缺血性卒中脑内IL-17A来源及其加重神经元缺血损伤的分子机制研究

As a pro-inflammatory cytokine, interleukin (IL)-17A plays an important role in the delayed neuronal injury after ischemic stroke. We previously reported that IL-17A content increased rapidly in peri-infart region and cerebrospinal fluid, while IL-17A only co-localized with astrocyte-specific marker GFAP in peri-infarct region of mice following middle cerebral artery occlusion (MCAO)-induced ischemic stroke; In the oxygen-glucose deprivation (OGD)-simulated cell ischemic model in vitro, IL-17A could enhance the neuronal ischemic injuries in a dose-dependent manner. Accordingly, in this project, the models of MCAO-induced mouse ischemic stroke in vivo and OGD-simulated cell ischemic injury in vitro, and the method of PLX5622-mediated microglia depletion were applied to further clarify the role of astrocytes in IL-17A production in ischemic stroked brain. With the multidisciplinary techniques of cell biology, biochemistry and molecular biology, the regulations of hypoxia inducible factor (HIF)-1alpha and miRNA-22-5p on IL-17A expression in astrocytes, as well as the detrimental effect of IL-17A on apoptosis, autophagy and pyroptosis of neurons and their related signal transduction mechanisms will be systematically explored. These achievements should expand our understanding of the molecular mechanism underlying neuronal ischemic injuries, and may provide experimental evidence for the biomarkers and therapeutic targets of ischemic stroke in clinical practice.

作为促炎因子，白细胞介素(IL)-17A在缺血性脑卒中神经元继发损伤中具有重要作用。我们曾发现，脑中动脉闭塞(MCAO)小鼠脑梗死周围区和脑脊液内IL-17A含量迅速增高，并只与星形胶质细胞特异标志物GFAP 共标；在离体细胞水平，IL-17A可剂量依赖性加重氧-糖剥夺(OGD)神经元缺血性损伤。据此，本课题拟利用MCAO小鼠和OGD细胞缺血模型，借助PLX5622耗竭脑内小胶质细胞，进一步明确星型胶质细胞在缺血性卒中脑内IL-17A产生的作用；借助细胞生物学、生物化学和分子生物学等学科技术方法，阐明缺血条件下低氧诱导因子(HIF)-1α和miRNA-22-5p对星型胶质细胞内IL-17A表达的调控，以及IL-17A加重细胞凋亡、自噬或焦亡等神经元缺血损伤的信号转导机制。所获成果将丰富人们对神经元缺血损伤机制的认识，并为缺血性脑卒中防治寻找生物标记物或治疗靶点提供实验依据。

白细胞介素(IL)-17A作为促炎因子参与了缺血性脑卒中神经元的继发损伤。前期研究我们发现星形胶质细胞是脑中动脉闭塞(MCAO)小鼠中枢神经系统内IL-17 的重要来源细胞之一。据此，本课题利用MCAO小鼠和OGD细胞缺血模型，进一步明确星型胶质细胞在缺血性卒中脑内IL-17A产生的作用;借助细胞生物学、生物化学和分子生物学等学科技术方法，阐明缺血条件下IL-17A加重细胞凋亡、自噬等神经元缺血损伤的信号转导机制。结果发现：1，IL-17A可以减轻 1 h OGD/R 24 h 诱导的原代皮层星形胶质细胞凋亡，抑制原代皮层星形胶质细胞经1 h OGD/R 24 h处理后下调的IL-17RA及IL-17RC膜转位水平，减少病灶周围区星形胶质细胞的激活。2， IL-17A通过Src-PP2B-mTOR途径介导的过度自噬加重缺血神经元损伤，通过Caspase-12依赖的内质网应激途径加重缺血神经元凋亡。3，侧脑室注射IL-17A单克隆抗体后可以通过抑制Caspase-12水解水平减少病灶周围区神经细胞凋亡，明显减少病灶周围区神经元丢失、减小脑梗死体积、改善MCAO小鼠神经功能预后。上述结果证明IL-17A 在中枢神经系统内的来源、参与脑缺血病灶周围区神经细胞凋亡、自噬的分子机制，为IL-17A有可能成为临床缺血性脑卒中的治疗靶点提供了科学实验依据。