查耳酮之新衍生物AN-07在抑制人類主動脈平滑肌細胞增生及細胞激素基因表達的作用及機制探討

The effect and molecular mechanisms of chalcone derivative AN-07 on the inhibition of human aorta smooth muscle cell proliferation and cytokine gene expression

張朕佳(Chen-Chia Chang)

指導教授 ： 卓夙航

高雄醫學大學/醫學院/醫學研究所/碩士(2009年)

摘要

動脈粥狀硬化為一種漸進性並且伴隨著發炎反應的疾病，會引起心肌梗塞、腦中風等病變，已經有研究證明氧化型低密度脂蛋白（ox-LDL）會刺激血管平滑肌細胞的增生及分泌細胞激素而造成動脈粥狀硬化。查耳酮（Chalcone）是類黃酮的其中一種，許多文獻表示類黃酮具有許多功能（抗氧化、抗發炎等）。本篇論文中想要探討利用天然植物萃取之chalcone類衍生物AN-07是否能夠抑制人類主動脈平滑肌細胞的增生及細胞激素的產生？是經由哪些機制所調控？
在本研究中使用WST-1 assay偵測細胞增生率、利用免疫螢光染色觀察核增生抗原(Ki-67)之表現、以西方墨點法觀察ERK 1/2訊息傳遞路徑及細胞週期蛋白的表現，用即時定量聚合酶連鎖反應觀察發炎基因的表現。結果發現30 ug/ml之ox-LDL處理人類主動脈平滑肌細胞，可以增加細胞增生率，增加核增生抗原之表現，透過活化ERK 1/2訊息傳遞路徑，增加細胞週期蛋白cyclin D1及cyclin D3表現使細胞增生；並且也可以觀察到細胞激素IL-6、IL-8及IL-1B的表現。當使用5 ug/ml AN-07處理細胞後，可以發現明顯的抑制細胞增生率，減少核增生抗原的表現，並且抑制被活化的ERK 1/2訊息傳遞路徑、降低細胞週期蛋白cyclin D1及cyclin D3表現，以及降低細胞激素IL-6、IL-8及IL-1B的表現；除此之外，研究結果中另外也發現到AN-07能夠增加PPARγ的基因和蛋白質表現量，並且在與PPARγ之ligand（Rosiglitazone）合併使用時會有好的抑制效果。
本研究的結論為在動脈粥狀硬化的過程中，查耳酮衍生物AN-07能經由抑制ERK 1/2的磷酸化、cyclin D1及cyclin D3蛋白質表現增加來預防人類主動脈平滑肌細胞的增生，並透過調控ERK 1/2及PPARγ來抑制細胞激素基因的表現，因而減少動脈粥狀硬化或是血管再狹窄的發生。

關鍵字

氧化型低密度脂蛋白 ； 人類主動脈平滑肌細胞 ； 細胞增生 ； 細胞激素 ； 查耳酮

並列摘要

Atherosclerosis is a progressive disease involved in inflammatory response, which will cause the myocardial infarction and stroke. Evidences demonstrate the oxidized LDL can induce vascular smooth muscle cell proliferation and inflammation. Chalcone is a kind of flavonoids. Many researches show the flavonoids have variety biological function such as antioxidant and anti-inflammatory effects. In the present study, we attempt to investigate the effect and the molecular mechanism of AN-07 on human aorta smooth muscle cell (HASMC) proliferation and inflammatory gene expression.
We examined cell proliferation by using WST-1 assay and by detecting the expression of nuclear proliferation marker (Ki-67). We measured ERK 1/2 phosphorylation and cell cycle protein expression by western blotting. We also detected the expression of inflammatory gene IL-6、IL-8、IL-1B by real-time PCR.
Ox-LDL (i.e. 30 ug/ml) induces cell proliferation, increases the Ki-67 expression in nuclear, activates Erk 1/2 signal pathway, upregulates cyclin D1 and cyclin D3 protein expression, and elevates gene expression of IL-6, IL-8, and IL-1B in HASMC. AN-07 (i.e. 5 ug/ml) could prevent the cell proliferation, reduce the Ki-67 expression in nuclear, inhibit the activation of ERK 1/2 signal pathway, decrease the cell cycle protein cyclin D1 and cyclin D3 expression, downregulate IL-6, IL-8, and IL-1B mRNA expression. In addition, we observe that AN-07 can upregulate the PPARγ expression at gene and protein levels. When HASMC treated with AN-07 combine the PPARγ ligand (Rosiglitazone, i.e. 5 ug/ml) indicates a better efficiency than treated with AN-07 or Rosiglitazone alone.
Our results suggest that AN-07 prevents HASMC cell proliferation via inhibition of ERK 1/2, cyclin D1 and cyclin D3, and reduces inflammatory gene expression via regulation of Erk 1/2 and PPARγ. AN-07 may be a new candidate for the prevention and treatment of atherosclerosis and restenosis.

並列關鍵字

ox-LDL ； smooth muscle cell ； proliferation ； cytokine gene ； chalcone ； MAPK pathway

參考文獻

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