建立斑马鱼动脉粥样硬化模型筛选平台评价理气活血方剂抗动脉粥样硬化的疗效^*

Establish a Zebrafish Atherosclerosis Model Screening Platform to Evaluate the Therapeutic Effects of Qi-Regulating and Blood-Activating Prescriptions on Atherosclerosis

摘要

目的 利用基于多品系apoeb基因敲除斑马鱼模型的抗动脉粥样硬化（arterial atherosclerosis, AS）中药复方筛选平台，系统筛选出具有抗AS潜力的理气活血功效的中药复方。方法 基于方剂大辞典数据库进行筛选，从933首活血化瘀方剂中筛选出复方丹参方、手拈散、金铃子散、理气活血汤。采用AB、Tg（mpeg1:EGFP）、Tg（lyzc:dsRed）、Tg（gata1a:mCherry）、Tg（flk1:EGFP）5种品系apoeb^-/-斑马鱼给予高脂饲料喂食2 d后构建AS模型，在此基础上给予DMSO（溶剂对照）、瑞舒伐他汀组（西药阳性对照）、复方丹参方组（中药阳性对照）、手拈散、金铃子散、理气活血汤药浴24 h，行油红O染色，检测血脂含量、氧化应激指标、血流速度、血管内皮细胞损伤情况以及炎症因子在斑马鱼体内表达情况，观察斑马鱼体内炎症细胞行为模式，综合评估理气活血方剂抗AS疗效。结果 与模型组比较，复方丹参方和手拈散可改善血管内脂质堆积，降低总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）含量，减少氧化应激反应，抑制中性粒细胞和巨噬细胞募集，修复血管内皮损伤，改善血流速度，降低炎症因子表达，从而减轻AS，差异有统计学意义；而金铃子散及理气活血汤对AS的改善效果有限。结论 该平台为抗AS中药复方的筛选和研究评价提供了一种高效性且靶向性较强的工具，加速推动抗AS中药复方的研究进程，为后续的药物开发和临床应用奠定了基础。

Abstract

Objective To systematically identify traditional Chinese medicine (TCM) compound formulas with anti-atherosclerosis (AS) potential using a screening platform based on multi-strainapoeb gene knockout zebrafish models for anti-atherosclerosis TCM compounds. Methods Screening was conducted using a formula dictionary database, and compound formulas such as Dan Shen Fang, Shang Ni San, Jin Lingzi San, and Li Qi Hua Liang Tang were selected from 933 blood-activating and stasis-resolving formulas. Five strains of apoeb^-/- zebrafish–AB, Tg(mpeg1:EGFP), Tg(lyzc:dsRed), Tg(gata1a:mCherry), and Tg(flk1:EGFP)-were fed a high-fat diet for 2 days, after which the AS model was established. DMSO (solvent control), rosuvastatin (western medicine positive control), compound Dan Shen Fang (TCM positive control), Shang Ni San, Jin Lingzi San, and Li Qi Hua Liang Tang were administered by bathing for 24 hours. Oil red O staining was used to detect lipid content, oxidative stress indicators, blood flow velocity, vascular endothelial cell damage, and the expression of inflammatory factors in zebrafish. The behavior patterns of inflammatory cells in zebrafish were observed, and the anti-AS efficacy of the Li Qi Hua Liang formulas was comprehensively evaluated. Results Compared with the AS model group, compound Dan Shen Fang and Shang Ni San improved lipid accumulation in the vascular lumen, reduced total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) levels, decreased oxidative stress response, inhibited the recruitment of neutrophils and macrophages, repaired vascular endothelial damage, improved blood flow velocity, and reduced the expression of inflammatory factors, thereby alleviating AS, with statistically significant differences. Jin Lingzi San and Li Qi Hua Liang Tang had limited effects on AS. Conclusion This platform provides an efficient and targeted tool for the screening and research evaluation of anti-atherosclerosis TCM compound formulas, accelerates the research progress of anti-atherosclerosis TCM compound formulas, and lays a foundation for subsequent drug development and clinical application.

关键词

动脉粥样硬化 / 斑马鱼 / 药物筛选 / 中药复方

Key words

Atherosclerosis / Zebrafish / Drug screening / Chinese medicine compound formulas

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基金资助

*山东省自然科学基金(ZR2020KH034)

山东省中医药科技项目(Z-2022045)