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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。
AIM: To investigate the susceptibility of vascular endothelial cells to oxidative low density lipoprotein (LDL) stimulation and the regulatory mechanism of downstream oxidative stress signaling pathway under hypoxic-hypoglycemic impairment. Methods: The cells were divided into normal control group, OGD group for 6h, ox-LDL group for 100μg / ml ox-LDL treatment, OGD 6h + 100μg / ml ox-LDL Stimulation 24h group (OGD + ox-LDL group), separately treated rat cerebral vascular endothelial cells (RBECs). Western blot was used to detect the changes of caveolin-1 and lectin-like oxidized low density lipoprotein receptor (LOX-1) in cerebrovascular endothelial cells and the effects of downstream oxidative stress MAPK signal pathway and endothelial nitric oxide Synthase (eNOS). Fluorescence intensity of Dil-ox-LDL (Dil-ox-LDL) in RBIs was detected by immunofluorescence. The expression of inflammatory factors, namely nitric oxide (NO), interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) were detected by colorimetric method and ELISA. Results: OxLDL stimulation could increase the Dil-ox-LDL fluorescence intensity of LOX-1 and RBECs and decrease the expression of Cav-1. Compared with normal control group, LOX-1 expression and Dil-ox-LDL fluorescence intensity in OGD group were not significantly increased, but Cav-1 expression decreased, however, LOX-1 expression and Dil-ox- LDL fluorescence intensity increased, and compared to ox-LDL group, there was a significant difference. In addition, except for the normal control group, the activation of p38, ERK1 / 2 and JNK oxidative stress pathways and the release of NO, IL-1β and TNF-αin MAPK signaling pathway were observed in all RBECs, and OGD + ox- LDL group compared with OGD group, ox-LDL group were significantly different. CONCLUSION: RBECs are susceptible to the stimulation of ox-LDL under the stimulation of OGD and ox-LDL, which can increase the fluorescence intensity of Dil-ox-LDL in cells and induce the downstream oxidative stress. The mechanism may be related to Cav- 1 regulation of oxidative stress response, this study may provide a scientific basis for lipid metabolism related diseases such as ischemic stroke represented by OGD cell model.