环氧化酶2/前列腺素E2在血管紧张素Ⅱ刺激巨噬细胞表达细胞外基质金属蛋白酶诱导因子中的作用
Effect of Angiotensin Ⅱ on Extracellular Matrix Metalloproteinase Inducer Expression in Macrophages via AT1R/COX-2/PGE2 Pathway
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摘要: 目的了解血管紧张素Ⅱ(AngⅡ)在动脉粥样硬化不稳定斑块中的作用及其相关信号传导通路,探讨AngⅡ对人单核细胞株(THP-1)巨噬细胞表达细胞外基质金属蛋白酶诱导因子(EMMPRIN)中的作用及其机制。方法以浓度为5μg/L的佛波醇诱导THP-1成巨噬细胞后,用浓度为10-6mol/L AngⅡ刺激巨噬细胞。RT-PCR及Western blot测AngⅡ刺激后的巨噬细胞中EMMPRIN基因及蛋白的表达,用ELISA监测刺激后上清中的前列腺素E2(PGE2)的变化。为进一步研究其具体机制,分别用血管紧张素1型受体(AT1R)拮抗剂(10-5mol/L)、血管紧张素2型受体(AT2R)拮抗剂(10-5mol/L)、环氧化酶2(COX-2)抑制剂(10-5mol/L)及PGE2(10-7mol/L)探讨其信号传导机制。结果AngⅡ可明显诱导巨噬细胞内的EMMPRIN基因及蛋白的表达,刺激后6 h可见明显增加,12 h达到最高,24 h后下降。相比于对照组(RPMI-1640培养基组),AngⅡ刺激12 h后基因表达量约增加至5倍,蛋白的表达约增加至4倍,而在AngⅡ刺激后引起的COX-2、PGE2与EMMPRIN的蛋白改变基本一致。进一步的信号通路研究实验结果显示AT1R拮抗剂及COX-2抑制剂可明显降低AngⅡ的诱导作用,再加入PGE2这种抑制作用可被反转,而AT2R拮抗剂则无影响。结论在佛波醇诱导后的THP-1巨噬细胞中,AngⅡ可通过AT1R/COX-2/PGE2途径上调巨噬细胞中EMMPRIN的表达,而AT2R则无影响。Abstract: Objective To explore the expression of extracellular matrix metalloproteinase inducer(EMMPRIN) in human leukemic cell line(THP-1) macrophages produced by angiotensin Ⅱ(Ang Ⅱ) stimulation and its possible mechanisms.Methods THP-1 monocyte were cultured with 5 μg/L PMA and transformed into macrophages by 10-6 mol/L Ang Ⅱ.EMMPRIN gene and its protein were measured by real-time PCR and Western blot.PGE2 expression was assayed by ELISA angiotensin type 1 receptor(AT1R,10-5 mol/L)antagonist,angiotensin type 2 receptor(AT2R,10-5 mol/L)antagonist,cyclooxygenase-2(COX-2) inhibitor,NS-398,were used to inhibit the effect of Ang Ⅱ,and prostaglandin E2(PGE2) was added to delineate the mechanism of Ang Ⅱ-induced EMMPRIN expression.Results Ang Ⅱ induced the EMMPRIN expression obviously in macrophages,enhanced at 6 h,peaked at 12 h and declined after 24 h.Compared to the control group(RPMI-1640 group),gene expression of EMMPRIN was increased to 5 times by Ang Ⅱ(10-6 mol/L) stimulation after 12 h;protein expression was increased to 4 times.In addition,the tendency of enhancement of COX-2 and PGE2 by Ang Ⅱ stimulation were coincident with the expression of EMMPRIN.AT1R antagonist and COX-2 inhibitor,but not AT2R antagonist,PD123319 inhibited the effect of Ang Ⅱ.Conclusion Ang Ⅱ up-regulate the EMMPRIN expression in THP-1 macrophages via AT1R/COX-2/PGE2 signal transduction pathway.
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