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目的观察黄芪、川芎中的有效成分黄芪甲苷、川芎嗪、阿魏酸、毛蕊异黄酮、藁本内酯的正交组合配伍对SD乳鼠脑微血管内皮细胞(rBMVECs)缺氧损伤的保护作用,并找出较优组合。方法 rBMVECs原代传代培养,并建立缺氧模型,4h后观察细胞形态,MTT法测定各成分的最大耐药质量浓度,以L_(18)(3~5)正交表设计安排成分配伍给药,采用黄嘌呤氧化酶显色法检测细胞超氧化物歧化酶(SOD)活性,流式细胞仪检测细胞凋亡率及细胞周期,RT-PCR法测定caspase-3与caspase-8基因在细胞中的表达。采用极差分析法分析正交试验结果。结果药物正交配伍组合可显著改善缺氧rBMVECs的形态,有效增强SOD活性,有效抑制缺氧诱导的rBMVECs发生Gl/S期阻滞,明显减少了细胞的凋亡,减弱细胞中caspase-3和caspase-8基因表达。正交试验结果分析显示其较优组合为藁本内酯5μg/mL、川芎嗪10μg/mL、阿魏酸20μg/mL、毛蕊异黄酮10μg/mL、黄芪甲苷10μg/mL。结论黄芪、川芎有效成分组合配伍对内皮细胞缺氧损伤有明显的保护作用。
Objective To observe the protective effects of the orthogonal combination of astragaloside, ligustrazine, ferulic acid, calycosin and ligustilide on the injury of cerebral microvascular endothelial cells (rBMVECs) And find a better combination. Methods The primary culture of rBMVECs was subcultured, and hypoxia model was established. After 4 hours, the cell morphology was observed. The maximum drug concentration of each component was determined by MTT assay. The components were designed and arranged with L_ (18) (3 ~ 5) The activity of superoxide dismutase (SOD) was detected by xanthine oxidase colorimetric assay. The apoptosis rate and cell cycle were detected by flow cytometry. The expressions of caspase-3 and caspase-8 were detected by RT- expression. Analysis of Orthogonal Test Results by Range Analysis. Results Orthogonal test showed that the morphological changes of rBMVECs were significantly improved and the activity of SOD was effectively increased. The inhibition of G1 / S arrest induced by hypoxia could effectively inhibit the apoptosis of rBMVECs and reduce the apoptosis rate of caspase-3 and caspase-8 gene expression. Orthogonal test results showed that the optimal combination of ligustilide 5μg / mL, ligustrazine 10μg / mL, ferulic acid 20μg / mL, calycosin 10μg / mL, Astragaloside 10μg / mL. Conclusion Radix Astragali and Rhizoma Chuanxiong combined with the active ingredients have a significant protective effect on endothelial cell hypoxia injury.