髓样细胞激活受体2调控氧糖剥夺/复氧模型小鼠小胶质细胞向M2型极化

doi:10.16098/j.issn.0529-1356.2021.03.001

摘要/Abstract

摘要：

目的探讨TREM2调控氧糖剥夺/复氧（OGD/R）模型小鼠小胶质细胞向M2型极化的机制。方法体外培养N9小胶质细胞系，采用TREM2过表达慢病毒（LV-TREM2）转染N9细胞，空载病毒LV-scramble作为对照组，并建立OGD/R模型。将OGD/R细胞随机分为OGD/R组、OGD/R+LV-scramble组和OGD/R+LV-TREM2组。Real-time PCR检测TREM2 mRNA在OGD/R 组细胞复氧72 h内表达情况。免疫荧光染色观察慢病毒LV-scramble 和LV-TREM2在正常N9小胶质细胞内转染情况，Real-time PCR和Western blotting验证慢病毒转染效率，Real-time PCR和ELISA检测小胶质细胞M1型标志物肿瘤坏死因子 α（TNF-α）、白细胞介素1β（IL-1β）和诱生型一氧化氮合酶（iNOS）及M2型标志物精氨酸酶 1（Arg-1）和IL-10 mRNA 和蛋白表达情况。Western blotting检测磷酸化-磷脂酰肌醇3激酶（p-PI3K）、PI3K、磷酸化蛋白激酶B（p-Akt）、Akt、磷酸化核因子κB抑制蛋白α（p-ⅠκBα）和ⅠκBα蛋白含量，免疫荧光分析核因子κB P65（NF-κB P65）蛋白在细胞内分布情况。结果复氧72 h内，OGD/R 组细胞TREM-2 mRNA含量明显增加，高峰位于24 h和48 h；慢病毒LV-TREM2可有效促进OGD/R组细胞TREM2 mRNA和蛋白表达（P<0.001，P<0.01）。与OGD/R组相比，OGD/R+LV-TREM2组TNF-α、IL-1β和iNOS mRNA和蛋白明显降低，而Arg-1和IL-10 mRNA和蛋白含量明显升高（P<0.05）；此外，OGD/R+LV-TREM-2组p-PI3K/PI3K和p-Akt/Akt比值明显升高（P<0.05），p-ⅠκBα/ⅠκBα比值显著下降（P<0.001），且NF-κB P65蛋白核转位明显被削弱。结论 TREM-2过表达可促进OGD/R 模型小胶质细胞向M2型转化从而发挥抗炎作用，该作用与TREM-2调控小胶质细胞PI3K/Akt和NF-κB信号通路有关。

关键词: 氧糖剥夺/复氧, 髓样细胞激活受体2, 磷脂酰肌醇-3激酶, 蛋白激酶B, 核因子κB, 免疫印迹法, 小胶质细胞

Abstract:

Objective To investigate the mechanism of TREM2 modulating the polarization of M2 microglia treated by oxygen-glucose deprivation/reoxygenation (OGD/R). Methods Mouse N9 microglial cells were cultured in vitro. N9 cells were transfected with lentivirus for TREM-2-overexpression (LV-TREM2), and LV-scramble acted as control group. OGD/R model was established. The OGD/R cells were randomly divided into OGD/R, OGD/R+LV-scramble and OGD/R+LV-TREM2 groups. Real-time PCR was used to detect the expression of TREM2 mRNA in OGD/R N9 cells within 72 hours after re-oxygenation. Immunofluorescence was applied to observe transfection of lentivirus LV-scramble and LV-TREM2 for normal N9 microglia, and Real-time PCR and Western blotting were used to verify the efficiency of lentivirus transfection. The mRNA and protein contents of M1 microglial markers tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS), M2 microglial markers arginase-1 (Arg-1) and interleukin-10 (IL-10) were detected by Real-time PCR and ELISA. The expressions of phosphorylated-phosphatidylinositol 3-kinases (p-PI3K), PI3K, phosphorylated protein kinase B （p-Akt), Akt, phosphorylated inhibitor of nuclear factor-κB (NF-κB)α (p-ⅠκBα) and ⅠκBα protein were detected by Western blotting. The distribution of NF-κB P65 (NF-κB P65) protein in N9 cells was analyzed by immunofluorescence method. Results TREM2 mRNA content in the OGD/R group cells increased significantly within 72 hours after re-oxygenation, and peaked at hour 24 and hour 48. Lentivirus LV-TREM2 effectively promoted the expression of TREM2 mRNA and protein of N9 cells in OGD/R model (P<0.001, P<0.01). Compared with the OGD/R group, the mRNA and protein content of TNF-α, IL-1β and iNOS decreased significantly, while Arg-1 and IL-10 in OGD/R+LV-TREM2 group increased significantly(P<0.05). Besides, the ratios of p-PI3K/PI3K and p-Akt/Akt increased obviously (P<0.05), the ratio of p-ⅠκBα/ⅠκBα decreased significantly in OGD/R+LV-TREM2 group (P<0.001), and the nuclear translocation of NF-κB P65 protein was obviously weakened. Conclusion TREM-2 overexpression exerts anti-inflammatory effect by modulating the polarization of microglia from M1 to M2 type, which is associated with PI3K/Akt and NF-κB signaling pathways regulated by TREM2 in N9 microglia with OGD/R model.

Key words: Oxygen-glucose deprivation/re-oxygenation, Triggering receptor expressed on myeloid cell 2, Phosphatidylinositol 3-kinase, Protein kinase B, Nuclear factor κB, Western blotting, Microglial cell

中图分类号:

R392.12

胥虹贝罗勇. 髓样细胞激活受体2调控氧糖剥夺/复氧模型小鼠小胶质细胞向M2型极化[J]. 解剖学报, 2021, 52(3): 329-336.

XU Hong-bei LUO Yong. Triggering receptor expressed on myeloid cell-2 modulating the polarization of mouse M2 microglia with oxygen-glucose deprivation/re-oxygenation model[J]. Acta Anatomica Sinica, 2021, 52(3): 329-336.

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