Pheophytin a抑制內毒素刺激之巨噬細胞Nitric Oxide Synthase-2合成

Inhibition of Endotoxin-Induced Nitric Oxide Synthase-2 Synthesis by Pheophytin a in Macrophages

林俊祐(Chun-Yu Lin)

指導教授 ： 陳彥旭

高雄醫學大學/醫學院/醫學研究所/碩士(2009年)

https://doi.org/10.6832/KMU.2009.00014

摘要

敗血症，是指因感染症而導致的全身系統性發炎反應，它的發生率與死亡率都很高。導致敗血症的微生物，最常見的是革蘭氏陰性菌，其細胞壁富含脂多醣體，又稱內毒素，是啟動與媒介敗血症相關發炎反應的重要物質。根據已往的研究發現，敗血症患者體內的氧化壓力明顯增加，其中，一氧化氮更是具有強大氧化能力的物質；而第二型一氧化氮合成酶，乃是使一氧化氮大量產出的重要酵素。因此，如何使敗血症患者體內一氧化氮的產生下降，進而減低氧化壓力，來改善患者的預後，是治療敗血症的一項合理策略。
　　根據初步的研究結果，我們發現一種綠茶非多酚類的萃取物pheophytin a，可以減少受到脂多醣體刺激而活化的巨噬細胞產生第二型一氧化氮合成酶，因而減少一氧化氮的生成。第二型一氧化氮合成酶的基因表現，主要在基因轉錄與轉錄後的層面調控。轉錄因子NF-κB可以結合在第二型一氧化氮合成酶基因的上游，並促進其表現。我們發現，pheophytin a並不能影響NF-κB轉位進入細胞核的情況，但是pheophytin a卻能影響第二型一氧化氮合成酶之訊息RNA的穩定性，最終減少訊息RNA的量，並降低第二型一氧化氮合成酶的生合成。另外，我們也進一步發現，pheophytin a可以增加ERK pathway磷酸化的情形，這樣的效應可能會負向地調控NF-κB相關的基因表現。這兩者都是pheophytin a可能的作用機轉。
　　透過我們的實驗，pheophytin a的安全性，與其抑制受到脂多醣體刺激的巨噬細胞產生一氧化氮的效果，得到證實，且其作用的分子機轉也得到部份的澄清。

關鍵字

敗血症 ； 第二型一氧化氮合成酶pheophytin a ； NF-κB ； ERK pathway ； 訊息RNA穩定性

並列摘要

Sepsis is a systemic inflammatory response to infection. Its incidence and mortality rate were high. The leading pathogen of sepsis is Gram-negative bacilli, which had lipopolysaccharide (endotoxin) in its cell wall. Endotoxin is an important mediator to induce inflammation associated with sepsis and septic shock. The oxidative stress increased significantly in patients with sepsis and nitric oxide (NO) is a potent substrate of oxidative reaction. The nitric oxide synthase-2 (NOS2) is the major enzyme leading to high-output NO production. It is a reasonable strategy to decrease NO production and oxidative stress in patients with sepsis and septic shock.
Pheophytin a, a substance in the non-polyphenolic fraction of green tea (Camellia sinensis), can inhibit the synthesis of NOS2 protein in endotoxin-stimulated murine macrophage RAW264.7 cells, leading to decreased NO production. NOS2 is primarily regulated at the expression level by transcriptional and post-transcriptional mechanisms. The transcriptional factor, NF-κB, regulated NOS2 expression primarily. Pheophytin a can not decrease NF-κB translocation from cytosol into nucleus, but can impact the NOS2 mRNA stability and finally decrease the mRNA and protein synthesis. Pheophytin a can also increase ERK phosphorylation, which might negatively regulate NF-κB-dependent gene expression. These are possible mechanisms of pheophytin a.
In conclusion, pheophytin a is a safe and potent substance which can inhibit NO production in endotoxin-stimulated macrophage. We also analyzed its molecular mechanism partially.

並列關鍵字

sepsis ； nitric oxide synthase-2 ； pheophytin a ； NF-κB ； ERK pathway ； mRNA stability

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