沉默TREM-1通过调控NF-κB信号通路抑制氧化三甲胺介导的血管平滑肌细胞炎症

Silencing triggering receptor expressed on myeloid cells-1 inhibits vascular smooth muscle cell inflammation mediated by trimethylamine N-oxide by regulating the nuclear factor-kappa B signaling pathway

摘要

目的 探讨髓系细胞触发受体-1（triggering receptors expressed on myeloid cells-1，TREM-1）在氧化三甲胺（trimethylamine oxide，TMAO）介导的大鼠血管平滑肌细胞（vascular smooth muscle cells，VSMCs）炎性反应中的作用及其可能机制。 方法 ①采用不同浓度（0、100、300、600、900、1 200、1 500 μmol/L）TMAO处理VSMCs 24 h；②将TREM-1基因siRNA干扰质粒（si-TREM-1）及其阴性对照干扰质粒（si-NC）转染至VSMCs中，采用600 μmol/L TMAO诱导VSMCs炎性反应，并联合核因子κB（nuclear factor-kappa B，NF-κB）激活剂佛波酯（phorbol myristate acetate，PMA）干预24 h。CCK-8检测细胞增殖活性；ELISA检测细胞上清中白细胞介素（interleukin，IL）-1β、IL-6及肿瘤坏死因子（tumor necrosis factor，TNF）-α水平；定量聚合酶链反应（quantitative polymerase chain reaction，qRT-PCR）检测细胞中TREM-1、环氧化酶-2（cyclooxygenase-2，COX-2）、细胞间黏附分子-1（intercellular adhesion molecule-1，ICAM-1）以及IL-1β、IL-6、TNF-α mRNA表达水平；Western blot检测细胞中TREM-1、COX-2、ICAM-1以及NF-κB p65、p-NF-κB p65（Ser536）蛋白表达水平。 结果 不同浓度TMAO处理对VSMCs细胞增殖活性无明显影响（P=0.375），但可明显上调细胞上清中IL-1β、IL-6、TNF-α水平以及细胞中TREM-1、COX-2、ICAM-1 等mRNA和蛋白表达水平（均P<0.01），且呈浓度依赖性。沉默TREM-1基因可明显抑制TMAO诱导的VSMCs上清液中IL-1β、IL-6、TNF-α水平的增加，以及细胞中COX-2、ICAM-1、IL-1β、IL-6、TNF-α mRNA和p-NF-κB p65/NF-κB p65蛋白比值的上调（均P<0.01）。然而，PMA干预可明显逆转沉默TREM-1基因对TMAO诱导VSMCs炎性反应的改善作用。 结论 沉默TREM-1基因可抑制TMAO诱导的VSMCs炎性反应，其机制可能与抑制NF-κB通路活化有关。

Abstract

Objective To investigate the role and possible mechanism of triggering receptor expressed on myeloid cells-1（TREM-1） in the inflammatory response of rat vascular smooth muscle cells（VSMCs） mediated by trimethylamine N-oxide（TMAO）. Methods VSMCs were treated with TMAO at different concentrations（0，100，300，600，900，1 200，1 500 μmol/L） for 24 hours. VSMCs were transfected with the siRNA interference plasmid targeting the TREM-1 gene（si-TREM-1） and its negative control interference plasmid（si-NC），and 600 μmol/L TMAO was used to induce inflammatory response in VSMCs，combined with the intervention with the nuclear factor-kappa B（NF-κB） activator phorbol myristate acetate（PMA） for 24 hours. CCK-8 assay was used to measure cell proliferative activity；ELISA was used to measure the levels of interleukin-1β（IL-1β），interleukin-6（IL-6），and tumor necrosis factor-α（TNF-α） in cell supernatant；qRT-PCR was used to measure the mRNA expression levels of TREM-1，cyclooxygenase-2（COX-2），intercellular adhesion molecule-1（ICAM-1），IL-1β，IL-6，and TNF-α in cells，and Western blot was used to measure the protein expression levels of TREM-1，COX-2，ICAM-1，NF-κB p65，and p-NF-κB p65（Ser536） in cells. Results Treatment with TMAO at different concentrations had no significant impact on the proliferative activity of VSMCs（P=0.375），but it significantly upregulated the levels of IL-1β，IL-6，and TNF-α in supernatant and the mRNA and protein expression levels of TREM-1，COX-2，and ICAM-1 in cells（all P<0.01） in a concentration-dependent manner. Silencing of the TREM-1 gene significantly inhibited the increases in the levels of IL-1β，IL-6，and TNF-α in the supernatant of VSMCs induced by TMAO，and it also suppressed the upregulation of the mRNA expression levels of COX-2，ICAM-1，IL-1β，IL-6，and TNF-α and the ratio of p-NF-kB p65/NF-kB p65 in VSMCs（all P<0.01）. However，PMA intervention significantly reversed the role of silencing the TREM-1 gene on TMAO-induced inflammatory response in VSMCs. Conclusion Silencing of the TREM-1 gene can inhibit TMAO-induced inflammatory response in VSMCs，possibly by inhibiting the activation of the NF-κB pathway.

关键词

氧化三甲胺 / 血管平滑肌细胞 / 炎性反应 / 髓系细胞触发受体-1 / 核因子κB通路

Key words

trimethylamine N-oxide / vascular smooth muscle cells / inflammatory response / triggering receptor expressed on myeloid cells-1 / nuclear factor-kappa B pathway

基金资助

河北省卫健委医学科学研究课题计划资助项目(20201151)