Lyu Yanni, Fu Longsheng, Li Yanming, Wen Jinhua, Wei Xiaohua, Qian Yisong
Department of Pharmacy, First Affiliated Hospital, Nanchang University, Nanchang 330006, China. *Corresponding author, E-mail:
Department of Pharmacy, First Affiliated Hospital, Nanchang University, Nanchang 330006, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2018 Mar;34(3):211-217.
Objective To study the effect of the administration of the circulating microvesicles (MV) obtained from lipopolysaccharide (LPS)-stimulated microglia supernatant into rat brain endothelial cells (RBECs) on the injury of tight junction in RBECs under the condition of oxygen-glucose deprivation (OGD) as well as the underlying mechanism. Methods The circulating MV was isolated from the supernatant of microglia stimulated with LPS 1 mg/L for 24 hours and subjected to morphological identification. The expression of miR-27a in MV was detected by real-time PCR. RBECs were randomly divided into control group, MV-RBECs control group, OGD 6-hour group and OGD-MV group. The expressions of occludin and claudin-5 were detected by immunofluorescence staining in the four groups. Western blot analysis was used to investigate the expressions of occludin, claudin-5, Toll-like receptor 4 (TLR4), NF-κBp65 and p38 proteins in RBECs, and ELISA was applied to detect the levels of interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) in RBECs. Results The shape of MV was approximately circular double membrane vesicles, with an average diameter of 150 nm, in accordance with the morphological characteristics of MV. Under LPS stimulation, the level of miR-27a in the circulating MV was abnormally elevated. Compared with the control group, RBECs were not obviously influenced by the incubation of MV; under OGD condition, tight junction of RBECs was damaged with the decreasing expressions of occluding and claudin-5. The degree of injury was further damaged after the treatment with MV. Fluorescence intensity of occludin and claudin-5 were further reduced. Meanwhile, Western blot analysis showed the levels of occludin and claudin-5 proteins decreased in the OGD group after MV treatment, which was consistent with immunofluorescence staining. Compared with the control group, the expression of TLR4 protein and the phosphorylation of NF-κBp65 and p38 proteins increased in OGD group; after MV treatment, the level of TLR4 protein and the phosphorylation of NF-κBp65 and p38 proteins further increased, and the release of IL-1β and TNF-α increased as well. Conclusion Treatment with the circulating MV containing miR-27a obtained from LPS-stimulated microglia supernatant damages the tight junction of RBECs under the OGD condition. The mechanism may be related to up-regulation TLR4 and phosphorylation of NF-κBp65 and p38.
目的 研究将脂多糖（LPS）刺激的小胶质细胞上清液中获得的循环微泡（MV）注入大鼠脑内皮细胞（RBECs）对氧糖剥夺（OGD）条件下RBECs紧密连接损伤的影响及其潜在机制。方法 从用1 mg/L LPS刺激24小时的小胶质细胞上清液中分离循环MV并进行形态学鉴定。通过实时PCR检测MV中miR-27a的表达。将RBECs随机分为对照组、MV-RBECs对照组、OGD 6小时组和OGD-MV组。采用免疫荧光染色检测四组中闭合蛋白和紧密连接蛋白-5的表达。用蛋白质印迹法检测RBECs中闭合蛋白、紧密连接蛋白-5、Toll样受体4（TLR4）、核因子κB p65（NF-κBp65）和p38蛋白的表达，并用酶联免疫吸附测定法检测RBECs中白细胞介素-1β（IL-1β）和肿瘤坏死因子α（TNF-α）的水平。结果 MV的形态为近似圆形的双膜囊泡，平均直径为150 nm，符合MV的形态特征。在LPS刺激下，循环MV中miR-27a水平异常升高。与对照组相比，MV孵育对RBECs无明显影响；在OGD条件下，RBECs的紧密连接受损，闭合蛋白和紧密连接蛋白-5的表达降低。MV处理后损伤程度进一步加重，闭合蛋白和紧密连接蛋白-5的荧光强度进一步降低。同时，蛋白质印迹分析显示MV处理后OGD组中闭合蛋白和紧密连接蛋白-5的蛋白水平降低，与免疫荧光染色结果一致。与对照组相比，OGD组中TLR4蛋白表达及NF-κBp65和p38蛋白磷酸化增加；MV处理后，TLR4蛋白水平及NF-κBp65和p38蛋白磷酸化进一步增加，IL-1β和TNF-α的释放也增加。结论 用从LPS刺激的小胶质细胞上清液中获得的含miR-27a的循环MV处理会在OGD条件下损伤RBECs的紧密连接。其机制可能与TLR4上调及NF-κBp65和p38磷酸化有关。
