摘要
目的探究PEG化甲氧基雌二醇(polyethylene glycol-2-methoxyestradiol,PEG-2ME_(2)/2ME_(2))纳米粒对糖氧剥夺/再灌注(oxygen glucose deprivation/reperfusion,OGD/R)后脑血管内皮细胞RBE4的影响。方法通过溶剂挥发法构建PEG-2ME_(2)/2ME_(2)纳米粒,通过透射电镜(transmission electron microscope,TEM)和激光粒度分析仪检测纳米粒表征;通过CCK-8法检测PEG-2ME_(2)/2ME_(2)纳米粒对RBE4的细胞毒性;使用OGD/R体外模拟缺血再灌注,通过CCK-8法检测细胞活性,采用Western blot法和免疫荧光法对紧密连接蛋白的表达情况进行检测,以及通过建立体外血脑屏障(blood-brain barrier,BBB)模型和检测荧光探针渗漏情况综合评估脑血管内皮细胞的屏障功能;利用活性氧(reactive oxygen species,ROS)探针检测RBE4细胞内ROS水平;采用Western blot法检测过氧化物歧化酶判断RBE4细胞内氧化还原情况。结果透射电镜结果显示,PEG-2ME_(2)/2ME_(2)纳米粒球形形态稳定;激光粒度分析仪结果显示,PEG-2ME_(2)/2ME_(2)纳米粒大小均一;CCK-8法检测结果显示,PEG-2ME_(2)/2ME_(2)纳米粒中2ME_(2)浓度小于5μmol/L对RBE4细胞无明显的细胞毒性;CCK-8法检测结果显示,PEG-2ME_(2)/2ME_(2)纳米粒较2ME_(2)单药能显著挽救OGD/R后RBE4细胞的细胞活性,能遏止紧密连接蛋白表达下调,能有效恢复脑血管内皮细胞的屏障功能;ROS探针检测和Western blot法检测结果显示,PEG-2ME_(2)/2ME_(2)纳米粒较2ME_(2)单药能明显降低细胞内ROS水平,恢复超氧化物歧化酶的表达。结论PEG-2ME_(2)/2ME_(2)纳米粒大小、形态均一稳定,对RBE4细胞起到保护作用,有效恢复血脑屏障的完整性和屏障功能,有望成为OGD/R后脑血管内皮屏障损伤治疗的潜在药物。
Objective To explore the effects of polyethylene glycol-2-methoxyestradiol(PEG-2ME_(2)/2ME_(2))nanoparticles on cerebral vascular endothelial RBE4 cells after oxygen glucose deprivation/reperfusion(OGD/R).Methods To construct the PEG-2ME_(2)/2ME_(2) nanoparticles by solvent evaporation method,and the characterization of nanoparticles was detected by transmission electron microscope(TEM)and laser particle size analyzer;the cellular cytotoxicity of PEG-2ME_(2)/2ME_(2) nanoparticles was detected by CCK-8.Using OGD/R to simulate the process of ischemia-reperfusion in vitro,to detect the cell activity by CCK-8;Western blot and immunofluorescence were used to detect the expression of tight junction proteins,and the barrier function of cerebrovascular endothelial cells was comprehensively evaluated by establishing blood-brain barrier(BBB)model in vitro and measuring fluorescence probe leakage;reactive oxygen species(ROS)probe was used to detect the ROS level of RBE4 cells,and Western blot was used to detect superoxide dismutase to identify the redox status of RBE4 cells.Results The results of TEM showed that the spherical morphology of PEG-2ME_(2)/2ME_(2) nanoparticles was stable,and the results of laser particle size analyzer showed that the size of PEG-2ME_(2)/2ME_(2) nanoparticles was uniform;the results of CCK-8 assay showed that the PEG-2ME_(2)/2ME_(2) nanoparticles had no significant cytotoxicity to RBE4 cells within 5μmol/L of 2ME_(2);the results of CCK-8 assay showed that PEG-2ME_(2)/2ME_(2) nanoparticles rescued the cellular activity of RBE4 cells after OGD/R compared to the 2ME_(2) alone,inhibited the downregulation of tight junction protein expression,and effectively restored the barrier function of cerebral vascular endothelial cells;the detection results of ROS probe and Western blot showed that PEG-2ME_(2)/2ME_(2) nanoparticles reduced intracellular ROS levels,restored the expression of superoxide dismutase compared to the 2ME_(2) alone.Conclusion PEG-2ME_(2)/2ME_(2) nanoparticles have uniform size and stable morphology,which can protect RBE4 cells against OGD/R,effectively restore the integrity and barrier function of the blood-brain barrier.The PEG-2ME_(2)/2ME_(2) nanoparticles are expected to become potential drugs for the treatment of cerebrovascular endothelial barrier injury after OGD/R.
作者
夏艺洋
王心涛
邹晨明
崔德荣
XIA Yiyang;WANG Xintao;ZOU Chenming(Department of Anesthesiology,Shanghai Sixth People′s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine,Shanghai 200233,China;不详)
出处
《医学研究杂志》
2024年第9期32-37,6,共7页
Journal of Medical Research
基金
国家自然科学基金资助项目(81974284,82202425)
上海交通大学医工交叉基金资助项目(YG2021GD03)。
关键词
糖氧剥夺/再灌注
血脑屏障
氧化应激
Oxygen glucose deprivation/reperfusion
Blood-brain barrier
Oxidative stress
