sFRP3蛋白对脑出血后炎症反应的调节作用及机制

Intracerebral hemorrhage (ICH) is accompanied by high mortality and morbidity. After ICH, the cellular toxicity of hemoglobin and activation of microglia induce secondary cerebral injury which lacks effective therapy. Recent study has revealed that RhoA high expression is associated with microglia activation after ICH. RhoA is a downstream target of the noncanonical Wnt pathway. Secreted frizzle-related protein 3(sFRP3) is a competitive antagonist that binds directly to Wnt lignads in the extracellular space. It is unknown whether sFRP3/Wnt signaling regulates RhoA to affect ICH injury and recovery. In our pilot studies, we found that sFRP3 expression level increases in the ipsilateral striatum at 4h after ICH; sFRP3 deletion increases neuronal death in organotypic hippocampal slice cultures exposed to hemoglobin; sFRP3 deletion aggravates brain lesion volume and behavioral deficits on day 3 after ICH; RhoA expression is elevated in hemoglobin-exposed, cultured astrocytes that lack sFRP3 expression. These data strongly support the involvement of sFRP3 in ICH injury and led us to hypothesize that sFRP3 regulates inflammatory response through Wnt/Rho/Rho-kinase signaling after ICH. To test this hypothesis, we propose to carry out in vivo and in vitro. We will use use wild-type and sFRP3 knockout mice for the standard collagenase-induced ICH model and apply genetic, cellular, molecular, MRI and flow cytometric approaches that have been established in the lab. These studies will illuminate an important and unrecognized role of sFRP3 in neuroinflammation and provide fresh insight into the role of Wnt signaling in inflammatory response after ICH. Closely regulating the sRFP3-Wnt signaling pathway could help the brain defend itself against acute brain injury and promote recovery. This proof-of-concept work is novel, has translational potential, and will stimulate more preclinical studies on sFRP3 in acute and chronic brain diseases.

脑出血具有高发病率和高死亡率。研究表明脑出血后高表达的RhoA蛋白与小胶质细胞的炎性活化相关。分泌性Frizzled相关蛋白3（the secreted frizzled-related protein 3, sFRP3)作为Wnt信号的可溶性调控因子，sFRP3/Wnt信号通路是否在脑出血后通过RhoA调控炎症反应的作用尚不明确。我们的预试验结果表明脑出血后sFRP3的表达上调；敲除sFRP3增加损伤后胶质细胞RhoA的表达水平；并加重脑损伤和神经功能缺陷的行为学表现。为此，我们提出如下假说：sFRP3可能通过Wnt/Rho/Rho-kinase信号通路调节脑出血后的炎症反应。本研究拟从体内及体外实验，运用细胞、分子生物学及流式细胞仪、MRI影像学方法探讨其中机制。此课题是首次研究神经系统内sFRP3参与调控炎症的作用，将有助于形成sFRP3功能的新理论，并为探索临床治疗提供新思路。

脑出血(intracerebral hemorrhage, ICH)，是由于脑部血管破裂而引起脑组织损伤的疾病。脑血管破裂后，血液在短时间内在脑实质局部的快速蓄积，导致颅内高压及脑水肿形成，造成了脑出血后的初级脑损伤；此后，血红蛋白、铁离子的细胞毒性作用，以及清除出血过程中免疫细胞的激活均可进一步加重脑出血后的脑损伤，这些称为脑出血后的次级脑损伤。因此，以减轻炎症反应、抑制过度的免疫应答来缓解脑出血愈后的研究越来越受到人们的关注。sFRP3 (secreted frizzled-related protein 3)作为Wnt信号通路的调控因子，其是否通过调控Wnt/β-catenin经典途径或通过Wnt/RhoA非经典途径参与脑出血后的炎症调控过程，目前尚不清楚。项目负责人在前期研究已发现sFRP3 RNA表达水平在脑出血后的4小时开始升高，且与小鼠的脑损伤面积和神经缺陷的行为学表现相关。因此本课题拟进一步阐明sFRP3参与调节脑出血后炎症过程的机制。本项目的主要研究发现如下：（1）发现脑出血后脑内sFRP3的表达水平的变化主要来源于星形胶质细胞；（2）发现源于星形胶质细胞的sFRP3对小胶质细胞炎症反应具有调节作用；（3）对sFRP3 参与调节脑出血后炎症反应的信号通路进行了探讨：（4）发现sFRP3对脑出血晚期神经再生的调控效应：（5）发现膜受体CASPR1通过调节Na+/K+-ATP酶的活性影响谷氨酸穿越血脑屏障的转运。这部分实验结果已经发表于 J Biol Chem (2019; 294(16): 6375-6386) (SCI 收录， IF 4.238)。