢測，發現 Bcl-2 家族中抑凋亡蛋白 Bcl-2, Bcl-XL 表現減少，促凋亡蛋白 Bad 表現增加。MAPK 家族相關蛋白 ERK, JNK, p38 磷酸化均上升。自噬相關蛋白 LC3, p62, Beclin 1 表現量增加，並在 p62 蛋白發現異常累積現象，表示 1-U 引發 RAW264.7 細胞自噬反應失調。根據研究結果顯示，1-U 首先導致細胞氧化壓力上升並且影響抗氧化酶表現，隨後造成 RAW264.7 細胞發生細胞凋亡，此外 1-U 會誘導自噬作用失調的現象發生，通過這些原因最終造成 RAW264.7 細胞的細胞毒性導致其死亡。

關鍵字

細胞凋亡 ； 細胞自噬 ； Bcl-2 家族 ； MAPK 家族

並列摘要

1-Undecanol (1-U) appears as a water-white liquid with a floral citrus-like odor. It is used as a flavoring ingredient in foods. 1-U could be synthesis together with oily fumes during cooking. 1-Undecanol, oleic acid, and 9-fluorenone were found to be potential markers for grilling and deep-frying which are widely applied in the Western-style cooking method. To explore the effect of 1-U on macrophages. In this research used mouse macrophages RAW264.7 cells to observe the toxicity mechanism of 1-U. It was found that the expression of ROS was induced by 1-U. The activity of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH) was affected by 1-U. The cytotoxicity and apoptosis of RAW264.7 cells was be induced by 1-U. And caspase 3, 8, 9 activity was also increased. According to lyso green assay, we found autophagosome induced by 1-U. The protein expression was detected by western blot. The Bcl-2 family was affected by 1-U. Phosphorylation of the MAPK family was increased. The expression of autophagy-related proteins LC3, p62, and Beclin 1 was raised. And we found abnormal accumulation of p62, indicating that the dysregulation of autophagy in RAW264.7 cells was induced by 1-U. According to the results of this study, 1-U led to oxidative stress and affected the expression of antioxidant enzymes. Apoptosis of RAW264.7 cells was induced. In addition, 1-U induced autophagy dysregulation. Consequently, caused cytotoxicity of RAW264.7 cells.

並列關鍵字

1-Undecanol ； Apoptosis ； Autophagy ； Bcl-2 family ； MAPK family

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