抗炎因子白介素-37抑制内皮细胞TLR2/4-ICAM-1介导中性粒细胞移行的研究

The endothelial inflammatory response includes the expression of cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) that allow circulating neutrophils to adhere to sites of endothelial injury, which was involved in the pathogenesis and development of atherosclerosis. Recent studies has found that Toll-like receptors (TLRs), an inflammatory receptor, activation could induce endothelial inflammatory responses and then followed by the adhesion and transmigration of the neutrophils, while this transmigration could be reduced when TLRs inhibited. IL-37, a new found anti-inflammatory cytokine, is uncompletely understood of the impact and mechanisms in endothelial inflammatory response. We already found that TLR2/4 activation could increase the IL-37 expression, while the expression of ICAM-1 could be decreased in the mouse coronary microvascular endothelial cells isolated from the IL-37 transgenic mice, which may be due to the reduction of NF-kB phosphorylation at the same time. Expression of IL-37 markedly reduced infarct size and improved LV function. The protective effects of IL-37 were associated with reduced IKK phosphorylation, NF-kB intranuclear translocation, MCP-1 production and mononuclear cell accumulation in the ischemic myocardium. The similar results has been proved in the aortic valve interstitial cells stimulated with TLR2/4 agonist. So we hypothesized that IL-37 could decrease the ICAM-1 expression in human coronary artery endothelial cells (HCAECs) stimulated with TLR2/4 activation, followed by the reduction of adhesion and transmigration of neutrophils. For these purposes, we are going to test the inflammatory responses in HCAECs stimulated with TLR2/4 agonist when NF-kB and/or IL-37 overexpression and inhibition. In the methods, we are going to test ICAM-1 and IL-37 mRNA expression by PCR, NF-kB phosphorylation by Westernblot at 1-8 hour, ICAM-1 expression by Westernblot and ICAM-1 distribution on cells by flow cytometry at 24 or 48 hours. Neutrophils isolated from healthy donors will be cocultural with the HCAECs treated as described above, we test the transmigration of neutrophils by immunofluorescence confocal microscopy and staining at 0.5, 1 and 2 hours of co-cultural.

内皮细胞炎症反应-ICAM-1释放-中性粒细胞粘附移行是动脉粥样硬化的重要病理进程。近来发现TLRs在其中起了重要作用，其受到抑制后减少了中性粒细胞的粘附。IL-37是新发现的抗炎症因子，我们前期发现TLRs激活可诱导转基因小鼠心脏内皮细胞中IL-37表达增加，ICAM-1和NF-κB磷酸化减少，缺血心肌组织IKK磷酸化和白细胞浸润也减少，并在人主动脉瓣膜细胞中得到证实。我们假设IL-37可以减少TLR2/4激活导致的人冠状动脉内皮细胞表达ICAM-1，抑制中性粒细胞移行进入内皮下，机制可能与NF-κB磷酸化受抑制有关。拟对内皮细胞NF-κB，IL-37的表达进行预处理（过表达或者抑制），再激活TLR2/4诱导内皮细胞的炎症反应，检测ICAM-1，IL-37mRNA表达和NF-κB磷酸化，ICAM-1表达和细胞表面分布。将受干预的内皮细胞与中性粒细胞共培养，检测中性粒细胞侵入内皮下的改变。

（1）研究背景：IL-37是新发现的抗炎因子，而内皮细胞损害和炎症反应分泌的因子可以引起中性粒细胞的移行迁入内膜下，促进动脉粥样硬化的发生。我们预测IL-37可以减轻这个炎症反应的进程。.（2）主要研究内容：利用冠状动脉内皮细胞在Toll样受体（TLR）2/4受体激活的炎症反应作为研究对象，使用RNA干扰技术抑制IL-37，IL-37b脂质体转染高表达IL-37，观察对于内皮细胞内促炎因子NF-κB和炎症因子ICAM-1的影响，以及对于中性粒细胞粘附的影响。另外，利用急性心肌梗死PCI术后患者的血清和白细胞检测，探讨IL-37可能在哪种细胞中表达改变。在高葡萄糖培养的人脐带内皮细胞中，检测ox-LDL诱导的炎症反应是否发生改变。.（3）重要结果和关键数据.1. IL-37表达增强后内皮细胞的炎症反应减弱。在TLR2/4激活的炎症反应中，NF-κB和ICAM-1表达增加，而提高IL-37b的表达时这些因子的表达明显受到抑制。IL-37受到抑制时这些结果则恰恰相反。.2.急性心肌梗死后PCI术后的患者中白细胞上IL-37和ICAM-1表达增强。在急性心肌梗死早期的12h两者的表达同步增加，48h时显著减少。.3.在高糖浓度培养的内皮细胞中，使用ox-LDL激活炎症反应可以导致炎症因子的改变，但是TLR2的表达没有发生变化。即使敲除了TLR2，ox-LDL激活的炎症反应也没有发生变化。.（4）科学意义.修复或者减轻内皮细胞的炎症反应，可能减少中性粒细胞的侵蚀。本研究结果部分回答了这个问题。.1.IL-37的表达对于内皮细胞的炎症反应控制有一定作用。IL-37表达受抑制可以增强内皮细胞促炎因子和炎症因子的表达，而增强其表达可以明显减轻这些因子的表达。这些表明IL-37可能在抑制内皮细胞的炎症反应方面起到一定作用，从而可能减少中性粒细胞的侵蚀。.2.IL-37在急性心肌梗死外周中性粒细胞上的表达增加。急性心肌梗死是动脉粥样硬化斑块破裂后发生的病理情况，炎症反应参与了其中。我们发现PCI术后外周血液中性粒细胞ICAM-1和IL-37表达的增加表明，炎症反应和抗炎作用是同步发生的。IL-37不仅可以在内皮细胞中起到减少炎症反应的效果，在中性粒细胞也同样如此。.3. ox-LDL诱导的炎症反应不通过TLR2起作用。说明TLR2很可能不是ox-LDL的作用受体。