星形胶质细胞源性HMGB1介导脑缺血再灌注损伤大鼠血管新生的机制研究

Angiogenesis is the main style of new blood vessels formation under pathological condition in adult. It can be interpreted as a natural defense mechanism helping to restore oxygen and nutrient supply to the ischemic brain tissue. Despite the devastating role of high-mobility group box-1 protein (HMGB1) in the acute stage of ischemic stroke, astrocytic HMGB1 has been proven to promote endothelial progenitor cell-mediated neurovascular remodeling in the delayed phases of stroke recovery. A recent study suggested that HMGB1 accelerated angiogenesis in RA through enhancement of HIF-1α activation. Our previous study has proved that treatment of enriched environment (EE) promoted post-stroke angiogenesis along with an increased expression of VEGF and its receptor, which may contribute to the improved neurological outcome. However little was known about how individual EE variables (such as social interactions, physical exercise) might differentially impact on specific downstream biological mechanisms. Pair housing (PH) environment was built to investigate the social interaction variable. We proposed that PH treatment could promote the secretion of HMGB1 from astrocytes. Astrocytic HMGB1 might up-regulate the level of HIF-1α by binding to its receptor RAGE via ERK signaling, which could regulate the transcription and activation of its target gene VEGF and eventually enhance angiogenesis in the later phase of ischemic stroke. This research could provide theoretical evidence for optimization of rehabilitation environment for stroke patients, and enrich the neurorestorative mechanism mediated by HMGB1 in cerebral ischemia/reperfusion injury.

血管新生是脑缺血再灌注损伤(I/R)后神经功能重建的重要途径，星形胶质细胞来源的高迁移率族蛋白 B1(HMGB1)在I/R慢性期可通过介导内皮细胞与星形胶质细胞之间的相互作用调控内皮细胞分化促进血管形成。近期研究发现HMGB1的促血管新生作用可能依赖于低氧诱导因子-1α(HIF-1α)的调控。我们前期研究表明丰富环境(EE)可以促进I/R大鼠的血管新生，上调血管内皮生长因子(VEGF)的表达，但EE的有效作用元件及促血管新生具体机制仍需深入研究。我们提出学术假设：I/R慢性期，代表EE中社交刺激因素的配对居住环境可促进星形胶质细胞来源HMGB1的表达与释放，HMGB1通过ERK途径与RAGE结合上调HIF-1α的表达，后者通过调控VEGF的转录与活化促进血管新生进而改善神经功能。本研究将为临床优化脑卒中患者的康复治疗提供科学依据，并为I/R慢性期HMGB1介导的神经修复机制研究提供新思路。

本项目原研究计划内容为：星形胶质细胞源性HMGB1介导脑缺血再灌注损伤大鼠血管新生的机制研究。在研究过程中发现，我们的实验条件下模型已进入恢复期，HMGB1始终处于静息态而非我们设想中的激活态。近些年来，越来越多细胞死亡方式被定义，如细胞焦亡、铁死亡、坏死性凋亡、自噬依赖性细胞死亡等。本课题组前期研究表明，丰富环境可以抑制神经细胞凋亡从而改善脑卒中后大鼠功能。丰富环境是否可以影响非凋亡性质的细胞死亡从而发挥促神经功能恢复的效应尚不明确。因此我们将研究转向新的细胞死亡方式上，并重点研究了丰富环境对脑卒中后神经细胞焦亡和铁死亡的影响及其机制。我们发现丰富环境能促进脑缺血再灌注损伤大鼠血管生成和功能恢复。与标准环境组大鼠相比，丰富环境组大鼠GSDMD-N和炎性小体相关蛋白（NLRP1、NLRP3、caspase 1）的表达水平明显降低，炎症因子IL-1β、IL-18表达水平及分泌水平也明显降低，说明丰富环境可通过抑制炎性小体活化和NF-κB p65 信号通路从而抑制神经细胞焦亡。我们进一步探究了丰富环境抑制缺血再灌注损伤后神经细胞铁死亡的潜在机制。通过检测铁和丙二醛含量，GPX4，普鲁士蓝染色等铁死亡相关指标，我们发现与标准环境组大鼠相比，丰富环境组大鼠神经细胞铁死亡发生水平明显降低。此外，丰富环境组大鼠ACSL4表达水平明显降低，炎症因子IL-1β、TNF-α、IL-6表达水平也明显降低，说明丰富环境通过抑制ACSL4表达从而抑制神经细胞铁死亡及炎症因子的表达。同时，丰富环境组大鼠Hif-1α水平明显升高，提示丰富环境抑制神经细胞铁死亡可能是通过Hif-1α/ ACSL4这一经典通路实现的。我们的研究表明，丰富环境作为一种简单易行的干预手段，在促进卒中后的功能恢复中表现出显著的作用，其作用机制涉及促进血管新生以及抑制神经细胞焦亡和铁死亡。本研究对丰富环境作用机制进行了探究，进一步阐明了卒中后神经功能的恢复过程，并有助于将丰富环境临床转化为丰富康复训练并在脑卒中康复过程中进行深入应用。