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腺苷酸活化蛋白激酶活化对单核细胞-内皮细胞黏附的影响及其机制

白洪波, 王昀, 张玉华, 张园

广州医科大学基础医学院生理教研室，广州 511436；广州军区总医院心血管内科，广州 510010

摘要

本研究旨在探讨腺苷酸活化蛋白激酶(AMP-activated protein kinase, AMPK)活化对单核细胞与内皮细胞黏附的影响及其分子机制。用不同剂量的AMPK激动剂5-氨基咪唑-4-甲酰胺核糖核苷酸(AICAR, 0~2 mmol/L)或AMPK抑制剂compound C (10 mmol/L)处理肿瘤坏死因子α (tumor necrosis factor α, TNFα, 10 ng/mL)诱导的人主动脉内皮细胞(human aortic endothelial cells, HAECs)，用TNFα诱导过表达活性型或显性抑制型AMPK蛋白的HAECs。用荧光染色法观察AMPK对荧光标记的单核THP-1细胞与HAECs黏附的影响。用荧光定量PCR检测血管细胞黏附分子1 (vascular cell adhesion molecule-1, VCAM-1)和细胞间黏附分子1 (intercellular cell adhesion molecule-1, ICAM-1) mRNA表达水平，用ELISA法检测二者的蛋白分泌量；用Western blot检测核因子-kappa B (nuclear factor-kappa B, NF-κB) p65的211位点赖氨酸乙酰化水平，用ELISA法检测NF-κB p65 DNA结合活性，并用试剂盒检测p300乙酰转移酶活性。通过小干扰RNA抑制HAECs组蛋白乙酰转移酶p300蛋白表达后，检测TNFα对NF-κB p65 DNA结合活性、黏附分子ICAM-1、VCAM-1的表达及单核细胞黏附率的影响。结果显示，AICAR显著抑制TNFα诱导的单核细胞与HAECs的黏附，在HAECs中下调TNFα诱导的ICAM-1、VCAM-1的mRNA水平上调和蛋白分泌。AICAR的效应可以被AMPK抑制剂compound C完全阻断。转染活性型AMPKα显著抑制TNFα诱导的ICAM-1、VCAM-1 mRNA表达和分泌，以及单核细胞-内皮细胞黏附，而转染显性抑制型AMPKα则无明显影响。RNAi干预抑制p300活性显著抑制TNFα诱导的黏附分子表达和单核-内皮细胞黏附。AMPK激活可抑制TNFα诱导的p300乙酰转移酶活性，抑制NF-κB p65的211位赖氨酸的乙酰化，降低NF-κB p65 DNA结合活性。以上结果提示，AMPK激活抑制单核细胞-内皮细胞黏附，作用机制可能与其降低p300酶活性，下调NF-κB p65转录活性密切相关。

关键词： 腺苷酸活化蛋白激酶; 内皮细胞 ; 细胞黏附分子 ; 核因子-κB ; p300

分类号：R32

AMP-activated protein kinase activation regulates adhesion of monocytes to vascular endothelial cells and the underlying mechanism

BAI Hong-Bo, WANG Yun, ZHANG Yu-Hua, ZHANG Yuan

Department of Physiology, School of Basic Science, Guangzhou Medical University, Guangzhou 511436, China； Department of Cardiology, General Hospital of Guangzhou Military Command of People’s Liberation Army, Guangzhou 510010, China

Abstract

The present study was aimed to explore the effect of AMP-activated protein kinase (AMPK) on monocyte adhesion to vascular endothelial cells and underlying molecular mechanism. Tumor necrosis factor α (TNFα)-activated human aortic endothelial cells (HAECs) were treated with different concentrations of AMPK agonist 5-Aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) or AMPK inhibitor compound C. And other HAECs were overexpressed with constitutive active or dominant negative AMPK protein and then treated with TNFα. The rates of monocytes adhering to endothelial cells were detected by fluorescent staining. Intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA levels and protein secretions were detected by quantitative PCR and ELISA, respectively. Acetylation of NF-κB p65 at lysine 221 site was assessed by Western blot. NF-κB p65 DNA binding activity was analyzed by an ELISA-based method. By using small interfering RNA based strategy, p300 expression in HAECs was down-regulated and then cells were incubated with TNFα. NF-κB p65 DNA binding activity, ICAM-1 and VCAM-1 expressions and adhesion rates were detected, respectively. The activity of p300 was also detected by ELISA. The results showed that AICAR treatment significantly reduced monocyte-endothelial adhesion rate, as well as ICAM-1 and VCAM-1 mRNA levels and protein secretions, in TNFα-activated HAECs. Moreover, transfection of constitutive active AMPKα but not dominant negative AMPKα strongly diminished TNFα-induced upregulation of ICAM-1 and VCAM-1 mRNA expressions and secretions, as well as monocyte-endothelial adhesion. Furthermore, AMPK activation decreased TNFα-mediated acetylation of NF-κB p65 at Lys221 site and reduced NF-κB p65 DNA binding activity. Silencing p300 by siRNA significantly abolished the effect of TNFα- induced adhesion molecules expression and monocyte-endothelial adhesion. Blocking AMPK activation by compound C almost completely reversed the effect of AICAR exerted on HAECs. These results suggest AMPK activation suppresses monocyte-endothelial adhesion, and the underlying mechanism is relevant to the inhibition of p300 activity and NF-κB p65 transcriptional activity.

Key words: AMP-activated protein kinase; endothelial cell ; intercellular cell adhesion molecule-1 ; vascular cell adhesion molecule-1 ; NF-κB

收稿日期：2015-08-11　　录用日期：2015-11-25

通讯作者：张园　　E-mail: yuanzhang_1980@163.com

引用本文：

白洪波, 王昀, 张玉华, 张园. 腺苷酸活化蛋白激酶活化对单核细胞-内皮细胞黏附的影响及其机制[J]. 生理学报 2016; 68 (1): 41-49.

BAI Hong-Bo, WANG Yun, ZHANG Yu-Hua, ZHANG Yuan. AMP-activated protein kinase activation regulates adhesion of monocytes to vascular endothelial cells and the underlying mechanism. Acta Physiol Sin 2016; 68 (1): 41-49 (in Chinese with English abstract).