黄连解毒汤有效成分对缺氧/复氧时脑微血管内皮细胞核因子-κB的影响

Effect of effective components of Huanglian Jiedu decocting on NF-κB in cultured rat cerebral microvascular endothelial cells injury induced by hypoxia and reoxygenation

目的:研究栀子苷、黄芩苷和小檗碱对缺氧/复氧损伤时大鼠脑微血管内皮细胞的保护机制。方法:建立离体大鼠脑微血管内皮细胞缺氧4 h复氧12 h损伤模型。用0.128,0.064,0.032μmol.mL-1栀子苷,0.028,0.014,0.007μmol.mL-1黄芩苷和0.024,0.012,0.006μmol.mL-1小檗碱分别作用于损伤的细胞,采用免疫细胞化学方法和图象定量分析技术检测核因子-κB(NF-κB)亚单位p65的表达,计算平均吸光度和平均面积,比较NF-κB蛋白表达的变化,计算核移位百分率和细胞核与细胞浆的透光度比值比较NF-κB核移位的变化。结果:模型组NF-κB蛋白表达和核移位显著高于正常组(P<0.01)。所有给药组的平均吸光度值低于模型组,但差异无统计学意义。所有给药组的平均面积均低于模型组(P<0.01)。所有给药组的核移位百分率低于模型组且高于正常组(P<0.01,P<0.01)。所有给药组细胞核与细胞浆的透光度比值高于模型组(P<0.01)。结论:栀子苷、黄芩苷和小檗碱等黄连解毒汤有效成分能够保护缺氧/复氧损伤的大鼠脑微血管内皮细胞,其保护机制之一是抑制NF-κB蛋白表达和核移位。

Objective： To investigate the protective mechanism of geniposide, baicalin and berberine on hypoxia and reoxygenation injury in cultured rat cerebral microvascular endothelial cells. Method： To establish a model of hypoxia four hours and reoxygenation twelve hours injury in rat cerebral microvascular endothelial cells in vitro. The injured cells were treated with geniposide （0. 128, 0. 064, 0. 032 μ mol·mL^-1）, baicalin （0. 028, 0. 014, 0. 007 μmol·mL^-1） and berberine （0. 024, 0. 012, 0. 006 μmol·mL^-1）. The expression of p65 subunit of NF-κB was detected by immunocytochemical assay and techniques of image quantitative analysis. The protein expression of NF-κB was calculated with the mean optical density and mean area. The nuclear translocation of NF-κB was calculated with the percentage of positive cells and ratios of light transmittance of cytoplasm and cell nucleus. Result： Compared with the normal group, both the protein expression and the nuclear translocation of NF-κB of model group were significant increased （P〈0. 01）. Compared with the model group, the mean optical density of all treated groups was decreased ,but these was no significant difference between them. As compared with model group, the mean area of all treated groups was significant decreased （P〈0. 01）. The percentage of nuclear translocation of all treated groups is not only lower than that of the model group but higher than that of the normal group （P〈0. 01）. Compared with the model group, the ratios of light transmittance of cytoplasm and cell nucleus of all treated groups was significantly elevated （P〈0. 01）. Conclusion： The results suggesed that geniposide, baicalin and berberine could protect hypoxia/reoxygenation injuried rat cerebral microvascular endothelial cells injury. One of the mechanism may lie in inhibiting both the protein expression and the nuclear translocation of NF-κB.