Susceptibility and mechanism of brain microvascular endothelial cells injury with ox-LDL stimulation under oxygen-glucose deprivation condition
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摘要: 目的:观察缺氧缺糖性损伤条件下, 血管内皮细胞对氧化型低密度脂蛋白刺激易感性的考察, 以及下游氧化应激信号通路的调控机制。方法:将细胞分为正常对照组、氧糖剥夺 (OGD) 组6h、100μg/ml氧化型低密度脂蛋白ox-LDL处理24h组 (ox-LDL组)、OGD 6h+100μg/ml ox-LDL刺激24h组 (OGD+ox-LDL组), 分别处理分离的大鼠脑血管内皮细胞 (RBECs)。Western blot检测脑血管内皮细胞小凹蛋白1 (Cav-1)、凝集素样氧化型低密度脂蛋白受体 (LOX-1) 变化, 及考察下游氧化应激MAPK信号通路和内皮型一氧化氮合酶 (eNOS)。免疫荧光检测RBECs中Dil细胞膜荧光标记的ox-LDL (Dil-ox-LDL) 荧光强度的变化。比色法和ELISA法检测炎症因子即一氧化氮 (NO)、白细胞介素1β (IL-1β) 和肿瘤坏死因子-α (TNF-α) 的表达。结果:oxLDL刺激可对增加LOX-1表达和RBECs的Dil-ox-LDL荧光强度, 并同时降低Cav-1表达。OGD组LOX-1表达和Dil-ox-LDL荧光强度相较于正常对照组无明显增加, 然而Cav-1表达有所降低, 然而在OGD+ox-LDL组LOX-1表达和Dil-ox-LDL荧光强度增加, 且相较于ox-LDL组, 有显著性差异。另外, 除正常对照组外, 各组RBECs均观察到MAPK信号通路中p38、ERK1/2、JNK氧化应激通路激活和NO、IL-1β、TNF-α炎症因子释放增加, 并且OGD+ox-LDL组相较于OGD组、ox-LDL组均有显著性差异。结论:RBECs在OGD和ox-LDL共同刺激条件下, 对ox-LDL的刺激具有易感性, 可在增加细胞内Dil-ox-LDL荧光强度, 并且诱导下游氧化应激反应, 机制可能与Cav-1调控氧化应激反应相关, 本研究可为以OGD细胞模型为代表的缺血性脑卒中等疾病相关脂质代谢研究提供科学根据。Abstract: Objective:To investigate the susceptibility of brain microvascular endothelial cells injury with oxLDL stimulation under oxygen-glucose deprivation condition, and the regulating mechanism of downstream oxidative stress signaling pathway.Method:The brain microvascular endothelial cells were divided into control group, OGD (Oxygen-glucose deprivation) 6 htreatment group, 100μg/ml ox-LDL (oxidatively modified low density lipoprotein) 24 h treatment group.OGD 6 h+100μg/mL ox-LDL 24 h treatment group were separately subjected to deal with vascular endothelial cells.Western blot was applied to detect Cav-1 and LOX-1 changes, oxidative stress MAPK sighaling pathway and eNOS expression in brain microvascular endothelial cells.Immunohistochemistry was used to detect the intensity of Dil-ox-LDL fluorescence changes in RBECs.Chemocolorimetry and ELISA method were applied to investigate the release of NO, IL-1β and TNF-αinflammatory cytokines.Result:ox-LDL could stimulate the increased expression of LOX-1 and the enhanced fluorescence intensity of Dil-ox-LDL in RBECs, and downregulated the expression of Cav-1.The expression of LOX-1 and Dil-ox-LDL in RBECs under OGD condition had no significant increase compared with control group, however, with the decreasing expression of Cav-1.Meanwhile, under the coupling stimaluation of both OGD and ox-LDL, the expression of LOX-1 and oxLDL got both increasing, and had significant difference compared with ox-LDL group.Also, except for the control group, p38, ERK1/2 and JNK in oxidative stress MAPK sighaling pathway and NO, IL-1β and TNF-α inflammatory cytokines were activated in all the groups, while the activated indexes in LOX-1 and ox-LDL group had significant difference compared with ox-LDL group.Conclusion:Vascular endothelial cells had the susceptibility to ox-LDL stimulation under OGD+ox-LDL condition, with the enhanced fluorescence intensity in RBECs, and the activation of related oxidative stress responses, while had the relationship with the oxidative stress response regulated with Cav-1.This study could provide the scientific basis for the representative diseases research in related lipid metabolism in OGD condition model.
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