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小胶质细胞是参与防御脑缺血再灌注损伤的主要免疫细胞,在中枢神经系统修复和神经发生中发挥重要作用。在缺血性中风发生后,小胶质细胞表型往往会发生改变[1-2]。小胶质细胞经活化后会极化为促炎M1型和抗炎M2型两种表型[3-4]。抗炎型小胶质细胞能通过减轻炎症反应、清除细胞碎片和释放神经营养因子来促进大脑的恢复。然而小胶质细胞除了具有神经保护作用外,还是大脑中促炎细胞因子的主要产生者。促炎型小胶质细胞能通过启动炎症级联反应释放大量的炎性细胞因子引起炎症反应,阻碍大脑恢复过程[5-6]。
乌帕替尼(upadacitinib)是一种口服的、可逆的JAK激酶(Janus kinase)抑制剂,对JAK1有较强的选择性,已被批准用于治疗类风湿关节炎[7-9],目前临床研究正在评估几种免疫介导的炎症性疾病、克罗恩病、溃疡性结肠炎等炎症相关疾病[10-11]。有报道称小胶质细胞的极化与JAK/STAT信号通路有关[12-13],却没有对于JAK1选择性抑制剂乌帕替尼在神经系统应用方面的报道。本研究通过使用BV2小胶质细胞OGD/R模型,探讨乌帕替尼对BV2小胶质细胞极化的影响及其可能的作用机制。
Effects of upadacitinib on BV2 cells after oxygen–glucose deprivation/recovery
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摘要:
目的 研究乌帕替尼对氧糖剥夺再复氧(OGD/R)后BV2小胶质细胞极化及炎症的影响,并探讨其作用机制。 方法 实验分对照组、OGD组和乌帕替尼组3组。BV2细胞经OGD/R处理后,噻唑蓝试剂(MTT)检测细胞生存率,划痕实验观察细胞迁移能力,实时荧光定量多聚核苷酸链式反应(qPCR)检测BV2细胞M1型极化标志物(CD11b、CD32、iNOS)和M2型极化标志物(Arg-1、IL-10、CD206)的mRNA水平,酶联免疫吸附测定(ELISA)检测培养基中IL-1β、IL-6、TNF-α含量,蛋白质印迹法(Western Blot)检测JAK1/STAT6通路相关蛋白表达水平。 结果 乌帕替尼能增加OGD/R后BV2细胞的生存率(P<0.05),能减少BV2细胞向M1型极化(P<0.05)。乌帕替尼可明显降低OGD/R诱导的BV2细胞的迁移能力(P<0.05),减少OGD/R诱导BV2细胞分泌的炎症因子:IL-1β、IL-6、TNF-α(P<0.05)。乌帕替尼提高共培养PC12细胞的生存率(P<0.05)。乌帕替尼可显著抑制由OGD/R诱导激活BV2细胞p-JAK1和p-STAT6蛋白的表达水平(P<0.05)。 结论 乌帕替尼可减少OGD/R诱导BV2细胞向M1型极化和减少炎症反应,与JAK1/STAT6通路有关。 -
关键词:
- 乌帕替尼 /
- 氧剥夺/复氧 /
- 极化,炎症,酪氨酸激酶/信号传导及转录激活因子
Abstract:Objective To investigate the effects of upadacitinib on the polarization and inflammation of BV2 microglia after oxygen glucose deprivation/recovery (OGD/R) and to explore its mechanism of action. Methods The experiment was divided into 3 groups: control group, OGD group and upadacitinib treatment group. After BV2 cells were treated with OGD/R, MTT was used to detect cell survival rate. Wound scratch assay was used to detect the cell migration ability. qPCR was used to detect mRNA levels of M1-type polarization markers (CD11b, CD32, iNOS) and M2-type polarization markers (Arg-1, IL-10, CD206) of BV2 cells. ELISA was used to detect the levels of IL-1β, IL-6, and TNF-α in the culture medium. Western blot was used to detect the expression levels of JAK1/STAT6 pathway-related proteins. Results Upadacitinib increased the survival of BV2 cells after OGD/R (P<0.05), reduced the polarization of BV2 cells to M1 type (P<0.05). Upadacitinib significantly decreased the migration ability of BV2 cells induced by OGD/R (P<0.05), reduced the inflammatory factors secreted by BV2 cells induced by OGD/R: IL-1β, IL-6, TNF-α (P<0.05). Upadacitinib increased the survival rate of co-cultured PC12 cells (P<0.05). Upadacitinib significantly inhibited the expression levels of p-JAK1 and p-STAT6 proteins in BV2 cells activated by OGD/R induction (P<0.05). Conclusion Upadacitinib decreases polarization of BV2 induced by OGD/R to M1 type and reduces inflammation, which is related to JAK1/STAT6 pathway. -
Key words:
- upadacitinib /
- OGD/R /
- polarization, inflammation, JAK/STAT
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