清开灵有效组分对大鼠脑微血管内皮细胞体外缺血再灌注损伤模型核转录因子-κB的影响

目的：建立大鼠脑微血管内皮细胞体外缺血再灌注损伤模型，探讨清开灵有效组分牛胆酸、猪胆酸、黄芩苷、栀子苷和珍珠母对其的保护作用是否与调节核转录因子-κB（nuclearfactor—kappa B，NF—κB）活化有关。 方法：体外培养大鼠脑微血管内皮细胞（microvascular endothelial cell，MVEC），氧糖剥夺法模拟缺血再灌注损伤，建立体外缺血再灌注损伤模型。随机分为模型组、尼莫地平组、猪胆酸组、黄芩苷组、栀子苷组、珍珠母组和牛胆酸组，并设正常MVEC为正常对照组，每组设6孔。使用免疫细胞化学法观察NF—κB蛋白表达情况并进行图像分析。 结果：光学显微镜下观察，正常组MVEC胞核的位置形成空腔，胞浆内见淡棕黄色均匀颗粒。模型组NF—κB细胞核的染色强度明显高于胞浆，活化后移位至细胞核；用药各组NF-κB在胞浆近胞核处有少量阳性表达，在胞核的表达明显弱于模型组。对各组MVECNF-κB表达强度的图像分析显示，模型组胞核与胞浆的透光度比值低于正常对照组，差异有统计学意义（P〈0．01），用药各组透光度比值显著高于模型组（P〈0．05，P〈0．01）。 结论：清开灵有效组分可能通过拮抗NF—κB活化抑制内皮细胞损伤。

Effects of Qingkailing effective components on nuclear factor-kappa B in an ischemia-reperfusion injury model of rat brain microvascular endothelial cells in vitro

Objective： To establish an ischemia-reperfusion injury model of rat cerebral microvascular endothelial cells （MVECs） in vitro, and to explore the relationship between nuclear factor-kappa B （NF-κB） and the protective effects of Qingkailing effective components （hyocholic acid, taurocholic acid, baicalin, jasminoidin, Pinctada martensii） on MVECs.
Methods： Brain MVECs of male rats were digested with trypsin and subcultured, then the content of MVECs was adjusted to 1×10^5/mL and the MVECs were divided into normal control group, untreated group, hyocholic acid group, taurocholic acid group, baicalin group, jasminoidin group, Pinctada martensii group and nimodipine group, with six holes in each group. Except for the normal control group, the MVECs in the other groups were exposed in oxygen and glucose deprivation （OGD） circumstance in vitro to simulate ischemia-reperfusion injury. Immunocytochemical staining and image analysis system were used to observe the expression of NF-κB protein.
Results： Under a light microscope, the nuclei of MVECs in the normal control group were blank. Staining intensity of NF-κB protein in the nucleus in the untreated group was much deeper than that in the endochylema, with NF-κB shifted to nucleus after activation; a small quantity of NF-κB protein were expressed in the border of nucleus next to endochylema in groups of Qingkailing effective components, and the NF-κB protein expression was weaker than that in the untreated group. With the image analysis, we found that transmittance of nucleus and endochylema in the untreated group was significantly lower than that in the normal control group （P〈0.01）. Transmittance of nucleus and endochylema in the treated groups was higher than that in the untreated group （P〈0.05, P〈0.01）.
Conclusion： Qingkailing effective components have significant effect in inhibiting NF-κB protein transferring from endochylema to nucleus in vitro.