脑通汤对缺氧-复氧损伤脑微血管内皮细胞的保护作用

Study on Protective Effects of Naotong Soup on Brain Microvascular Endothelial Cell Injury Induced by Hypoxia-reoxygenation

目的:探讨脑通汤对缺氧-复氧损伤脑微血管内皮细胞(BMECs)的保护作用及机制。方法:将大鼠原代BMECs培养传代至第二代,分为对照组、正常血清组、缺氧-复氧模型组、大、中、小剂量脑通汤含药血清组,后5组细胞均放入三气培养箱中持续通入95%N2和5%CO2混合气体缺氧24 h再复氧2 h制作脑缺血再灌注细胞模型;采用MTT法检测缺氧24 h和复氧2 h时BMECs的活性并计算细胞存活率,实时荧光定量PCR法检测BMECs中HIF-1α、VEGF、MMP-9 mRNA的表达。结果:缺氧24 h时,模型组的BMECs的活性及存活率较对照组明显下降(P<0.05),与正常血清组及各剂量含药血清组比较差异无统计学意义(P>0.05);复氧2 h时各剂量含药血清组BMECs的活性及存活率均较模型组明显升高(P<0.05),正常血清组与模型组差异无统计学意义(P>0.05);模型组HIF-1α、VEGF、MMP-9 mRNA的表达量均升高(P<0.05),与模型组比较,各剂量含药血清组的HIF-1α、VEGF mRNA的表达量明显升高,MMP-9 mRNA的表达量明显下降,脑通汤大、中剂量组变化更显著(P<0.01);正常血清组与模型组比较上述3种mRNA表达量差异无统计学意义(P>0.05)。结论:脑通汤可以保护缺氧-复氧损伤的BMECs,其机制可能与其能上调HIF-1α、VEGF mRNA表达以及下调MMP-9mRNA表达有关。

Objective： To investigate the protective effects of Naotong soup on brain microvascular endothelial cell（BMECs） injury induced by hypoxia-reoxygenation and its mechanism. Methods： Primary rat BMECs were identified and passaged to the second generation. BMECs were divided into control group, normal serum group, hypoxia-reoxygenation model group, large, medium and small dose of Naotong soup medicinal serum group, BMECs in the later 5 groups were put into the three gas incubator for continuous infusion of mixture gas （95% N2 and 5% CO2 ） for 24 h and reoxygenation for 2 h to establish the model of cerebral ischemia and reperfusion. The activity and the survival rate of BMECs at 24 hours after hypoxia and 2 hours after reoxygenation were evaluated by MTr assay. Real-time fluorescent quantitative PCR was adopted to detect the relative expression levels of HIF-1 or, VEGF and MMP-9 mRNA. Results： Compared with normal cell groups, BMECs activity and the survival rate of model groups was significantly lower after hypoxia for 24 h （ P 〈 0.05 ） in the later five groups, andthere was no significant difference among the later 5 groups （P 〉 0.05 ）. Compared with model group, different dose of Naotong soup medicinal serum groups were significantly increased after reoxygenation for 2 h（P 〈0.05）. The relative expression levels of HIF-1α, VEGF and MMP-9 mRNA increased in model group （ P 〈 0.05 ）. Compared with the model group, the relative mRNA expression levels of HIF-1α and VEGF increased and MMP-9 mRNA decreased in Naotong soup medicinal serum groups, the changes were more significant in large, medium Naotong soup medicinal serum groups（ P 〈 0.05 ）. However,there was no significant difference between normal serum group and model group （P 〉 0. 05 ）. Conclusions： Naotong soup can protect BMECs after hypoxia -reoxygenation injury. Its mechanism may relate to up-regulate HIF-1 α and VEGF mRNA levels and down-regulate MMP-9 levels.