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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48723

Title: 探討一氧化氮經由調節IKK與HSP90交互作用
Authors: Lee, Ming-Yung
李明勇
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: 一氧化氮;熱休克蛋白;氣喘;激酶IκB;轉錄因子NF-κB
Nitric oxide;Heat shock protein 90;Asthma;IκB kinase;Nuclear factor κB
Date: 2014
Issue Date: 2018-08-22T06:30:44Z
Abstract: 中文摘要 氣喘是一種現代常見的慢性呼吸道發炎疾病,不僅影響到生活品質,發作時可能相當嚴重甚至會致命,是世界各國重要的大眾健康問題。當氣喘病人遇到各種內生性或外源性的刺激時,便會導致過度的支氣管收縮以及支氣管黏膜的發炎反應。臨床上發現,氣喘的病患罹患肺炎的比例及吐氣一氧化氮(nitric oxide, NO)濃度相較一般人高。人體中一氧化氮扮演許多重要的生理的角色,而且具有免疫調節的功能。 巨噬細胞(macrophage)在先天免疫反應上有著重要的角色,在氣喘病患氣管沖洗液中巨噬細胞會明顯增加,也是氣喘呼吸道慢性發炎病因之一。Lipopolysaccharide (LPS)為葛蘭氏陰性菌細胞壁之成份,以LPS處理巨噬細胞,會誘導巨噬細胞發炎反應釋放發炎前趨物質。當巨噬細胞活化時會分泌許多物質參與免疫發炎反應,例如細胞激素及一氧化氮等。一氧化氮在先天免疫上是重要分子,也會調節LPS引發的巨噬細胞內的細胞核轉錄因子NF-κB(nuclear factor kappa B)路徑發炎反應,可能間接也會影響巨噬細胞的免疫抗菌能力。 熱休克蛋白Hsp (heat shock protein, HSP)功能大多是在於保護細胞內重要蛋白質。細胞在壓力下,如遭遇高溫、缺氧、發炎等情況,均會產生熱休克蛋白,此為細胞因應環境變化以求得生存的反應方式。同時熱休克蛋白Hsp90參與了LPS引發的NF-κB發炎反應的調控,IKK (IκB激酶)也需要Hsp90的協同作用,否則不穩定的IKK容易被蛋白酶體所分解而失去作用。研究發現在嚴重氣喘病患,Hsp90會明顯升高,這表示Hsp90蛋白與氣喘的發炎亦有相關。 本研究即在了解氣喘病患高感染率的可能原因,是否與巨噬細胞內的NF-κB發炎路徑受到高濃度一氧化氮抑制有關,或者是因一氧化氮影響巨噬細胞先天免疫反應所致。所以本實驗利用藥物LPS處理RAW 264.7巨噬細胞以及卵蛋白(ovalbumin)致敏氣喘鼠動物模式,探討一氧化氮影響LPS所導致NF-κB發炎反應的可能機轉。實驗結果發現使用一氧化氮生成藥物(SNAP)處理致敏氣喘鼠後,發現動物氣管沖洗液中的發炎細胞激素減少;然而使用一氧化氮合成酶抑制藥物(1400W)處理後,動物氣管沖洗液中的發炎細胞激素,反而增加,說明一氧化氮確實可以抑制實驗鼠的氣管發炎反應。為了解一氧化氮影響巨噬細胞發炎反應的分子機轉,我們使用LPS、SNAP及1400W處理RAW 264.7細胞。實驗發現一氧化氮可減少LPS引起的細胞發炎反應、抑制NF-κB路徑、及減少發炎前趨激素的分泌。藉由免疫沉澱法證實,一氧化氮可能藉由干擾細胞內Hsp90與IKK (IκB激酶)的交互作用,進而減少IKK的活化與表現,使得NF-κB表現受到抑制。在動物鼠肺部的組織免疫染色切片實驗亦證實,用SNAP處理動物,其組織切片中IKK呈色單核球數量減少,反而使用1400W處理,IKK呈色單核球數量增加,說明一氧化氮可減少實驗鼠肺部的發炎及IKK量。目前已知一氧化氮有數個抑制NF-κB路徑的機制,而我們首次提出一氧化氮可藉由減少Hsp90與IKK之交互作用,進而降低IKK的穩定性,導致其發炎反應受到抑制。這個實驗結果可進一步解釋一氧化氮抑制LPS所引發的巨噬細胞免疫發炎反應而影響了先天免疫能力,這有可能是導致氣喘病患肺部容易感染病菌的原因之一。 實驗結論說明在巨噬細胞及致敏氣喘鼠動物的模式中,一氧化氮可以藉由減少Hsp90與IKK之相互作用,進而抑制LPS誘導的發炎反應。 關鍵字:一氧化氮、熱休克蛋白、氣喘、激酶IκB、轉錄因子NF-κB
Abstract Asthma is a common disease of chronic airway inflammation and affects the life quality of the patients with this disease. Seriously, it can be life-threatening if attacks without an immediate and proper treatment. Therefore, asthma has become a worldly concerned issue in public health. Clinical evidence shows that patients with asthma have a higher possibility of lung infections and higher levels of exhale nitric oxide (NO) when compared to patients without asthma, and the level of NO is higher in accordance with asthma severity. Nitric oxide also suppressed NF-κB inflammatory reaction. Lipopolysaccharide (LPS) is a component of cell-wall of Gram-negative bacteria. Once infected, host may release a battery of inflammatory substances reacting to LPS from invading bacteria. In this scenario, macrophage plays an essential role in detecting and activating immune cascades. In vitro, pretreated LPS can activate macrophages to secret an array of inflammatory substances, including cytokines and NO. NO, an important effector molecule of innate immune system, can also regulate LPS induced NF-κB inflammatory pathway. The important function of heat shock protein (Hsp) is to protect key protein in cells. The level of Hsp increases when cells are in stressed condition, such as hot, anoxia and inflammation. This is the survival method of cells responding to stressed environment. Hsp90 is also a crucial regulator of LPS induced NF-κB inflammatory pathway. This study is to elucidate the relationship between high pneumonia morbidity in asthma and nitric oxide suppressed NF-κB pathway. In this study, we examined the role of NO in lipopolysaccharide (LPS)-induced inflammation in an ovalbumin (OVA)-challenged mouse asthma model and RAW 267.4 cells. To determine whether NO affected the LPS-induced response, an NO donor (S-nitroso-N-acetylpenicillamine, SNAP) or a selective inhibitor of NO synthase (1400W) was injected intraperitoneally into the mice before the LPS stimulation. Decreased levels of proinflammatory cytokines were demonstrated in the bronchoalveolar lavage fluid of mice treated with SNAP, whereas increased levels of cytokines were found in the 1400W-treated mice. To further explore the molecular mechanism of NO in inhibition of proinflammatory responses in macrophages, RAW 264.7 cells were treated with 1400W or SNAP before LPS stimulation. LPS-induced inflammation in the cells was attenuated by the presence of NO. The LPS-induced IκB kinase (IKK) activation and the expression of IKK were reduced by NO through attenuation of the interaction between Hsp90 and IKK in the cells. The IKK decrease in the lung immunohistopathology was verified in SNAP-treated asthma mice, whereas IKK increased in the 1400W-treated group. We report for the first time that NO attenuates the interaction between Hsp90 and IKK, decreasing the stability of IKK and causing the down-regulation of the proinflammatory response. Furthermore, the results suggest NO may repress LPS-stimulated innate immunity in asthma patients. Keywords: Nitric oxide, Heat shock protein 90, Asthma, IκB kinase, Nuclear factor κB
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0D97360004.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48723
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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