Metformin 與Rimonabant (SR141716)對於TNF-α 所引起
內皮細胞發炎反應之抑制作用與機轉

研究背景根據近年的基礎研究結果顯示，血管發炎反應在動脈粥狀硬化的病程發生中扮演著相當重要的角色。而在現今可取得的治療藥物當中，口服降血糖劑metformin與抗肥胖劑rimonabant (SR141716)，都有研究報告指出其改善病人的整體代謝功能。本篇研究之宗旨在於調查metformin和rimonabant對於血管內皮細胞是否具有抗發炎的功效，以及其中相關的分子機轉。實驗方法本篇採用人類臍靜脈內皮細胞，以及TNF-α處理細胞所引起之IL-6生成和NF-κB路徑之活化，作為發炎反應的研究模式，測試metformin和rimonabant對於此發炎反應所產生的影響，同時也利用調節相關路徑的方法來找出參與其中的訊息傳遞分子。實驗結果 TNF-α促使人類臍靜脈內皮細胞的IL-6生成量增加。此誘發量與TNF-α的濃度成正相關。而NF-κB抑制劑則會阻止該誘發反應。若以metformin (100-1000 umol/L)預處理內皮細胞，會抑制TNF-α所誘發之IL-6生成，IKKα/β磷酸化，以及IκB-α的分解。此預處理metformin而減弱IκB-α分解的效果，若同時以wortmannin (PI3K抑制劑)預處理則會抵消。Metformin使AMPK的磷酸化增加，而此增幅亦為wortmannin所消除。AMPK活化劑AICRA對於TNF-α所誘發之IL-6具有與metformin相似的抑制效果。利用轉染siRNA的方式減少內皮細胞的AMPK表現量，則可阻止metformin對其抑制作用，顯示AMPK在此抑制效果中的必要性。 Rimonabant是一種CB1 antagonist。無論是以1或10 umol/L的rimonabant，預處理15, 30或60分鐘，對於TNF-α所誘發之IL-6生成皆具有顯著的抑制效果。Rimonabant同時也抑制了誘發性的IKKα/β磷酸化，以及IκB-α的分解。若在rimonabant之前預處理CB1致活劑ACEA，則能阻止前述抑制效果。PKA抑制劑H-89亦能消除此抑制作用。Rimonabant增加了細胞內的cAMP含量以及PKA調節性次單位(PKA-RII)的磷酸化，顯示PKA的活化對於rimonabant作用之必要性。預處理wortmannin則不影響rimonabant抑制TNF-α所誘發IL-6生成的效果。結論在人類臍靜脈內皮細胞，TNF-α所誘發之IL-6生成、IKKα/β磷酸化、以及IκB-α的分解，皆為metformin與rimonabant所抑制。Metformin的作用機轉與AMPK的磷酸化相關，且此AMPK磷酸化反應具有PI3K依賴性。而另一方面，rimonabant的抗發炎功效則依賴CB1受體拮抗效果以及PKA的活化。

關鍵字

動脈粥狀硬化；單磷酸腺苷致活蛋白激脢；類大麻鹼受體拮抗劑； cAMP 依賴性蛋白激脢；血管內皮細胞；發炎反應； Metformin ； Rimonabant

並列摘要

Background Recent evidence in basic research has shown that vascular inflammation plays an important role in the pathogenesis of atherosclerosis. Among currently available therapeutic agents, the oral hypoglycemic metformin and the anti-obesity rimonabant (SR141716) have been reported to improve metabolic profile of patients. The purpose of the present study was to investigate the possible anti-inflammatory effects of metformin and rimonabant on vascular endothelial cells and the related molecular mechanisms. Methods Human umbilical vein endothelial cell (HUVEC) was used for the experiments. The effects of metformin and rimonabant on TNF-α-induced IL-6 production and NF-κB pathway were investigated. Modulation of related signal transduction pathway was also performed. Results TNF-α increased IL-6 secretion by HUVEC in a concentration-dependent manner but inhibitors of NF-κB abolished the TNF-α-induced IL-6 production. Pre-treatment with metformin (100–1000 μmol/L) also inhibited TNF-α-induced IL-6 production, phosphorylation of IKKα/β and IκB-α degradation. Metformin increased phosphorylation of AMPK but wortmannin, a PI3K inhibitor, negated its effects on AMPK phosphorylation and TNF-α-induced IκB-α degradation. AICAR, a direct AMPK activator, had inhibitory effects on TNF-α-induced IL-6 production, similar to that of metformin. Transfection of siRNA against α1-AMPK eradicated the inhibitory effects of metformin on TNF-α-induced IL-6, implying the essential role of AMPK. Rimonabant at 1 and 10 μmol/L significantly inhibited TNF-α-induced IL-6 production when added at 15, 30 and 60 minutes before TNF-α treatment. Rimonabant also inhibited TNF-α-induced phosphorylation of IKK α/β and IκB-α degradation. ACEA, a CB1 agonist, added before rimonabant abolished the former effects of rimonabant. H-89, an inhibitor of PKA, abolished the inhibitory effects of rimonabant on TNF-α induced IL-6 production. Rimonabant also increased cellular cAMP contents and the phosphorylation of PKA regulatory subunit II (PKA-RII), implying the essential role of PKA activation in the inhibitory effects of rimonabant. Treatment with wortmannin did not abolish the inhibitory effects of rimonabant on TNF-α induced IL-6 production. Conclusions Both metformin and rimonabant inhibited TNF-α-induced IKKα/β phosphorylation, IκB-α degradation and IL-6 production in HUVEC. This effect of metformin was related to PI3K-dependent AMPK phosphorylation. On the other hand, the anti-inflammatory effect of rimonabant was dependent on CB1 antagonism and PKA activation.

並列關鍵字

Atherosclerosis ； AMP-activated protein kinase ； Cannabinoid receptor antagonist ； cAMP-dependent protein kinase ； Vascular endothelial cells ； Inflammation ； Metformin ； Rimonabant

參考文獻

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Metformin 與Rimonabant (SR141716)對於TNF-α 所引起內皮細胞發炎反應之抑制作用與機轉

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