Jiang Run-Hao, Xu Xiao-Quan, Wu Chen-Jiang, Lu Shan-Shan, Zu Qing-Quan, Zhao Lin-Bo, Liu Sheng, Shi Hai-Bin
a Department of Radiology , the First Affiliated Hospital of Nanjing Medical University , Nanjing , P.R. China.
Neurol Res. 2018 Sep;40(9):717-723. doi: 10.1080/01616412.2018.1473075. Epub 2018 May 29.
The role of CD40/CD40 ligand (CD40L) in microvascular thrombosis is now widely accepted. However, the exact mechanisms linking the CD40/CD40L system and the soluble form of CD40L (sCD40L) with microvascular thrombosis are currently a topic of intensive research. The objective of this study was to assess the potential mechanisms in CD40/CD40L system-regulated microvascular thrombosis after focal ischemia/reperfusion (I/R).
Rats were subjected to 60-min transient middle cerebral artery occlusion (MCAO). The experiments were divided into three groups: sham operation, MCAO, and MCAO + CD40 antagonist. Dynamic changes of serum-free sCD40L levels for 0, 1, 3, 5, 6, and 12 h by ELISA detecting kit after focal I/R were observed, and the CD40 expression levels in both platelet surface and vascular endothelial cell surface were measured by flow cytometry and immunofluorescence, respectively. Cerebral infarct volume was analyzed 12 h after reperfusion. mTOR/S6K signaling was determined by Western blot.
A comparison of thrombus formation between MCAO and CD40 antagonist treatment rats revealed a role for CD40 and/or CD40L in the inflammation-enhanced thrombosis responses in both of the platelet and vascular endothelial cell. MCAO rats yielded an acceleration of thrombus formation that was accompanied by increased CD40 levels in serum. The brain infarction was significantly decreased in CD40 antagonist treatment group compared to MCAO model group. The mTOR/S6K signaling was activated in MACO model than that of CD40 antagonist treatment group.
Our findings indicate that CD40/CD40L system contributes to microvascular thrombosis and brain infarction induced by MCAO and reperfusion. The mTOR/S6K signaling pathway is involved in the regulation of cerebral microvasculature after focal I/R by CD40/CD40L.
AKT: protein kinase B; CD40L: CD40 ligand; CSF: cerebrospinal fluid; FITC: fluorescein isothiocyanate; I/R: ischemia/reperfusion; MCAO: middle cerebral artery occlusion; mTOR: mechanistic target of rapamycin; PE: P-phycoerythrin; sCD40L: soluble form of CD40L; TNF-a: tumor necrosis factor-alpha; WT: wild type.
CD40/CD40配体（CD40L）在微血管血栓形成中的作用现已得到广泛认可。然而，将CD40/CD40L系统及CD40L的可溶性形式（sCD40L）与微血管血栓形成联系起来的确切机制，目前仍是深入研究的课题。本研究的目的是评估局灶性缺血/再灌注（I/R）后CD40/CD40L系统调节微血管血栓形成的潜在机制。
将大鼠进行60分钟的短暂大脑中动脉闭塞（MCAO）。实验分为三组：假手术组、MCAO组和MCAO + CD40拮抗剂组。采用ELISA检测试剂盒观察局灶性I/R后0、1、3、5、6和12小时血清中游离sCD40L水平的动态变化，分别通过流式细胞术和免疫荧光法检测血小板表面和血管内皮细胞表面的CD40表达水平。再灌注12小时后分析脑梗死体积。通过蛋白质印迹法测定mTOR/S6K信号传导。
比较MCAO组和CD40拮抗剂治疗组大鼠的血栓形成情况，发现CD40和/或CD40L在血小板和血管内皮细胞炎症增强的血栓形成反应中发挥作用。MCAO组大鼠血栓形成加速，同时血清中CD40水平升高。与MCAO模型组相比，CD40拮抗剂治疗组的脑梗死明显减少。与CD40拮抗剂治疗组相比，MACO模型组的mTOR/S6K信号被激活。
我们的研究结果表明，CD40/CD40L系统促成了MCAO和再灌注诱导的微血管血栓形成和脑梗死。mTOR/S6K信号通路参与了局灶性I/R后CD40/CD40L对脑微血管的调节。
AKT：蛋白激酶B；CD40L：CD40配体；CSF：脑脊液；FITC：异硫氰酸荧光素；I/R：缺血/再灌注；MCAO：大脑中动脉闭塞；mTOR：雷帕霉素靶蛋白；PE：藻红蛋白；sCD40L：CD40L的可溶性形式；TNF-α：肿瘤坏死因子-α；WT：野生型
