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標題: | 研究Eupafolin和山苦瓜對腫瘤壞死因子刺激肺臟發炎之影響及相關機轉 To study the protective effects of eupafolin and wild bitter gourd on TNF-α-induced lung inflammation and the related mechanisms |
作者: | Hsin-Ching Sung 宋欣錦 |
指導教授: | 陳玉怜(Yuh-Lien Chen) |
關鍵字: | eupafolin,山苦瓜,發炎,細胞黏附因子-1,MAPKs,miR-221/-222,PI3K/AKT,NF-κB, eupafolin,WBGE,inflammation,ICAM-1,MAPKs,miR-221/-222,PI3K/AKT,NF-κB, |
出版年 : | 2017 |
學位: | 博士 |
摘要: | 在肺臟相關的發炎疾病中,白血球會經由與呼吸道上皮細胞的細胞黏附因子黏著而移動,因此細胞黏附因子對發炎疾病具有重要的功能。Eupafolin是一種類黃酮,也是鴨舌癀(Phyla nodiflora)中的主要活性物質,具有抗發炎的能力。另外,山苦瓜萃取物也具有許多藥理上的活性。本文主要目的在探討,在經過TNF-α刺激肺泡上皮細胞、C57BL/6小鼠以及miRNA-221/222基因剔除小鼠中,eupafolin及山苦瓜萃取物對細胞黏附因子表現的影響。研究中首先利用西方點墨法及免疫螢光染色法,觀察eupafolin及山苦瓜萃取物對TNF-α刺激A549細胞後ICAM-1及相關蛋白表現的影響。另外小鼠以腹腔注射eupafolin 3天(第一部分),或是給予C57BL/6小鼠及miRNA-221/222基因剔除小鼠口服5天山苦瓜萃取物(第二部分)後, 再以插管方式給予TNF-α 1天後取出肺臟。接著再以西方點墨法及組織免疫染色觀察ICAM-1表現的改變。第一部分實驗結果顯示,eupafolin確實可降低因TNF-α刺激引起的ICAM-1表現,而此作用是經由抑制ERK1/2、JNK、p38和AKT/PI3K的磷酸化。然而,加入p38和PI3K的抑制劑並不會改變ICAM-1的表現。再者,eupafolin同時降低了TNF-α 所引起之NF-κB的活化及核轉移。在小鼠肺臟組織中,受TNF-α刺激而表現量增加的ICAM-1會受到eupafolin的抑制。第二部分實驗結果顯示,在A549細胞中,山苦瓜萃取物確實可經由抑制PI3K/AKT/NF-κB/IκB的磷酸化作用而減緩因TNF-α引起的ICAM-1表現,並且減少了白血球的黏附作用。除此之外,山苦瓜萃取物也會降低內生性的ICAM-1表現且會增加miRNA -221/-222的表現。讓細胞過度表現miRNA 222也可降低PI3K/AKT/NF-κB/IκB的磷酸化及ICAM-1表現量與白血球的黏附作用。另外,在小鼠肺臟組織中,山苦瓜萃取物可抑制TNF-α刺激或沒刺激而表現的ICAM-1以及增加miRNA -221/-222的表現;但並不影響miRNA-221/222基因剔除小鼠之miRNA-221/-222但些微影響ICAM-1的表現。此結果顯示,eupafolin及山苦瓜萃取物在細胞及動物實驗中皆可降低ICAM-1的表現。Eupafolin可降低因TNF-α引起的ICAM-1表現以及白血球的黏附作用,且是經由抑制AKT/ERK1/2/JNK的磷酸化作用以及NF-κB的核轉移所致;而山苦瓜萃取物則是經由miR-221/-222/PI3K/AKT/NF-κB這條路徑來控制。因此,eupafolin及山苦瓜萃取物也提供了另一種治療肺臟發炎相關疾病的藥物的另一種新選擇。 The deregulation of cell adhesion molecules associated with the epithelium-leukocyte interaction plays the important role in the pathogenesis of lung airway inflammatory disorders. Eupafolin, a major bioactive compound found in Phyla nodiflora, has been reported to have the anti-inflammatory property. In addition, the extracts from wild bitter gourd fruit (WBGE) also possess numerous pharmacological activities. The purpose of this study was to investigate the effects of eupafolin or WBGE on intercellular adhesion molecule-1 (ICAM-1) expression in epithelial cells, C57BL/6 wild-type (WT) mice or microRNA (miR)-221/-222 knockout (KO) mice with or without tumor necrosis factor-α treatment and the related mechanisms. At first, the effects of eupafolin and WBGE on ICAM-1 expression and the related signals in A549 cells were examined by western blot and immunofluorescent staining. The part of the manuscript is the mice were injected intraperitoneally with or without eupafolin and then left untreated or injected intratracheally with TNF-α. The part section of them, WT mice and miR-221/-222 KO mice were orally treated with or without WBGE and then left untreated or injected intratracheally with TNF-α. The expression levels and patterns of ICAM-1 in the lung tissues were examined by western blot and immunohistochemical staining. In part one, eupafolin pretreatment reduced the TNF-α-induced ICAM-1 expression and also the ERK, JNK, p38, and AKT/ PI3K phosphorylation. However, the increase in ICAM-1 expression with TNF-α treatment was unaffected by p38 and PI3K inhibitors. Moreover, eupafolin decreased the TNF-α-induced NF-κB p65 activation and its nuclear translocation. Furthermore, eupafolin reduced ICAM-1 expression in the lung tissues of TNF-α-treated mice. In part two, WBGE significantly decreased the TNF-α-induced ICAM-1 expression in A549 cells through the inhibition of PI3K/ AKT/ NF-κB /IκB phosphorylation and decreased leukocyte adhesion. In addition, WBGE reduced endogenous ICAM-1 expression and upregulated miR-221/-222 expression. The overexpression of miR-222 decreased PI3K/AKT/NF-κB/IκB phosphorylation and ICAM-1 expression, which resulted in reducing monocyte adhesion. Moreover, WBGE reduced ICAM-1 expression in lung tissues of WT mice with or without TNF-α treatment and upregulated miR-221/222. WBGE did not affect the miR-221/-222 level and had little effect on ICAM-1 expression in miR-221/-222 KO mice. These results suggest that eupafolin and WBGE reduced ICAM-1 expression both under in vitro and in vivo conditions. The protective effects of eupafolin were mediated via AKT/ERK1/2/JNK phosphorylation and nuclear translocation of NF-κB. Furthermore, WBGE were mediated partly through the miR-221/-222/PI3K/AKT/NF-κB pathway. Therefore, eupafolin and WBGE may represent novel therapeutic agents targeting epithelial activation in lung inflammation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/2439 |
DOI: | 10.6342/NTU201701930 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 解剖學暨細胞生物學科所 |
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