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,4 h后观察细胞形态,MTT法测定各成分的最大耐药质量浓度,以L₁₈(3⁵) 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G₁/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5 μg/mL、川芎嗪10 μg/mL、阿魏酸20 μg/mL、毛蕊异黄酮10 μg/mL、黄芪甲苷10 μg/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 effect of orthogonal compatibility of astragaloside IV (A), ligustrazine (B), ferulic acid (C), calycosin (D), and ligustilide (E), the main components from Chuanxiong Rhizoma and Astragali Radix, on the brain micro vascular endothelial cells (rBMVECs) of SD neonatal rat damaged by hypoxia, and find out an optimized combination. Methods To prepare the normal primary rBMVECs from SD rats, to establish the hypoxia model, and to observe cellular morphology after 4 h; The maximum concentration of drug resistance was determined by MTT method. The compatibility of components was arranged by L₁₈ (3⁵) orthogonal design; The levels superoxide dismutase (SOD) was tested as kits instruction, the cells apoptosis rates and DNA stages were assayed with flow cytometry, and the expressions of caspase-3 and caspase-8 in cells were tested by using the method of real-time polymerase chain reaction (RT-PCR). Analyze the results of orthogonal test by using range analysis method. Results The orthogonal compatibility of reagents could play an significant roles against the hypoxia damage of rBMVECs, improve the cellular morphology obviously, enhance the activity of SOD, reduce the apoptosis of cells apparently, restrain the retardant of G₁/S stage induced by hypoxia effectively, and decrease the expression of caspase-3 and caspase-8 in cells. The analysis of test results shows that the combination with ligustilide 5 μg/mL, ligustrazine 10 μg/mL, ferulic acid 20 g/mL, calycosin 10 μg/mL, and astragaloside 10 μg/mL is the superior one. Conclusion The protective effect of the compatibility with the main components from Chuanxiong Rhizoma and Astragali Radix on hypoxia-damaged rBMVECs is obvious.

国家自然科学基金资助项目(81202636,81274176,81374053);浙江省自然科学基金资助项目(LR12H27001);浙江省中医药(中西医结合)重点学科(2012-XK-A06)