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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 factorα,TNFα,10 ng/m L)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)m RNA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 p65DNA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m RNA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-1m RNA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。
This study was designed to investigate the effects of AMPK activation on the adhesion of monocytes to endothelial cells and its molecular mechanisms. Tumor necrosis factor-α (TNF-α) was treated with various doses of AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotides (AICAR, 0-2 mmol / L) or AMPK inhibitor compound C factor α, TNFα, 10 ng / m L) induced human aortic endothelial cells (HAECs). TNFα was used to induce HAECs overexpressing active or dominant inhibitory AMPK protein. Fluorescent staining was used to observe the effect of AMPK on the adhesion of fluorescent labeled mononuclear THP-1 cells to HAECs. The expression levels of vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) m RNA were detected by real-time PCR. ; Western blot was used to detect the lysine acetylation at position 211 of nuclear factor-kappa B (NF-κB) p65; NF-κB p65 DNA binding activity was detected by ELISA; Kit detects p300 acetyltransferase activity. The effect of TNFα on the NF-κB p65 DNA binding activity, the expression of ICAM-1 and VCAM-1, and the adhesion rate of monocytes were detected by using small interfering RNAs to inhibit the expression of histone acetyltransferase p300 in HAECs. The results showed that AICAR significantly inhibited TNFα-induced adhesion of monocytes to HAECs and down-regulated TNFα induced ICAM-1 and VCAM-1 m RNA levels and protein secretion in HAECs. The effect of AICAR can be completely blocked by the AMPK inhibitor compound C. Transfection of active AMPKα significantly inhibited TNFα-induced ICAM-1, VCAM-1m RNA expression and secretion, as well as monocyte-endothelial cell adhesion, whereas transfection of dominant-negative AMPKα had no significant effect. Inhibition of p300 activity by RNAi intervention significantly inhibited TNFα - induced adhesion molecule expression and monocyte - endothelial cell adhesion. AMPK activation can inhibit the TNFα-induced p300 acetyltransferase activity, inhibit the acetylation of 211-position lysine of NF-κB p65, and decrease the NF-κB p65 DNA binding activity. These results suggest that AMPK activation inhibits monocyte - endothelial cell adhesion, the mechanism may be related to its activity of reducing p300 and down-regulating NF-κB p65 transcriptional activity.