川芎嗪与黄芪甲苷配伍对过氧化氢诱导人脐静脉内皮细胞氧化损伤的保护作用及机制

Protective Effect and Mechanism of Ligustrazine Combined with Astragaloside on Oxidative Stress Injury of Human Umbilical Vein Endothelial Cells Induced by Hydrogen Peroxide

目的研究川芎嗪、黄芪甲苷及二者不同浓度配伍对过氧化氢(H_2O_2)诱导人脐静脉内皮细胞(HUVECs)氧化损伤模型的保护作用及机制。方法建立H2O2诱导HUVECs损伤模型,将细胞分为17组:空白对照组,模型组(H_2O_20.2 mmol·L^(-1))、川芎嗪低、中、高(40,80,160μg·m L^(-1))剂量组,黄芪甲苷低、中、高(10,20,40μg·m L^(-1))剂量组,川芎嗪+黄芪甲苷两两配伍组。噻唑蓝(MTT)法检测不同浓度川芎嗪、黄芪甲苷及其配伍对氧化损伤HUVECs增殖的影响,筛选出最佳配伍比例。检测最佳配伍组丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性,Annexin V-FITC/PI双染法检测细胞凋亡率,Western Blot检测p-Akt/Akt蛋白的表达,RT-PCR检测Akt m RNA的表达。结果与模型组相比,川芎嗪中剂量组、黄芪甲苷各剂量组及其配伍组均能不同程度提高细胞存活率(P<0.05,P<0.01,P<0.001),且川芎嗪(80μg·m L^(-1))+黄芪甲苷(40μg·m L^(-1))剂量组细胞存活率最高,选为最佳配伍组;川芎嗪(80μg·m L^(-1))+黄芪甲苷(40μg·m L^(-1))剂量组能降低细胞MDA含量(P<0.001),提高细胞SOD活力(P<0.001),降低细胞凋亡率(P<0.01),上调p-Akt/Akt蛋白及Akt基因m RNA的表达(P<0.001)。结论川芎嗪(80μg·m L^(-1))+黄芪甲苷(40μg·m L^(-1))剂量组对H_2O_2诱导的HUVECs氧化损伤有保护作用,能缓解氧化应激诱导的HUVECs凋亡,其机制与PI3K/Akt信号通路有关。

Objective To study the protective effect and mechanism of ligustrazine（Lig）combined with astragaloside（As） on H2O2 injury of human umbilical vein endothelial cells（HUVECs） induced by hydrogen peroxide（H2O2）.Methods The HUVECs model of oxidative injury was established by H2O2. The HUVECs for pharmacodynamics study were divided into 17 groups, blank control group, model group（H2O20.2 mmol·L-1）, there Lig groups（40,80,160 μg·m L-1）,there As groups（10,20,40 μg·m L-1） and 9 Lig＋As compatibility groups. The morphological changes in cell proliferation of each group after oxidative stress injury were detected by MTT assay,and the optimal compatibility proportion was confirmed after the evaluation of the cell proliferation. The content of malondialdehyde（MDA）and superoxide dismutase（SOD）activity in the supernatant of the optimal compatibility groups were detected.The apoptotic rate of HUVECs was analyzed by Annexin V-FITC/PI staining under fluorescence microscope. Western blot was used to examine the protein expression of p-Akt and Akt,and RT-PCR was used to detect the expression of Akt m RNA. Results Compared with that in the model group, cell viability of Lig-As compatibility groups was significantly increased（P〈0.05,P〈0.001,P 0.0001）,and the difference of cell viability in Lig（80 μg·m L-1）＋As（40 μg·m L-1）combined group was obviously significant（P〈0.001）. The level of MDA was reduced（P〈0.001）,SOD activity was enhanced（P〈0.001）,apoptotic rate was decreased（P〈0.01）,and p-Akt/Akt protein expression as well as Akt m RNA expression was up-regulated（P〈0.001）in Lig（80 μg·m L-1）＋AS（40 μg·m L-1）compatibility group. Conclusion The combination of Lig（80 μg·m L-1） and As（40 μg·m L-1） can protect HUVECs from the oxidative stress injury induced by H2O2,and its mechanism may be correlated with the PI3K/Akt signaling pathway.