Notch1与TLR2相互作用调控CD14^+单核细胞功能在冠状动脉粥样硬化性心脏病中的作用

Interaction between Notch1 and TLR2 regulates CD14^+ monocytes function in patients with coronary atherosclerotic heart disease

目的观察Notch1/2分子和Toll样受体(TLR)在冠心病患者中的表达变化,探讨Notch信号通路与TLR2的相互作用对CD14^+单核细胞的调控作用。方法本研究入组稳定性心绞痛(SA)22例、不稳定性心绞痛(UA)34例、急性心肌梗死(AMI)27例,同时入组31例健康对照者。实时定量PCR法检测PBMC中Notch1/2以及TLR1-10 mRNA的相对表达量。分选CD14^+单核细胞,应用Notch信号通路抑制剂DAPT和/或TLR2激动剂Pam3Csk4刺激培养24 h,检测TLR2和Notch信号通路相关分子的表达变化,ELISA法检测上清中促炎细胞因子的表达变化,Western blot法检测细胞中抗髓样分化因子88(MyD88)和TIR结构域接头分子(TRIF)的水平。结果 Notch1 mRNA的相对表达量在AMI组显著升高,Notch2 m RNA的相对表达量在各组间的差异无统计学意义。TLR4、TLR7、TLR9 mRNA的相对表达量在SA组、UA组和AMI组中均显著高于NC组,TLR2 mRNA的相对表达量在AMI组中显著高于NC组、SA组和UA组。DAPT刺激可降低CD14^+单核细胞中TLR2 m RNA的相对表达量,对TLR4、TLR7和TLR9 mRNA的表达无显著影响。Pam3Csk4刺激可升高AMI患者CD14^+单核细胞中Notch1 mRNA的相对表达量,Notch信号通路相关分子Hes1和Hes5 mRNA的相对表达量亦显著升高。DAPT刺激还可抑制AMI患者中TLR2介导的炎症信号通路的应答,降低Pam3Csk4刺激后AMI患者CD14^+单核细胞培养上清中白细胞介素(IL)-6、IL-8和肿瘤坏死因子-α的表达,这一过程主要通过影响TIR结构域接头分子表达及核因子-κB的磷酸化而实现。结论 Notch1和TLR2的相互作用可能发挥调控冠心病患者CD14^+单核细胞功能的作用。

Crosstalk between Notch signaling pathway and Toll-like receptor (TLR) contributes to the inflammatory response in infectious and inflammatory diseases. Coronary atherosclerotic heart disease (CAHD) is also a chronic inflammatory disease. Thus, this study was designed to investigate the expressions of Notch receptor and TLRs in CAHD patients, and to assess modulatory activity of Notch signaling pathway and TLR2 to CD14^+ monocytes. Total of 22 cases of stable angina (SA), 34 cases of unstable angina (UA), and 27 cases of acute myocardial infarction (AMI), and 31 normal controls (NC) were enrolled. The mRNA expression of Notch1/2and TLR1-10 were measured by real-time PCR; CD14^+ monocytes were purified, and stimulated with Notch signaling inhibitor DAPT and/or TLR agonist Pam3Csk4 for 24 hours. The expression of TLR2 and Notch signaling related molecules were measured; proinflammatory cytokines production in the supernatants was measured by ELISA, while myeloid differentiation factor 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF) expression in cultured CD14+ monocytes were measured by Western blotting. Data showed that Notch1 mRNA expression was significantly elevated in AMI group, while there was no remarkable difference of Notch2 mRNA among the groups; the mRNA levels of TLR4, TLR7, and TLR9 were notably elevated in SA, UA, and AMI groups in comparison with NC group, while TLR2 mRNA was increased in AMI patients compared with NC, SA, or UA groups. DAPT stimulation down-regulated TLR2 mRNA expression in cultured CD14^+ monocytes, however, did not affect TLR2, TLR7, or TLR9 mRNA level. Pam3Csk4 stimulation elevated Notch1 mRNA expression in cultured CD14+ monocytes from AMI patients, which was accompanied by elevation of Hes1 and Hes5 mRNA expressions. DAPT stimulation also inhibited TLR2-mediated inflammatory signaling pathway in AMI patients, which presented as the reduction of interleukin (IL)-6, IL-8, and tumor necrosis factor-α production in CD14+ monocytes from AMI patients in response to Pam3Csk4 stimulation. This process mainly affected TRIF expression and nuclear factor-κB phosphorylation. Taken together, the crosstalk between Notch1 and TLR2 might modulate CD14+ monocyte function and inflammatory responses in CAHD patients.