Beraprost抑制老鼠主動脈平滑肌細胞增生分子機制之研究

Anti-proliferative effects of rat aortic smooth muscle cells by Beraprost, a PGI2 analogue, through the induction of PPARδ and its downstream effector of iNOS

李嘉玲(Ja-Ling Lee)

指導教授 ： 阮淑慧

臺北醫學大學/醫學院/醫學科學研究所/碩士(2006年)

摘要

前列環素（prostacyclin, PGI2）已有文獻指出，不論是在人類或者是動物的模式上，對於改善動脈粥狀硬化有其有益的作用。我們利用具有保護血管功能的貝前列素鈉（beraprost sodium, BPS）（一種前列環素的相似物）在老鼠主動脈平滑肌細胞中來探討PGI2抑制動脈粥狀硬化的分子機轉。將細胞以BPS處理，利用同位素標定來觀察細胞增生，結果顯示BPS可以抑制由TPA所誘導的平滑肌細胞增生情形。分別再以反轉錄?-聚合?連鎖反應（RT-PCR）、西方墨點法和亞硝酸鹽分析（nitrite assay）的方法分析，則發現BPS會增加過氧化體增生活化受體δ（peroxisome proliferator- activated receptor, PPARδ）和誘導型一氧化氮生成?（inducible nitric oxide synthase, iNOS）的表現以及一氧化氮（nitric oxide, NO）的生成。此外，若以PPARδ的拮抗劑和iNOS抑制劑處理，則可以反轉由BPS誘導PPARδ及iNOS的表現，抑制細胞增生情形。有趣的是，PPARδ的拮抗劑會降低iNOS的表現，因此我們假設iNOS的表現可能會受到PPARδ的調節。除此之外，由luciferase的活性分析結果發現BPS可以促進iNOS基因啟動子的活性表現。另外，刪除iNOS基因啟動子區域上PPARδ的結合位置-1245 ~ -1222，則會降低luciferase的活性。這個結果顯示，位於老鼠iNOS啟動子上PPARδ的結合位置將是藉由PGI2調節iNOS基因表現的一個很重要的機制。綜合以上結果，我們發現PPARδ可藉由BPS而活化，調控iNOS的表現，並涉及ㄧ連串的訊息傳導路徑，達到抑制平滑肌細胞的增生，為臨床上預防動脈粥狀硬化的發生，提供一個很好的治療方向與目標。

關鍵字

前列環素 ； 貝前列素鈉 ； 過氧化體增生活化受體δ ； 誘導型一氧化氮生成?

並列摘要

Prostaglandin I2 (prostacyclin, PGI2) has been reported to exhibit beneficial effects on atherosclerosis in both human and animal models. To clarify the underlying molecular mechanism, we investigated the vasoprotective effects of beraprost sodium (a PGI2 analogue) in primary culture of rat aortic smooth muscle cells (RASMC). Cells treated with beraprost sodium can reverse the effect of TPA-induced cell proliferation by [3H]thymidine incorporation experiments and increased the PPARδ and iNOS expression as assessed by RT-PCR, Western blots, and NO production by the Griess method. Additionally, PGI2, in accompanied with the induction of PPARδ and iNOS is involved in the anti-proliferative effects of PGI2 since PPARδ antagonists and NO inhibitors abolished the anti-proliferative effects of PGI2. Interestingly, PPARδ antagonists inhibited the induction of iNOS, suggesting PPARδ regulates iNOS expression. Additionally, beraprost sodium increased the promoter activity of rat iNOS gene, as determined by luciferase assays. Deletion of PPARδ binding site located at -1245 to -1222 in the promoter region strongly reduced luciferase activity. The presence of functional PPARδ sites within rat iNOS promoter may represent a novel mechanism for regulating iNOS gene expression by PGI2.Accrodingiy, we investigated that PPARδ up-regulate iNOS expression by BPS activation , and it involved a series of signal transduction pathway to have inhibitory effect on RASMC proliferation. It will provide clinically a new direction and therapeutic target to prevent the incidence of atherosclerosis.

並列關鍵字

prostacyclin ； beraprost sodium ； PPARδ ； iNOS

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