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该研究主要探讨3种桂枝汤苯丙烯类化合物(桂皮醇、桂皮醛和桂皮酸)对氧化低密度脂蛋白(ox-LDL)诱导人脑微血管内皮细胞(HBMEC)氧化应激损伤的保护作用。实验通过MTT活力试验确定3种桂枝汤苯丙烯类化合物和ox-LDL对HBMEC的增殖和损伤浓度,将处于对数生长期HBMEC分为空白对照组(DMSO)、模型对照组(ox-LDL)、叔丁基对苯二酚(t-BHQ)组、桂皮醇组、桂皮醛组和桂皮酸组。模型对照组予以终浓度50 mg·L~(-1)的ox-LDL,孵育24 h造成细胞损伤模型,再予以终浓度20μmol·L~(-1)的t-BHQ,桂皮醇,桂皮醛和桂皮酸,孵育24 h后,MTT法检测细胞存活率,激光共聚焦扫描显微镜观察活性氧族(ROS)荧光强度,ELISA测定各组细胞内内源性氧化应激指标(SOD,MDA,NO,eNOS)的含量,qRT-PCR检测Nrf2 mRNA表达量。实验结果发现:ox-LDL能够诱导HBMEC内源性氧化应激损伤,3种桂枝汤苯丙烯类化合物对正常HBMEC细胞活性无抑制作用。与模型对照组比较,3种桂枝汤苯丙烯类化合物可显著或极显著的改变HBMEC氧化应激状态,上调损伤细胞Nrf2 mRNA表达量(P<0.05,P<0.01),其综合效应:桂皮醛>特丁基对苯二酚>桂皮酸>桂皮醇,表明3种桂枝汤苯丙烯类化合物(20μmol·L~(-1))对ox-LDL(50 mg·L~(-1))诱导的HBMEC氧化应激损伤有较好的保护作用,其机制可能与核转录因子Nrf2调控其下游抗氧化酶基因表达相关。
The aim of this study was to investigate the protective effects of three kinds of Guizhi Tang compounds (cinnamic alcohol, cinnamaldehyde and cinnamic acid) on the oxidative stress injury induced by ox-LDL in human brain microvascular endothelial cells (HBMEC) . The MTT assay was used to determine the proliferation and injury of HBMEC induced by three kinds of Guizhi Tang compounds and ox-LDL. The logarithmic growth phase was divided into two groups: control group (DMSO), model group (ox-LDL ), T-butyl hydroquinone (t-BHQ), cinnamyl alcohol, cinnamaldehyde and cinnamic acid. The model control group was treated with ox-LDL at a final concentration of 50 mg · L -1 for 24 h, then the cells were induced to undergo a cell injury model by t-BHQ, cinnamaldehyde and cinnamaldehyde at a final concentration of 20 μmol·L -1 And cinnamic acid. After incubation for 24 h, the cell viability was detected by MTT assay. The fluorescence intensity of reactive oxygen species (ROS) was observed by laser scanning confocal microscopy. The levels of endogenous oxidative stress (SOD, MDA, NO , eNOS). The expression of Nrf2 mRNA was detected by qRT-PCR. The results showed that: ox-LDL can induce endogenous HBMEC oxidative stress injury, three kinds of Guizhitang benzene compounds on normal HBMEC cell activity was not inhibited. Compared with the model control group, three kinds of Guizhi Tang benzene compounds can significantly or very significantly change the oxidative stress state of HBMEC, up-regulate the expression of Nrf2 mRNA in injured cells (P <0.05, P <0.01) Aldehyde> t-butyl hydroquinone> cinnamic acid> cinnamic alcohol, which indicated that the effect of three kinds of Guizhi Soup (20μmol·L -1) on ox-LDL (50 mg · L -1) ) Induced HBMEC oxidative stress injury. The mechanism may be related to the regulation of downstream antioxidant enzyme gene expression by nuclear transcription factor Nrf2.