Recombinant FGF21 as a novel approach for treating ischemic stroke in type 2 diabetes

项目摘要 糖尿病中风患者的死亡率更高，神经预后更差。新兴的临床和 实验数据表明，血脑屏障紊乱、神经炎症和中风恢复障碍 在T2D中加重。因此，该项目的目标是研究能够针对 卒中中这些特定的T2D机制。成纤维细胞生长因子-21是一种循环内分泌 主要在肝脏表达的荷尔蒙。FGF21具有多效性代谢作用，但也有组织 保护和修复角色。在这里，我们将检验外源重组人的总体假设 FGF21(RFGF21)激活FGFR1Klotho--Klotho复合体，可能改善T2D影响机制 卒中通过：(1)激活PPAR以保护血脑屏障，(2)抑制核因子B介导的但激活PPAR 对于神经炎症的调节，(3)促进AMPK/Nrf2介导的血管/白质重塑 促进中风后的神经恢复。 目的1.观察rFGF21对T2D卒中小鼠血脑屏障完整性的影响。我们将检查FGF21 特异性受体FGFR1磷酸化，PPAR活性，血脑屏障完整性，出血性转化， 血管炎症分子和紧密连接蛋白的表达在3天后进行评估 Db/db T2D小鼠卒中(DMCAO)模型和脑内皮细胞/星形胶质细胞共培养的T2D卒中体外模型。 目的2.探讨重组成纤维细胞生长因子21(RFGF21)对T2D卒中神经炎症的影响。我们将检查NFB和PPAR 活化及其与小胶质细胞/巨噬细胞活化、促炎因子表达的相关性 逆转录-聚合酶链式反应、免疫组织化学、免疫印迹和流式细胞术检测大鼠局灶性大脑中动脉梗死后的病变面积 T2D小鼠，体外原代培养小胶质细胞。目的3.研究rFGF21对血管/白质细胞的影响 T2D卒中的物质重塑。我们将检测AMPK和Nrf2的激活，营养因子的表达 通过免疫印迹分析、免疫组织化学和免疫组织化学方法分离脑微血管片段和脑组织。 MRNA微阵列。我们还将测试随着时间的推移血管和白质重塑的生物标记物 T2D小鼠缺血性卒中后的免疫组织化学、RT-PCR和Western blotts及体外模型 原代人脑内皮细胞培养和少突胶质细胞培养。

Project Summary Diabetic stroke patients have higher mortality and worse neurological outcomes. Emerging clinical and experimental data suggest that BBB disruption, neuroinflammation, and stroke recovery impairment are exacerbated in T2D. Hence, the goal of this project is to investigate therapeutic approaches that can target these specific T2D mechanisms in stroke. Fibroblast Growth Factor-21 (FGF21) is a circulating endocrine hormone that is primarily expressed in the liver. FGF21 has pleiotropic metabolic actions, but also has tissue protective and repair roles. Here, we will test the overall hypothesis that exogenous recombinant human FGF21 (rFGF21) activates FGFR1--klotho complex that may ameliorate T2D-affected mechanisms after stroke via: (1) activation of PPAR for BBB protection, (2) inhibition of NFB-mediated but activation of PPAR for neuroinflammation modulation, (3) promotion of AMPK/Nrf2-mediated vascular/white matter remodeling to boost neurorestoration after stroke. Aim 1. Investigate rFGF21 effects on blood-brain barrier integrity in T2D stroke mice. We will examine FGF21 specific receptor FGFR1 phosphorylation, PPAR activity, BBB integrity, hemorrhagic transformation, expression of vascular inflammatory molecules and tight junction proteins, are assessed up to 3 days after stroke (dMCAO) in db/db T2D mice, and T2D stroke in vitro model of brain endothelial/astrocytes co-cultures. Aim 2. Investigate rFGF21 effects on neuroinflammation in T2D stroke. We will examine NFB and PPAR activation, and correlation with microglia/macrophage activation, pro-inflammatory factor expression in peri- lesion area by RT-PCR, immunohistochemistry, western blots, and flow cytometry after focal dMCAO stroke in T2D mice, and in primary microglia cultures in vitro. Aim 3. Investigate rFGF21 effects on vascular/white matter remodeling in T2D stroke. We will examine activation of AMPK and Nrf2, expression of trophic factors in isolated brain microvascular fragments and brain tissues by western blot analysis, immunohistochemistry, and mRNA microarray. We will also test biomarkers of vascular and white matter remodeling over time by immunohistochemistry, RT-PCR and western blots after ischemic stroke of T2D mice, and in vitro models of primary human brain endothelial cultures and oligodendrocyte cultures.