20-HETE抑制剂在新生猪缺氧缺血性脑病中延长亚低温治疗窗的作用机制

Hypoxic ischemic encephalopathy remains a significant cause of mortality and long-term disability in infants.Due to the limited therapeutic window,infants especially with severe encephalopathy or those who lose the chance of hypothermia within the therapeutic window are less likely to benefit from treatment.20-HETE, the product of arachidonic acid(AA), is the key regulator of cerebral vascular function and novel therapeutic target for cardiovascular diseases and neurological disorders.We have firstly found that the 20-HETE-synthesis inhibitor provides neonatal neuroprotection and strengthens the effect of mild hypothermia.Apoptosis mediated by endoplasmic reticulum stress is related to Calpain which form Capn-InsP3R by activating IP3R1 of endoplasmic reticulum.We suppose that 20-HETE-synthesis inhibitor may extend the therapeutic window of hypothermia.To verify the hypothesis,we will observe the injuries of white matter by DWI and count apoptosis neurons by immunofluorescence staining with the HIE modle of new born piglets.We will verify the role of m-Calpain in the process of Ca2+ overload induced by ERS with RNA interference technique on oxygen glucose deprivation/reoxygenation model.We will apply patch clamp to measure the concentration of Ca2+ and the channel function of Ca2+and IP3R1.The goals of the present proposal are to further investigate the mechanism of 20-HETE-synthesis inhibitor on extending the therapeutic window of hypothermia and provide the new strategies for preventing hypoxic ischemic encephalopathy.

缺氧缺血性脑病是新生儿致死、致残重要原因，亚低温治疗是目前唯一临床证实安全并有效降低病死率和致残率的方法，但是受限于治疗窗，许多患儿尤其是重症患儿疗效甚微。花生四烯酸代谢产物20-HETE是脑血管功能重要调节因子。我们首次发现20-HETE抑制剂HET0016对发育期神经元有保护作用，并可以增强亚低温治疗效果。内质网应激启动的凋亡与Calpain激活内质网IP3R1形成Capn-InsP3R1有关，本研究拟通过建立新生仔猪缺氧缺血脑病模型，应用磁敏感加权成像动态观察缺氧缺血脑病仔猪脑白质的损伤情况及免疫荧光染色法计数凋亡神经元，验证HET0016能够延长亚低温治疗窗这一猜想；建立离体氧糖剥夺/复氧模型，采用RNA干扰技术，验证m-Calpain在内质网应激导致钙超载过程中所起的作用，并用全细胞膜片钳测定神经元内IP3R1及钙离子通道功能改变进一步探讨HET0016延长亚低温治疗窗作用机制。

新生儿缺氧缺血脑病（hypoxic-ischemic encephalopathy，HIE）是新生儿围生期窒息导致的脑缺氧缺血（hypoxic-ischemic ，HI）性损害，病情重，病死率高，并可能导致永久性神经功能障碍,是新生儿围生期死亡和严重伤残的主要原因,发生率为1-7‰。如何能够降低新生儿缺氧缺血脑病的病死率和致残率一直是大家关注的热点，但是目前仍然没有一种药物被证明能够安全有效的广泛应用于临床。亚低温是目前唯一被公认为是安全有效的临床治疗手段，但是受限于6小时时间窗，临床很难有效实施。本课题组前期工作通过建立新生仔猪脑缺氧缺血模型，首次发现给予20-HETE抑制剂HET0016可以通过抑制NMDA受体NR1亚基pSer896、Na+／K+-ATP酶α亚基pSer23 的磷酸化及ERK1/2通路减轻氧化应激反应，减少神经元凋亡并能改善脑缺氧缺血后神经功能评分,证实20-HETE抑制剂HET0016对发育期神经元有保护作用 。为了证明20-HETE抑制剂HET0016是否能够增强亚低温的脑保护作用，我们给予缺氧缺血脑病新生仔猪20-HETE抑制剂HET0016并联合实施亚低温治疗，建立新生仔猪缺氧缺血脑病模型，静脉给予HET0016联合不同时点的全身亚低温，进行神经系统功能评分，磁敏感加权成像探测脑白质损伤情况，HE染色后显微镜下观察神经元细胞形态。 培育胎鼠原代海马神经元，建立氧糖剥夺/复氧模型。测定在Calpain基因抑制前后，HET0016联合不同时点的亚低温干预后，神经元m-Calpain表达的情况并镜下计数海马神经元的凋亡。采用Wester-Blot法对复氧后不同时间点capn-InsP3R1进行定性定量分析，结合钙离子成像测定神经元游离钙的浓度，分析capn-InsP3R1对钙稳态的影响。我们发现20-HETE抑制剂HET0016可以通过抑制m-Calpain的表达，增强6小时实施亚低温的脑保护作用。