- 學位論文
探討Oxazolone對人類單核球細胞株THP-1凋亡及發炎反應之機制
Study on the Mechanism of Oxazolone Induced Apoptosis and Inflammation in Human Monocytic THP-1 Cell Line
摘要
Oxazolone (4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one)是一種用來誘發接觸性皮膚炎、異位性皮膚炎及結腸炎等疾病的強效致敏性物質。我們研究發現Oxazolone可誘導人類角質細胞株(HaCaT)產生過敏反應，但對發炎反應尚不清楚，因此我們將使用人類單核球細胞株THP-1探討Oxazolone在先天免疫反應中所扮演的角色，並透過觀察細胞毒殺、細胞分化、氧化壓力、能量代謝、細胞凋亡，進一步說明Oxazolone對於THP-1細胞的影響。 實驗結果顯示Oxazolone濃度高於0.5 mM時，會降低人類單核球細胞株THP-1的存活率且明顯改變細胞形態，此外，qPCR結果顯示與M1及M2巨噬細胞相關之CD86、IL-1β、IFN-γ、TNF-α mRNA與CD163、IL-4、IL-10 mRNA的表現量增加，表示Oxazolone可誘導 THP-1細胞分化成M1或M2巨噬細胞。而在氧化壓力試驗結果顯示，Oxazolone會使活性氧化物質Hydrogen peroxide (H2O2)的產量增加時抗氧化酵素相關基因Glutathione peroxidase (GPx) mRNA表現量下降，但GCLC及GCLM mRNA表現量上升。然而調節抗氧化酵素的轉錄蛋白Nrf2的表現量下降，及抗氧化物Glutathione (GSH)含量下降情形，會造成抗氧化物及活性氧化物質之間的不平衡而最終導致氧化壓力。在能量代謝試驗發現Oxazolone所誘導的氧化壓力，會造成粒線體的損壞，使粒線體膜電位(ΔΨm)下降、ATP合成減少，並走向內在途徑的凋亡方式，且經由西方墨點法得知Cytochrome c及Bax的表現量增加及Bcl-xL、Pro-caspase 9、Pro-caspase 3的表現量下降。 本研究結果發現Oxazolone可以促進人類單核球細胞株THP-1分化成M1或M2巨噬細胞並且引起發炎反應，且透過增加細胞內氧化壓力，破壞粒線體功能，最終導致內生性途徑的細胞凋亡。
並列摘要
Oxazolone (4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one) is a hapten allergen with strong sensitizing potential to induce contact hypersensitivity, atopic dermatitis and Th2-mediated colitis. In addition, our previous study showed oxazolone could stimulate allergy response of the human keratinocyte cell line (HaCaT). Up to now, the effects of oxazolone on inflammation remain unclear; thus, we designed experiments to explore the role of oxazolone in immune-response by using human monocytic cell line THP-1 in which we analyzed the cytotoxicity, cell differentiation, oxidative stress, energy metabolism and cell apoptosis of oxazolone-treated THP-1 cells. Results showed that oxazolone at dose higher than 0.5 mM could change THP-1 cell morphology and significantly decrease cell viability. In addition, oxazolone increased the M1 and M2 associated inflammatory cytokines’ mRNA expressions including CD86, IL-1β, IFN-γ, TNF-α, and CD163, IL-4, IL-10 suggesting oxazolone might induce THP-1 cells to differentiate into M1 and M2 macrophages. Moreover, oxazolone also augmented oxidative stress by increasing H2O2 production and GCLC and GCLM mRNA expressions and decreasing glutathione peroxidase (GPx) mRNA expression, Nrf2 protein expression and glutathione (GSH) levels. Simultaneously, it decreased mitochondrial membrane potential (ΔΨm) and ATP levels. Eventually, intrinsic apoptosis was induced by accompany with the increase in cytochrome c and Bax as well as the decrease in Bcl-xL, pro-caspase 9 and pro-caspase 3 expressions. This study indicate that oxazolone induces monocyte differentiation into macrophages, stimulates inflammation, increases oxidative stress, damages mitochondria, and causes intrinsic apoptosis of THP-1 cells suggesting monocytic cells may play inflammatory role in the oxazolone-induced contact allergic response.