锌指蛋白A20在灵仙新苷减轻血脑屏障炎性损伤中的作用及其机制研究

Based on previous research and references, we proposed that Clematichinenoside could protect blood brain barrier (BBB) inflammatory injury through upregulation of zinc finger protein A20 and inhibition of NF-κB. Clematichinenoside was one monomer of clematis chinensis osbeck that could obviously inhibit rheumatoid arthritis (RA). Also in our early stage work, BBB integrity protection in RA rats and zinc finger protein A20 upregulation in brain microvessel endothelial cells were observed after administration or inbubation with Clematichinenoside. In this project, zinc deficiency combine with cerebral ischemia model of rats will be established to study zinc supplement and BBB injury inhibition effect of Clematichinenoside. In vitro experiments, brain microvessel endothelial cells were served as in vitro model of BBB. Culture medium with high Zn2+ or Zn2+ deprivation to verify Zn2+ deficiency could mediate NF-κB oevrexpression and inflammation which induced by A20 supression. The results will reveal that mechanism of Clematichinenoside on upregulation A20 was depending on zinc supplement. Furthermore, we will apply gene transfection and western technology to illustrate the molecular mechanism of Clematichinenoside on inducing A20 expression and negatively inhibiting NF-κB through TNF-α→TNFR1→NF-κB signal pathway. The outcomes of this project will provide experiments basis for clinical use of Clematichinenoside, and will offer reference for anti inflammation target research based on zinc finger protein A20.

基于前期工作及文献提出上调锌指蛋白A20表达、负反馈抑制NF-κB，是灵仙新苷减轻血脑屏障炎性损伤的重要机制。灵仙新苷是威灵仙的单体成分，前期研究表明有显著的抗类风湿性关节炎作用；我们进一步研究发现，灵仙新苷能减轻关节炎大鼠血脑屏障损伤，且能上调TNF-α刺激的脑微血管内皮细胞A20表达。本项目在前期工作基础上，建立锌缺乏合并脑缺血模型，确证补锌作用是灵仙新苷保护血脑屏障完整性的物质基础；并通过体外试验验证锌缺乏致脑微血管内皮细胞A20表达抑制、介导炎症反应的作用，进而阐明灵仙新苷上调A20表达的机制；在此基础上，运用基因转染、Western等分子生物学技术，阐明灵仙新苷通过上调A20表达、负反馈抑制TNF-α→TNFR1→NF-κB信号通路、抑制血脑屏障炎性损伤的分子机制。研究成果将为灵仙新苷的临床应用提供依据，并对基于锌指蛋白A20的抗炎靶点研究具有借鉴作用。

脑卒中是严重危害人类健康的重大疾病，系统性炎症患者可破坏血脑屏障，进而加剧脑缺血/再灌注损伤。因此，炎症抑制将是治疗脑缺血的潜在靶点。. 本课题首先建立慢性全身炎症（长期高脂饲料喂养）合并大鼠脑缺血/再灌注损伤。结果表明，与正常饮食组大鼠比较，高脂饲料喂养组大鼠脑梗死面积增大，脑水肿加剧，血脑屏障通透性升高。炎性介质MMP-9、ICAM-1过度表达。因此，慢性系统性炎症反应恶化脑缺血/再灌注损伤的可能机制为介导炎症因子过度表达，破坏血脑屏障，加剧炎性浸润。. 课题组进一步建立急性炎症（LPS腹腔注射）合并大鼠脑缺血/再灌注模型，并在体外原代培养脑微血管内皮细胞，LPS合并氧糖剥夺致细胞损伤，研究抗炎药物灵仙新苷保护血脑屏障的作用及其机制。结果表明灵仙新苷能够显著降低炎症合并脑缺血/再灌注损伤所致的血脑屏障通透性升高、脑水肿加剧及其脑梗死加大；抑制过度的炎症因子释放，减轻炎症细胞浸润。体外实验表明，灵仙新苷显著升高细胞存活率，降低细胞通透性，下调NF-κB、iNOS、ICAM-1，TNF-α及IL-1β表达。siRNA沉默A20，可阻断灵仙新苷对脑微血管内皮细胞的保护作用。因此，灵仙新苷通过上调A20，负反馈抑制NF-κB及其下游信号通路，抑制炎症反应，保护血脑屏障完整性，进而减轻脑缺血/再灌注损伤。