观察丹参-川芎有效成分（丹参素、原儿茶醛、川芎嗪、阿魏酸）配伍对体外原代培养的海马神经元细胞缺糖缺氧（氧糖剥夺）损伤的保护作用，并优选较佳组合。方法 原代培养乳鼠海马神经元细胞，免疫组化法进行神经元特异性烯醇化酶（NSE）鉴定，并建立海马神经元细胞氧糖剥夺模型。MTT法确定丹参素、原儿茶醛、川芎嗪、阿魏酸及尼莫地平的非细胞毒性剂量范围，以L₉（3⁴）正交表设计安排成分配伍给药。将培养的细胞随机分为12组：对照组、模型组、尼莫地平阳性对照组及正交配伍1~9组。采用比色法、WST-1法、硫代巴比妥酸（TBA）法分别检测细胞上清液中乳酸脱氢酶（LDH）活力、超氧化物歧化酶（SOD）活性及丙二醛（MDA）水平，酶联免疫吸附试验检测细胞上清液中肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、IL-6水平变化，Hoechst33258荧光染色法观察海马神经元细胞凋亡情况，流式细胞术检测细胞早期凋亡率。采用极差分析法分析正交试验结果。结果 药物正交配伍组合均可显著改善缺氧海马神经元细胞的形态，显著减弱LDH活力，增强SOD的活性以及降低MDA的水平，显著抑制细胞TNF-α的释放，降低IL-1β和IL-6的水平，明显减少了细胞的凋亡。丹参-川芎有效成分对LDH活力的影响大小依次为丹参素 > 川芎嗪 > 原儿茶醛 > 阿魏酸，最佳配伍组合为丹参素（120 μg/mL）、原儿茶醛（120 μg/mL）、川芎嗪（80 μg/mL）、阿魏酸（20 μg/mL）。对SOD活性的影响主次因素依次为阿魏酸 > 川芎嗪 > 丹参素 > 原儿茶醛，最佳配伍组合为丹参素（120 μg/mL）、原儿茶醛（120 μg/mL）、川芎嗪（80 μg/mL）、阿魏酸（40 μg/mL）。对MDA水平的影响主次因素依次为丹参素 > 原儿茶醛 > 阿魏酸 > 川芎嗪，最佳配伍组合为丹参素（60 μg/mL）、原儿茶醛（60 μg/mL）、川芎嗪（80 μg/mL）、阿魏酸（20 μg/mL）。对TNF-α水平影响的主次因素依次为川芎嗪 > 原儿茶醛 > 丹参素 > 阿魏酸，最佳配伍组合为丹参素（60 μg/mL）、原儿茶醛（60 μg/mL）、川芎嗪（40 μg/mL）、阿魏酸（10 μg/mL）。影响IL-1β水平的主次因素依次为川芎嗪 > 阿魏酸 > 丹参素 > 原儿茶醛，最佳配伍组合为丹参素（30 μg/mL）、原儿茶醛（30 μg/mL）、川芎嗪（80 μg/mL）、阿魏酸（20 μg/mL）。影响IL-6水平的主次因素依次为原儿茶醛 > 川芎嗪 > 阿魏酸 > 丹参素，最佳配伍组合为丹参素（120 μg/mL）、原儿茶醛（120 μg/mL）、川芎嗪（80 μg/mL）、阿魏酸（10 μg/mL）。影响细胞早期凋亡率主次因素为阿魏酸 > 原儿茶醛 > 川芎嗪 > 丹参素，最佳配伍组合为丹参素（60 μg/mL）、原儿茶醛（30 μg/mL）、川芎嗪（20 μg/mL）、阿魏酸（40 μg/mL）。结论 丹参-川芎有效成分配伍对氧糖剥夺海马神经元细胞的保护作用机制可能与减轻氧化应激损伤、减轻炎症损伤和抑制细胞凋亡有关，可参考实验结果为临床不同病程需要更改组方配比，指导临床用药。

Objective To observe the protective effect of Salvia miltiorrhizae and Ligusticum chuanxiong effective constituents:danshensu, protocatechuic aldehyde, ligustrazine, and ferulic acid combination on primary cultured hippocampal neurons injured by oxygen glucose deprivation, and find out an optimized combination. Methods Primary cultured rats hippocampal neurons was chosen as research objects by adopting immunohistochemistry of the neuron-specific enolase IgG to authenticate, then the OGD model of the hippocampal neurons injured by oxygen glucose deprivation was established. The non-cytotoxic dose range of danshensu, protocatechuic aldehyde, ligustrazine, ferulic acid, and nimodipine was studied by MTT method. The compatibility of components was arranged by L₉ (3⁴) orthogonal design. Primary cultured rats hippocampal neurons was divided into 12 groups:control group, model group, Nimodipine positive control group, and orthogonal design 1-9 group. The activity of LDH was measured by colorimetry, the activity of SOD was tested by WST-1 and the levels of MDA were examined by TBA. The levels of TNF-α, IL-1β, and IL-6 in cell culture supernate were examined by ELISA, the apoptosis of hippocampal neurons was detected by Fluorochrome Hoechst33258 staining and the cell early apoptosis rate was detected with flow cytometry. The results of orthogonal test was analyzed by