Metformin對於TNF-α所誘發之內皮細胞發炎反應的抑制作用及其機轉探討

Abstract

背景：糖尿病是現今盛行率及致死率都非常高的文明病，而且將近80％的第二型糖尿病病人是死於心血管疾病或中風。治療糖尿病的口服用藥很多，其中metformin的作用機轉是增加細胞對胰島素的敏感度，而且metformin在臨床試驗中證實了可以減少大血管疾病。本研究之目的在研究metformin以及其他改善胰島素阻抗的藥物是否能改善內皮細胞的發炎反應。 方法與結果：發炎因子如TNF-α會引發內皮細胞的發炎反應，像是增加ICAM-1表現、IL-6釋放等等，在動脈粥樣硬化的過程當中扮演著啟動者的角色。本研究即以TNF-α引起人類臍靜脈內皮細胞的發炎反應為研究模式。在本實驗中發現，處理TNF-α 10 ng/ml 24小時後，內皮細胞的IL-6釋放量約是未經任何處理的15倍，而metformin能抑制TNF-α誘發的IL-6釋放量，在預處理metformin 100 μM後，抑制TNF-α誘發產生之IL-6的作用就已具有統計意義（p＜0.05）；metformin 500 μM可以抑制了接近一半的IL-6釋放量；metformin 1000 μM更可以更加抑制IL-6的釋放量。當使用了NF-κB的抑制劑- CAPE (caffeic acid phenethyl ester)（25 μg/ml）與PDTC (pyrrolidine dithiocarbamate)（20 μM）後，能部份的抑制TNF-α誘發產生的IL-6，因此TNF-α誘發產生IL-6的作用與NF-κB有關，而metformin（500 μM）也可以抑制TNF-α （10 ng/ml）所誘發的IκB-α分解作用及NF-κB p65細胞核位移作用， PI3K抑制劑wortmanin（100 nM）可以減弱metformin的抑制作用。由於PI3K與metformin磷酸化/活化AMPK有關，因此推論metformin的抑制作用可能與活化PI3K及其下游的AMPK有關。另外，metformin （30μM）亦能抑制TNF-α誘發的ICAM-1表現。同樣有增加胰島素敏感度作用的PPAR-γ受器親和劑15d-PGJ2，在本篇也發現能抑制TNF-α誘發的ICAM-1表現。而與胰島素阻抗有關的脂肪激素resistin，並沒有發現能明顯的增加內皮細胞的IL-6分泌或ICAM-1表現。 結論：降血糖藥物metformin可能是藉由PI3K的途徑磷酸化/活化AMPK後，抑制TNF-α所誘發的IκB-α分解作用，進而影響NF-κB p65位移到細胞核的作用，因此能抑制TNF-α所誘導產生的IL-6與ICAM-1。由於可見，metformin對於內皮細胞的發炎反應具有保護的作用，這將可以解釋metformin在臨床上可以減少大血管疾病。

Background：Type 2 diabetes is a disease with high morbidity and mortality in modern society, and nearly 80% of deaths in those with type 2 diabetes involve cardiovascular disease or stroke. Among many oral hypoglycemic drugs, metformin lowers blood glucose level through the improvement of insulin sensitivity. Clinical trials have demonstrated that metformin is unique in protecting patients with type 2 diabetes from macrovascular disease. The aim of the present study was to investigate whether metformin and other agents involved in insulin resistance modulated inflammatory reactions in endothelial cells. Methods and Results：The proinflammatory factor TNF-α can induce inflammatory reaction in endothelial cells and promote IL-6 secretion and ICAM-1 expression. Therefore, we used TNF-α induced inflammatory reactions in HUVECs as the experimental model. In our study, the secretion of IL-6 after TNF-α (10 ng/ml) treatment for 24hr was about 15 times that of basal level. Metformin concentration-dependently inhibited TNF-α induced IL-6 secretion. When pretreated with 100 μM metformin, the secretion of IL-6 induced by TNF-α was significantly reduced when compared with positive control (10 ng/ml TNF-α only). The secretion of IL-6 after 500 μM and 1000 μM metformin pretreatment were about 50% and 40% that of positive control respectively. We found that TNF-α induced IL-6 secretion was related to NF-κB activation because NF-κB inhibitors CAPE (pyrrolidine dithiocarbamate) (25 μg/ml) and PDTC (pyrrolidine dithiocarbamate) (20 μM) partially inhibited the IL-6 secretion induced by TNF-α. Moreover, metformin (500 μM) also inhibited TNF-α (10 ng/ml) induced IκB-α degradation and NF-κB p65 translocation to the nucleus, and these inhibitory effects were blocked by a PI3K inhibitor wortmanin (100 nM). Because the phosphorylation/activation of AMPK by metformin is PI3K- dependent, we speculated that the activation of AMPK might be concerned in the inhibitory effect of metformin. Similar to IL-6 secretion, metformin (30 μM) also inhibited TNF-α induced ICAM-1 expression. PPAR-γ agonist 15d-PGJ2 is an insulin sensitizer, too. We also found that 15d-PGJ2 inhibited TNF-α induced ICAM-1 expression. Resistin is an adipokine and is related to insulin resistant. We found that it did not change IL-6 secretion or ICAM-1 expression in HUVECs. Conclusions：In our study, metformin had inhibitory effects on TNF-α induced IL-6 secretion and ICAM-1 expression in HUVECs. We speculated that these effects were due to its ability to activate AMPK through a PI3K-dependent pathway and to inhibit TNF-α induced IκB-α degradation to prevent NF-κB p65 from translocation to the nucleus. We demonstrated that metformin had protective effects on endothelial inflammation. However, its precise mechanisms of action remain to be elucidated.