NOX4在缺血性卒中血管内机械再通后出血转化中的作用及机制

Patients with acute ischemic stroke (AIS) may benefit from mechanical thrombectomy with high recanalization rates by removal of large-vessel intracranial occlusions. Severe hemorrhagic transformation (HT) after thrombolysis and endovascular recanalization predicts a poor outcome in AIS patients. Our previous studies found that HT rate was nearly 40% in AIS patients after thrombectomy. The radiological finding of leukoaraiosis are related to the blood-brain barrier (BBB) dysfunction. In addition, leukoaraiosis in the deep white matter increases the risk of severe HT after thrombectomy. However, the mechanism of HT associated with mechanical thrombectomy in AIS is unknown. Our preliminary data indicate that the expression of NADPH oxidases 4 (NOX4) and matrix metalloproteinase 9 (MMP-9) in the ischemic brain might pertain to BBB disruption. It is hypothesized that HT after thrombectomy may result in reperfusion injury after reopening of the occluded large vessel, in which reactive oxygen species -generating NOX4 oxidative stress contribute to the neurovascular dysfunction, BBB disruption and apoptosis through MMP-9 pathway in ischemic brain. The goal of this grant is to gain a better understanding of the mechanisms of NOX4 in HT after mechanical recanalization using both in vivo and in vitro models, and to develop the potential therapeutic interventions to attenuate neurological deterioration post-thrombectomy. To test the hypothesis, a rat HT model with 6h tMCAO will be created. A canine ischemic-reperfusion model, a model more close to the pathophysiology of human stroke and reproducing the phenomena of endovascular recanalization will be also generated by endovascular MCA occlusion for 5h with microcoil. Stroke volume and HT will be quantified by MR techniques in this canine model, while expression of NOX4, MMP-9 and tight junction protein in ischemic brain tissue will be analyzed by immunofluorescent histochemistry, qRT-PCR and Western blot. Then, the mechanisms of neurovascular dysfunction responsible for HT in brain microvascular endothelial cells, neurons, astrocytes and microglia will be validated using oxygen-glucose deprivation/reoxygenation by silencing NOX4 gene. Finally, a therapeutic intervention study will be carried out on the rat and canine stroke model through intravenous or intraarterial injection NOX inhibitor to test the efficacy of inhibition of NOX4 to attenuate post-thrombectomy HT and ischemic damage. The proposed studies are designed to determine NOX4 as an important functional component associated with post-thrombectomy HT and suggest NOX4 as an appropriate target for therapeutic intervention to decrease HT injury.

血管内机械再通治疗缺血性卒中后可发生颅内出血转化(HT)。我们前期研究进一步表明BBB破坏相关的脑白质疏松表现与机械再通后HT发生有关。明确卒中机械再通后HT的发生机制有重要意义。我们在大鼠MCA缺血6h再通后HT的模型中，发现NOX4和MMP-9表达升高和BBB破坏有关。由此，我们推测NOX4氧化应激诱导MMP-9途径和BBB破坏可能是卒中机械再通后HT的新机制。本项目拟继续建立大鼠和犬MCA缺血再通模型，免疫荧光、RT-PCR、Western blot和MR研究，明确NOX4氧化应激和机械再通后HT有关。结合体外氧糖剥夺复氧研究,证实缺血再灌注时NOX4诱导神经血管细胞MMP-9上调、细胞凋亡和BBB破坏。进一步在大鼠和犬模型应用NOX抑制剂干预，观察防治HT的效果。本项目将阐明NOX4在卒中机械再通治疗后HT中的作用及机制,为确定NOX4作为防治机械再通后HT的新靶点提供科学依据。

卒中是中国人群死亡首位原因，血管内介入取栓成为急性缺血性卒中的重要治疗策略，减轻介入取栓治疗后再灌注损伤和颅内出血转化并提高疗效是卒中领域的关键科学问题。.本项目通过建立血管内机械再通后颅内出血转化的大鼠模型，发现机械再通后颅内出血转化的脑组织中NADPH氧化酶(NOX)的NOX2和NOX4表达增高。再通前30分钟静脉内给予NOX4抑制剂VAS2870，通过减轻氧化应激引起的血脑屏障损伤和抑制神经元凋亡的机制，减轻了机械再通治疗后的脑水肿和严重颅内出血转化，并且改善了缺血大鼠的神经功能和降低再灌注损伤及颅内出血引起的死亡率。采用microRNA芯片检测机械再通后颅内出血转化的大鼠脑组织，结合Target Scan等microRNA基因预测软件检索与NOX2和NOX4有结合位点的microRNA，发现与NOX4和NOX2有结合位点的下调表达microRNA分别为18个和12个。确定与NOX2和NOX4结合程度高的microRNAs进行qRT-PCR，证实了miR-29a、miR-136、miR-153、miR-337和miR-376a在颅内出血转化的大鼠脑组织中均显著降低。同时发现NOX4抑制剂VAS2870促进了miR-29a等多个NOX2和NOX4相关microRNAs的上调表达，从而降低了NOX2和NOX4的蛋白和mRNA水平表达，并抑制了NF-κβ信号通路的激活。.本研究结果发现了NOX4是缺血性卒中介入取栓再通治疗后颅内出血并发症的防治新靶点以及机械再通治疗后颅内出血并发症的microRNA表达谱，在动物模型上提出了机械再通联合NOX4抑制剂是防治介入取栓治疗后颅内出血并发症的新方法，发现了几个microRNA是脑缺血再灌注损伤的潜在防治新靶点，为临床上防治介入取栓后颅内出血和再灌注损伤并发症的转化应用提供了新策略。.取得的研究成果在国内外核心期刊发表6篇通讯作者论著，包括5篇SCI论著发表在Stroke、J Neurointerv Surg和Medicine杂志，累计影响因子18分，单篇最高引用25次。研究成果多次在国际学术会议上交流，培养了5名研究生，建立了一支脑血管病临床和转化医学研究团队。后续研究计划2017年获得国家自然科学基金重点国际合作项目基金资助。