目的 采用UPLC-Q-Orbitrap MS结合网络药理学技术及体内外实验，探究生甘草、炙甘草、炒甘草3种炮制品的抗心律失常药效差异，初步阐明其药效物质基础与作用机制，为临床优选提供依据。方法 使用4.2 μg·mL^-1氯化钡诱导斑马鱼心律失常模型，实验设置对照组、模型组、白藜芦醇（阳性药，30.0 μg·mL^-1）组、生甘草（生甘草黄酮提取物，28.6 μg·mL^-1）组、炒甘草（炒甘草黄酮提取物，26.7 μg·mL^-1）组及炙甘草（炙甘草黄酮提取物，31.4 μg·mL^-1）组，采用心率、静脉窦至动脉球（SV-BA）间距和心包水肿面积评价药效，吖啶橙（AO）染色法评估心肌细胞凋亡水平；通过UPLC-Q-Orbitrap MS分析3种甘草炮制品黄酮类化学成分并辨识差异化合物；利用网络药理学预测3种炮制品的差异活性成分，预测关键靶点与通路；分别用不同浓度（0、5、10、20、40、80、120 μmol·L^-1）的柚皮素与甘草查尔酮A处理H9C2细胞24 h，采用CCK-8法检测细胞活力，考察药物对细胞增殖的影响；使用4 μmol·L^-1阿霉素（Dox）与药物共同处理细胞24 h，评价药物对Dox诱导的细胞存活率的影响； Western blotting法检测H9C2细胞磷脂酰肌醇3-激酶（PI3K）-蛋白激酶B（Akt）通路及凋亡相关蛋白表达水平。结果 3种炮制品黄酮类成分能够逆转氯化钡诱导的心率降低、SV-BA间距和心包水肿面积增加，AO染色显示心包区域荧光强度降低（P＜0.05、0.01），提示心肌细胞凋亡情况改善，且炙甘草的心脏损伤修复率更高； UPLC-Q-Orbitrap MS分析表征了51个黄酮类成分，并筛选出不同炮制品间的21个差异成分；网络药理学筛选得到88个共同作用靶点，蛋白质-蛋白质相互作用（PPI）网络分析确定AKT1、肿瘤坏死因子（TNF）等15个核心靶点，锁定柚皮素、甘草查尔酮A为关键活性成分，富集分析推测其通过调控PI3K-Akt通路、抑制细胞凋亡发挥作用；体外细胞毒性实验表明，柚皮素在20 μmol·L^-1以下、甘草查尔酮A在40 μmol·L^-1以下时对细胞活力无明显影响，2者均可逆转Dox诱导的H9C2细胞损伤； Western blotting实验表明柚皮素与甘草查尔酮A处理能够逆转Dox诱导的PI3K、AKT1蛋白表达水平的下降，同时，能够下调裂解型半胱氨酸天冬氨酸蛋白酶-3（cleaved Caspase-3）蛋白表达水平，上调B淋巴细胞瘤-2基因（Bcl-2）/Bcl-2相关X蛋白（Bax）比值（P＜0.05、0.01），从而逆转细胞凋亡。结论 炙甘草是抗心律失常最佳炮制品，增效机制与炮制后特有成分柚皮素、甘草查尔酮A相关，二者通过激活PI3K-Akt通路、抑制心肌细胞凋亡实现心脏保护，为炙甘草增效的科学内涵提供理论与实验依据。

Objective To investigate the differences in the anti-arrhythmic efficacy of three processed products of Glycyrrhiza uralensis (raw, honey-fried, and stir-fried) by integrating UPLC-Q-Orbitrap MS, network pharmacology, and in vivo and in vitro experiments. This study aims to elucidate their pharmacodynamic material basis and mechanisms of action, providing a basis for optimal clinical selection. Methods A zebrafish arrhythmia model was induced using 4.2 μg·mL^-1 barium chloride. The experimental groups included a control group, a model group, a positive control group (resveratrol, 30 μg·mL^-1), and groups treated with G. uralensis flavonoid extracts of raw (28.6 μg·mL^-1), stir-fried (26.7 μg·mL^-1), and honey-fried (31.4 μg·mL^-1). Efficacy was evaluated by measuring heart rate, SV-BA distance, and pericardial edema area, while cardiomyocyte apoptosis was assessed using acridine orange (AO) staining. Flavonoid constituents in the three processed products were analyzed by UPLC-Q-Orbitrap MS to identify differential compounds. Based on these, active components were screened, and network pharmacology was employed to predict key targets and pathways. H9C2 cells were treated with various concentrations (0, 5, 10, 20, 40, 80, 120 μmol·L^-1) of naringenin and licochalcone A for 24 h, and cell viability was determined using the CCK-8 assay to examine the effects on cell proliferation. Cells were also cotreated with 4 μmol·L^-1 doxorubicin (Dox) and the compounds for 24 h to evaluate their effects on Dox-induced cell viability. The expression levels of phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) pathway and apoptosis-related proteins in H9C2 cells were detected by Western blotting. Results The flavonoid components from all three processed products reversed the barium chlorideinduced decrease in heart rate and increases in SV-BA distance and pericardial edema area. AO staining showed reduced pericardial fluorescence intensity, indicating attenuated cardiomyocytes apoptosis, with honey-fried G. uralensis demonstrating the highest cardiac injury repair rate. UPLC-Q-Orbitrap/MS analysis characterized 51 flavonoid components and identified 21 differential compounds among the products. Network pharmacology screening yielded 88 common targets. Protein-protein interaction (PPI) network analysis identified 15 core targets, including AKT1 and tumor necrosis factor (TNF). Naringenin and licochalcone A were pinpointed as key compounds. Enrichment analysis suggested their action involves regulating the PI3K-Akt pathway and inhibiting apoptosis. In vitro cytotoxicity tests showed that naringenin (≤ 20 μmol·L^-1) and licochalcone A (≤ 40 μmol·L^-1) had no significant effect on cell viability. Both compounds reversed Dox-induced H9C2 cell injury. Western blotting showed that naringenin and licochalcone A treatment reversed the Dox-induced downregulation of PI3K and AKT1 protein expression. Additionally, they reduce the expression level of cleaved Caspase-3 protein and upregulated the Bcl-2/Bax ratio, thereby counteracting apoptosis. Conclusion Honey-processed G. uralensis is the most effective processed product against arrhythmia. Its enhanced efficacy is associated with specific post-processing components, naringenin and licochalcone A, which confer cardioprotection by activating the PI3K-Akt pathway and inhibiting cardiomyocyte apoptosis. This study provides theoretical and experimental evidence for the scientific basis underlying the efficacy enhancement of honey-fried G. uralensis.

R284.1;R285.5

吉林省自然科学基金资助项目（YDZJ202401442ZYTS）