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目的:探讨特异性抑制溴结构域(bromodomain and extra-terminal,BET)蛋白对血管内皮细胞激活与早期动脉粥样硬化形成的作用及其分子机制。方法:1.原代分离培养脐静脉内皮细胞和小鼠心脏血管内皮细胞后用肿瘤坏死因α(TNFα)刺激模拟炎症过程,以小分子化合物JQ1特异性抑制BET蛋白,分组如下:(1)对照组;(2)TNFα(25 ng/m L)处理组;(3)TNFα+JQ1处理组。采用Realtime-PCR及流式细胞术检测各组细胞炎症因子m RNA及蛋白水平的表达,采用5XκB荧光素酶报告基因检测各组核转录因子kappa B(NF-κB)转录活性。2.LDL受体基因敲除(LDLR-/-)小鼠随机分为2组:JQ1组(n=8,JQ1腹腔注射,50 mg/kg,每天一次)和对照组(n=8,DMSO溶媒组),同时给予高胆固醇饮食8周,采用免疫组化方法检测主动脉弓部血管细胞黏附分子-1(VCAM-1)表达水平。结果:与对照组相比,TNFα组炎症因子m RNA、蛋白表达明显上调(P<0.01),使用JQ1干预后,炎症因子E选择素(E-selectin)、P选择素(P-selectin)、VCAM-1及白细胞介素-8(IL-8)m RNA及蛋白表达均明显下调(P<0.01)。LDLR-/-小鼠高脂饮食诱导8周后JQ1显著下调了主动脉弓部VCAM-1蛋白表达。5XκB荧光素酶报告基因结果显示,与TNFα(-)相比,TNFα(+)组荧光素酶报告基因活性增强,JQ1可以显著下调报告基因活性(P<0.01)。结论:BET蛋白通过调控NF-κB信号通路参与了血管内皮炎症基因转录;抑制BET蛋白下调了NF-κB目的基因表达从而减轻了内皮激活及高脂诱导的早期动脉粥样硬化病理改变。
Objective: To investigate the effect of bromodomain and extra-terminal (BET) protein on the activation of vascular endothelial cells and early atherosclerosis and its molecular mechanism. The primary culture of human umbilical vein endothelial cells and mouse heart vascular endothelial cells with tumor necrosis factor α (TNFα) stimulated mimic the inflammatory process, the small molecule compound JQ1 specific inhibition of BET protein, grouped as follows: (1) Control group; (2) TNFα (25 ng / m L) treatment group; (3) TNFα + JQ1 treatment group. Real-time PCR and flow cytometry were used to detect the mRNA and protein expression of inflammatory cytokines in each group. The transcription activity of nuclear factor kappa B (NF-κB) was detected by 5 × κB luciferase reporter assay. LDL receptor knockout (LDLR - / -) mice were randomly divided into two groups: JQ1 group (n = 8, JQ1 ip, 50 mg / kg once daily) and control group Vehicle group), and given high cholesterol diet for 8 weeks. The expression of VCAM-1 in the aortic arch was detected by immunohistochemistry. Results: Compared with the control group, the mRNA and protein expression of inflammatory cytokines in TNFα group were significantly increased (P <0.01). After intervention with JQ1, the expression of inflammatory cytokines E-selectin, P-selectin, The mRNA and protein expressions of VCAM-1 and IL-8 were significantly down-regulated (P <0.01). JQ1 significantly down-regulated VCAM-1 protein expression in the aortic arch after 8 weeks of high-fat diet induction in LDLR - / - mice. The results of 5XκB luciferase reporter assay showed that TNFα (+) luciferase reporter gene activity was enhanced compared with that of TNFα (-) and JQ1 significantly down-regulated the reporter gene activity (P <0.01). CONCLUSION: BET protein is involved in the transcription of vascular endothelial dysfunction by regulating NF-|ÊB signaling pathway. Inhibition of BET protein down-regulates the expression of NF-|ÊB target gene and attenuates endothelial activation and early atherosclerosis induced by hyperlipidemia.