茶鹼衍生物對大鼠氣管平滑肌細胞生長，轉移及細胞激素引起基質金屬蛋白酶表現之影響

EFFECTS OF GROWTH, MIGRATION AND MATRIX METALLOPROTEINASE BY A XANTHINE DERIVATE IN RAT TRACHEAL SMOOTH MUSCLE CELLS

許銘泰(Ming-Tai Hsu)

指導教授 ： 陳英俊

高雄醫學大學/醫學院/藥理學科研究所/碩士(2007年)

摘要

基質金屬蛋白酶（matrix metalloproteinase）九型以及十二型在呼吸道發炎的發展過程中，扮演了極為重要的角色。之前的研究已指出黃嘌呤衍生物 KMUP-1 具有抗前發炎作用 TNF-α 的能力，其主要是藉由抑制細胞生長激素引起的 iNOS 表現，並且具有活化 sGC 和 PKG 的作用。本篇論文目的在於研究在大鼠氣管平滑肌中，給予腫瘤壞死因子-α（TNF-α）所分別引起的基質金屬蛋白酶九型以及十二型的表現量。在西方點墨法的分析指出基質金屬蛋白酶九型以及十二型的表現能被 KMUP-1（1-100 μM）所減弱。
在線性傷痕分析（linear wound assay）實驗之中，我們也進行研究在給予不同的誘導因子如 TNF-α，interleukin-1β（IL-1β）和 sphingosine-1-phoshate（S1P）時，氣管平滑肌細胞的遷移（migration）情況；在大鼠氣管平滑肌的遷移實驗中，將 KMUP-1 與 Y-27632 進行比較，發現 KMUP-1 能有效的抑制由 TNF-α、IL-1β 和 S1P 所誘發的細胞遷移，但相反的，Rho kinase抑制劑 Y-27632 在相同濃度之下，不能達到與 KMUP-1 相同的抑制量，甚至有在細胞遷移早期時，有促進細胞遷移的現象產生。
此外，本實驗也證實 KMUP-1 能抑制 MAP kinase 的傳遞路徑包括了 p38，ERK1/2，JNK。在西方點墨的分析之中，我們研究 p38，ERK1/2，JNK 磷酸化的表現情況在給予單一劑量 TNF-α（100 ng/ml）5、15、30和60分鐘之後，並且選定每種蛋白表現量最多的時間點進行給藥組的實驗。而在給藥組的部份，KMUP-1 很明顯的削弱了 p38，ERK1/2，JNK磷酸化的表現情形。
綜合以上實驗發現，KMUP-1 能藉由抑制 MAP kinase 訊息傳遞路徑來抑制氣管平滑肌的遷移，且同時也能減少內生性細胞激素所引起的基質金屬蛋白酶的過度表現。由上述結果我們提出，由本實驗室所合成的 KMUP-1 ，擁有能抑制前發炎反應的作用，並且能抑制氣管平滑肌細胞因發炎介質所引起的細胞遷移。或許 KMUP-1 之後能發展成在臨床上用於治療慢性阻塞性肺病或是氣喘病人的用藥。

關鍵字

MMP-9 ； MMP-12 ； TNF-alpha ； IL-1beta

並列摘要

The matrix metalloproteinases (MMP)-9 and (MMP)-12 are involved in the development of airway inflammation. Our previous study has shown that KMUP-1 possessed the anti-proinflammation ability by inhibiting expression of cytokine-induced iNOS expression, in addition to activation of soluble guanylyl cyclase and protein kinase G (PKG). In this study, we further investigated that tumor necrosis factor-α (TNF-α) induced the expression of MMP-9 and MMP-12 in rat tracheal smooth muscle cells (TSMCs). The expression of MMP-9 and MMP-12 in cultured TSM cells were attenuated by KMUP-1 (1-100 μM), analyzed by Western blotting.
In linear wound assay, we also studied that different inducers such as TNF-α, interleukin-1β (IL-1β) and sphingosine-1-phoshate (S1P) induced migration are evident in TSMCs. KMUP-1 significantly inhibits TNF-α, IL-1β and S1P induced migration of rat TSMCs. However, Y27632, a Rho kinase inhibitor, did not reduce the migration as KMUP-1 in the same concentration, but enhanced the early migration in linear wound assay.
In addition, KMUP-1 suppressed mitogen-activate protein （MAP） kinase signals containing the p38, ERK 1/2, and JNK. In Western blotting, we also detected the level of phospho-p38, phospho-ERK 1/2, and phospho-JNK in early time period (5, 15, 30 and 60 min) induced by TNF-α (100 ng/ml). KMUP-1 significantly suppressed the levels of phosphorylated ERK 1/2, phosphorylated p38 and phosphorylated JNK.
From the results, in this study, it is proposed that KMUP-1 could suppress the migration of airway smooth muscle by inhibiting the MAP kinase signaling pathway, and also can diminish the cytokines-induced MMP-9 and MMP-12 expression. KMUP-1 possessed well capabilities to inhibit the proinflammation and airway smooth muscle migration. In conclusion, KMUP-1 may have the ability to become a valuable agent for the treatment of asthma or chronic obstructive pulmonary disease （COPD）.

並列關鍵字

基質金屬蛋白酶-9 ； 基質金屬蛋白酶-12 ； 腫瘤壞死因子-alpha ； 間白素-1beta

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