using range analysis method. Results The orthogonal compatibility of reagents played significant roles against the hypoxia damage of hippocampal neurons, improved the cellular morphology obviously, reduced the activity of LDH, increased the activity of SOD and decreased the content of MDA significantly, inhibited the release of TNF-α, and reduced the content of IL-1β and IL-6, reduced the apoptosis of cells apparently. The effect of active ingredients of S. miltiorrhiza and L. Chuanxiong on LDH activity was danshensu > ligustrazine > protocatechuic aldehyde > ferulic acid, and the best combination was danshensu (120 μg/mL), protocatechuic aldehyde (120 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (20 μg/mL). The effect on SOD activity was ferulic acid > ligustrazine > danshensu > protocatechuic aldehyde. The best combination was danshensu (120 μg/mL), protocatechuic aldehyde (120 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (40 μg/mL). The order of influence on MDA content was danshensu > protocatechuic aldehyde > ferulic acid > ligustrazine, and the best combination was danshensu (60 μg/mL), protocatechuic aldehyde (60 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (20 μg/mL). The effect on TNF-α content were:ligustrazine > protocatechuic aldehyde > danshensu > ferulic acid, and the best combination was danshensu (60 μg/mL), protocatechuic aldehyde (60 μg/mL), ligustrazine (40 μg/mL), and ferulic acid (10 μg/mL). The order of influence on the content of IL-1β was ligustrazine > ferulic acid > danshensu > protocatechuic aldehyde, and the best combination was danshensu (30 μg/mL), protocatechuic aldehyde (30 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (20 μg/mL). The effect on the content of IL-6 was protocatechuic aldehyde > ligustrazine > ferulic acid > danshensu, and the best combination was danshensu (120 μg/mL), protocatechuic aldehyde (120 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (10 μg/mL). The effect on the early apoptosis rate was ferulic acid > protocatechuic aldehyde > ligustrazine > danshensu, and the best combination was danshensu (60 μg/mL), protocatechuic aldehyde (30 μg/mL), ligustrazine (20 μg/mL), and ferulic acid (40 μg/mL). Conclusion The protective mechanism of the effective constituents of S. miltiorrhizae and L. chuanxiong were relied on reducing the oxidative damage, reducing inflammation damage, and antagonizing cell apoptosis. According to the experimental results, we need to change the prescription ratio and guide clinical medication for different clinical courses.

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国家自然科学基金资助项目（81630105）；国家自然科学基金资助项目（81874366）；浙江省自然科学基金资助项目（LZ17H270001）；浙江省自然科学基金资助项目（LZ18H270001）；浙江省自然科学基金资助项目（LQ19H270001）