脑中新发现的Matrilin-3抗缺血性脑中风作用及其依赖自噬的调节反应性星形胶质细胞的机制

Targeting ischemic stroke induced-reactive astrocytes and the formation of glial scar is becoming an important therapeutic strategy in stroke research field. matrilin-3 is an extracellular matrix, as an adaptor protein. Matrilin-3 is an essential component during cartilage development and potential osteoarthritis pathomechanisms. The role of matrilin-3 in brain is totally unknown. We firstly found that matrilin-3 was extensively expressed in neurons, astrocytes and different brain area. In a rat model of transient middle cerebral artery occlusion (tMCAO), we found that the protein levels of matrilin-3 were down-regulated in the ischemic core of cortex or striatum from 3 day of reperfusion, respectively, while in the peri-infarct area of glia scar formation, matrilin-3 was significantly up-regulated in astrocytes. Matrilin-3 is required for astrocytic survival, but not for neurons. Overexpression of matrilin-3 decreased the LDH release in OGD or OGD/reoxygenation (OGD/R)-induced glia scar formation in vitro. Overexpression of matrilin-3 also reduced tMCAO-induced brain injury both in the acute stage and the chronic stage associated with the inhibition of reactive astrocytes and the formation of glial scar in the peri-infarct area. Overexpression of matrilin-3 inhibited the BMP2-Smad1, 5, 9 signaling and induced the expression of IL-1Ra (IL-1 receptor antagonist) in reactive astrocytes and the formation of glial scar in vivo and in vitro. Autophagy regulated the protein levels of matrilin-3 and its secretion in the astrocytes. The serum and cerebrospinal fluid concentration of matrilin-3 were decreased at 4 weeks in rats after tMCAO, respectively. Based on these data, using in vivo transient middle cerebral artery occlusion (tMCAO) model and in vitro oxygen and glucose deprivation (OGD) or oxygen glucose deprivation and reoxygenation (OGD/R) model, we will investigate the antagonistic effects of matrilin-3 on ischemic stroke and its mechanisms of inhibition of BMP2 and IL-1β signaling and the linkage via the programmed necrosis key kinase RIP1K （serine/threonine kinase receptor interacting protein 1，RIP1, RIP1K）in the regulation of reactive astrocytes and glial scar formation in an autophagic-dependent manner; The ER to Golgi secretory pathway of matrilin-3 and the degradation mechanisms of matrilin-3 regulated through the autophagic mechanisms; The therapeutic window of recombinant human matrilin-3 protein; The measurement of the protein levels of MATN-3 in serum and Cerebro-Spinal Fluid (CSF) in rats with ischemic stroke. This project will provide a novel endogenous target of matrilin-3 for regulation of the reactive astrocytes and glial scar formation induced by ischemic stroke and the biomarker for evaluating the prognosis for ischemic stroke.

matrilin-3是非胶原性细胞外基质,发挥衔接蛋白作用,主要参与软骨发育和骨关节炎病理机制。预实验意外发现脑组织存在matrilin-3,并具有抑制局灶性脑中风诱导的反应性星形胶质细胞及胶质瘢痕形成作用,减轻急、慢性期脑损伤，与其调节BMP2及IL-1β信号有关；自噬调节星形胶质细胞matrilin-3水平及分泌。本课题在体内、外脑缺血诱导的胶质瘢痕模型上研究：matrilin-3抗缺血性脑中风作用及其依赖自噬的抑制反应性星形胶质细胞BMP2及IL-1β信号机制及其通过程序性坏死激酶RIP1K链接此两条信号通路的作用;自噬调节matrilin-3的内质网-高尔基体合成、分泌机制及其降解机制；重组matrilin-3的治疗窗；缺血性脑中风大鼠血清及脑脊液matrilin-3的动态变化。以发现调控反应性星形胶质细胞的内源性新靶点matrilin-3及评估脑中风预后的生物学指标。

Matrilin-3（MATN-3）是一种细胞外基质蛋白，主要在软骨组织表达，matn3缺陷小鼠表现出软骨细胞肥大和早期骨关节炎。但脑中是否有MATN-3蛋白表达尚无定论。本课题意外地发现脑组织表达MATN-3，首次系统性研究MATN-3在缺血性脑卒中的作用及其分子机制和临床意义。.利用matn3 -/-小鼠及慢病毒转染技术敲低或过表达脑中matn3基因，给予人重组MATN-3（rhMATN-3）等，在大、小鼠短暂性大脑中动脉阻塞模型(tMCAO)和氧糖剥夺再给氧(OGD/Re)诱导的原代星形胶质细胞或神经元细胞或人星形胶质细胞损伤模型上，发现：.（1）在大、小鼠和人类不同脑区、神经元和星形胶质细胞中均富含MATN-3，在动物和健康受试者脑脊液中亦存在MATN-3。.（2）脑缺血/再灌注诱导MATN-3在缺血脑组织和边缘区反应性星形胶质细胞中降低，在OGD/Re损伤的星形胶质细胞中亦降低，但MATN-3水平在缺血性神经元细胞中未见明显变化。.（3）在正常和缺氧缺糖条件下，MATN-3是星形胶质细胞生长、生存所必须的，但其可能不是神经元所必需的；MATN-3通过保护缺血性星形胶质细胞间接保护缺血性神经元细胞损伤。.（4）内源性和外源性MATN-3均具有保护脑缺血/再灌注损伤的作用，抑制反应性星形胶质细胞介导的神经炎症和其反应性增生及胶质瘢痕形成。.（5）MATN-3抑制反应性星形胶质细胞神经炎症的分子机制包括：.①细胞核机制：MATN-3在反应性星形胶质细胞质中与NF-κB p65结合成复合物进入细胞核，在细胞核阻断NF-κB p65的转录活性，抑制NF-κB p65调控的神经炎症靶基因IL-1β、IL-6和TNF-α转录。.②细胞外机制：MATN-3与细胞外BMP-2结合，抑制BMP-2与BMP-2膜受体结合，从而抑制BMP-2/Smads通路。.③抑制RIP1K：MATN-3通过降低脑缺血/再灌注诱导的反应性星形胶质细胞RIP1K水平升高，抑制RIP1K介导的BMP2/Smad通路激活和神经炎症因子水平升高。.（6）激活或抑制自噬分别增加或减少反应性星形胶质细胞中MATN-3的水平，并抑制或加重胶质瘢痕形成。.（7）缺血性脑卒中患者急性期血清MATN-3水平较健康志愿者显著下降，MATN-3可能成为早期诊断和判断预后的血清生物学指标物。