ANCA相关血管炎中mCRP放大中性粒细胞致病性活化的机制

Antineutrophil cytoplasmic antibodies (ANCA) can induce the activation of neutrophils and then injure the endothelial cells. Although this process has been considered as the causative factor of ANCA associated vasculitis (AAV), the adequate activation of neutrophils by ANCA needs the existence of some cofactors. Previous studies indicate that the injury of endothelial cells taken by neutrophils needs the existence of activated platelets. There are high levels of circulating pentameric C reactive protein (pCRP) in patients with active AAV. Our previous work which has been published recently demonstrates that the pCRP can dissociate into the monomeric form (mCRP) in AAV. These newly formed mCRP can bind and activate static platelets. The activated platelets then release high-mobility group protein 1 (HMGB-1), an activator of neutrophils. This indicates that mCRP can enlarge the ANCA-induced activation of neutrophils through stimulating platelets. We have also demonstrated that the binding between mCRP and platelets might relay on the aa 35-47 of mCRP and the lipid rafts of platelets. In the further study, we intend to investigate the characteristics and the signal pathway of the binding between mCRP and platelets with recombinant mCRP which has mutant aa 35-47. We also intend to investigate the influence of mCRP-activated platelets on the injury of endothelial cells induced by ANCA with flow chamber experiment, laser confocal imaging and in vivo imaging technology. After establishing the AAV rat models, we want to observe the renal protective effects of 1,6-bis(phosphocholine)-hexane, which can inhibit the dissociation form pCRP into mCRP. Our study may help to understand the mechanisms of the pathogenesis of AAV.

抗中性粒细胞胞浆抗体（ANCA）诱导中性粒细胞损伤血管内皮是ANCA相关血管炎（AAV）的病因，但既往研究表明中性粒细胞对内皮细胞的损伤依赖活化血小板的存在。AAV患者血中五聚体C反应蛋白（pCRP）明显升高。课题组近期发表的文章证明，ANCA可通过中性粒细胞诱捕网使pCRP解聚为单体（mCRP），mCRP可活化血小板并释放中性粒细胞激活因子HMGB-1，提示mCRP可以血小板为桥梁辅助ANCA诱导中性粒细胞的活化。初步证明这种作用是由mCRP通过aa35-47与血小板脂筏结合而实现的。本研究拟通过基因重组aa35-47突变mCRP研究mCRP与血小板脂筏结合特性及信号通路；利用流体小室、激光共聚焦及活体成像等技术研究mCRP活化的血小板对ANCA诱导中性粒细胞损伤血管内皮的影响；观察mCRP生成抑制剂1,6-bis(PC)-H对AAV模型大鼠的肾保护作用，为探索AAV发病机制提供新思路。

AAV发病机制复杂，ANCA可激活中性粒细胞释放多种炎症因子，但炎症水平与病情的严重程度并非严格相关。我们的研究发现AAV患者尿中mtDNA的水平明显增高，并且和肾脏损伤的严重程度有关。向富血小板血浆中加入激活剂可以激活血小板并促进游离mtDNA的释放，体系中加入mCRP可以增加血小板的激活及游离mtDNA的释放水平。PLA2抑制剂可以抑制游离mtDNA的释放。pCRP也可以增加已激活的血小板释放游离mtDNA，加入pCRP解离抑制剂1,6-bis(PC)可以抑制pCRP的作用但不能抑制mCRP的作用，加入脂筏破坏剂或是识别CRP35-47的抗体可以抑制mCRP的作用，加入NF-κB 通路抑制剂 Bay11-7082明显抑制mCRP的作用。将mCRP作用后的血小板上清与ANCA刺激的中性粒细胞共孵育可以增强中性粒细胞的激活及NETs释放水平，使用PLA2抑制剂预处理或是加入TLR9受体抑制剂可以减弱这种作用。因此AAV时mCRP可以刺激血小板释放mtDNA从而增强ANCA对中性粒细胞的激活。这种作用主要依赖于mCRP通过氨基酸35-47结合到血小板脂筏微区并激活NF-κB 通路使血小板激活并释放线粒体。补体旁路途径活化在AAV至关重要的作用，我们提取中性粒细胞并用ANCA激活，制作小鼠MPO-ANCA血管炎模型并注射CRP，观察CRP对小鼠病情的影响。肾活检病理显示CRP可解离单体形式，mCRP在肾小球及小管部位沉积，并与H因子呈部分共定位沉积，分离正常人中性粒细胞经ANCA刺激后，发现上清中存在高水平CRP时上清中炎症因子IL-8水平更高，但补体活化产物C5b-p水平更低，检测mCRP的沉积与H因子的沉积共定位。体外培养人脐静脉内皮细胞，经NETs刺激后诱导脐静脉内皮细胞凋亡，发现上清中存在高水平CRP时有更多H因子与mCRP共定位沉积。体外实验证实人mCRO可与小鼠H因子结合，制作小鼠MPO-ANCA血管炎模型，同时注射人mCRP的干预组较未干预组外周血炎症因子IL-8水平更高，但蛋白尿及血尿素氮水平更低，肾小球可见mCRP与H因子共沉积。同时注射CRP解离抑制剂可以部分抵消CRP的作用。因此AAV时高水平血清CRP可以辅助ANCA刺激中性粒细胞，增加炎症水平，但同时CRP解离为mCRP沉积后可以结合更多的H因子，抑制了补体旁路途径的激活。

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