目的 探讨狭基线纹香茶菜水提物（Isodon lophanthoides var. gerardianus aqueous extract，ILAE）的成分及其对乙醇诱导的酒精性肝病（alcoholic liver disease，ALD）斑马鱼的保护作用，并利用网络药理学分析其作用的潜在成分和靶点。方法 采用超高效液相色谱-四极杆飞行时间质谱技术（ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry，UPLC-Q-TOF/MS）鉴定ILAE的成分；以36、73、146 μg/mL ILAE与70 μg/mL水飞蓟素对受精后4 d（4 d post-fertilization，4 dpf）的野生型AB品系与转基因中性粒细胞斑马鱼Tg（lyz: DsReD）分别处理16 h，再以2%乙醇诱导32 h建立ALD模型，通过观察斑马鱼的发育情况、检测生化指标与行为学实验，考察ILAE的肝脏保护作用。运用网络药理学分析ILAE作用于ALD的潜在成分与靶点，通过分子对接与Western blotting实验进行验证。结果 共鉴定出ILAE中的成分56种,主要为黄酮类与酚酸类。ILAE可以明显改善乙醇诱导的斑马鱼的发育受阻，卵黄囊延迟吸收面积与肝脏肿大面积显著减小（P＜0.05、0.001），脂质积累显著减少（P＜0.001），肝脏病理损伤缓解，肝细胞排列有序、脂肪空泡减少，丙氨酸氨基转移酶（alanine aminotransferase，ALT）、天冬氨酸氨基转移酶（aspartate aminotransferase，AST）活性显著降低（P＜0.05、0.01、0.001），乙醇脱氢酶（alcoholdehydrogenase，ADH）活性和还原型谷胱甘肽（glutathione，GSH）水平显著升高（P＜0.05、0.01、0.001），活性氧（reactive oxygen species，ROS）、丙二醛（malondialdehyde，MDA）和三酰甘油（triglyceride，TG）水平显著降低（P＜0.05、0.01、0.001），中性粒细胞数目明显减少（P＜0.01、0.001），斑马鱼兴奋状态减轻，快速运动轨迹与总运动距离减少（P＜0.05、0.001）。ILAE与ALD相关的核心靶点为蛋白激酶Bα（protein kinase Bα，AKT1）、核因子-κB 1（nuclear factor-κB subunit 1，NF-κB1）、信号转导与转录激活因子3（signal transducer and activator of transcription 3，STAT3）等，核心成分为白杨素、异泽兰黄素、迷迭香酸等，且化合物与靶点可以紧密结合。京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）分析与基因本体（gene ontology，GO）分析显示，作用过程涉及脂质代谢、胰岛素抵抗、胆汁酸分泌与外源物质代谢等通路。Western blotting结果显示，ILAE可以显著降低乙醇诱导的斑马鱼体内NF-κB1、STAT3蛋白及其磷酸化形式的表达水平（P＜0.05、0.01、0.001），升高AKT1蛋白及其磷酸化形式的表达水平（P＜0.05、0.01）。结论 ILAE可能通过迷迭香酸、白杨素、咖啡酸、7-羟基香豆素等成分，作用于AKT1、NF-κB1、STAT3等靶点改善ALD。其保护作用可能与降低炎症反应、减少脂质积累与增强抗氧化活性有关。

Objective To investigate the components of Isodon lophanthoides var. gerardianus aqueous extract (ILAE) and its protective effects against ethanol-induced alcoholic liver disease (ALD) in zebrafish, and to analyze its potential active components and targets using network pharmacology. Methods Ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was employed to identify the components of ILAE. Wild-type AB strain and transgenic neutrophil zebrafish Tg (lyz: DsRed) at 4 d post-fertilization (4 dpf) were treated with 36, 73, 146 μg/mL ILAE or 70 μg/mL silymarin for 16 h, followed by induction of ALD with 2% ethanol for 32 h. The protective effects of ILAE on liver were assessed by observing developmental status, measuring biochemical indicators and conducting behavioral tests. Network pharmacology was used to analyze the potential components and targets of ILAE in treating ALD. Validation was performed through molecular docking and Western blotting. Results A total of 56 components were identified in ILAE, primarily flavonoids and phenolic acids. ILAE significantly alleviated ethanol-induced developmental impairment in zebrafish, the delayed absorption area of yolk sac and the area of liver enlargement were significantly reduced (P < 0.05, 0.001), lipid accumulation was significantly reduced (P < 0.001), liver pathological damage was relieved, liver cells were arranged in an orderly manner, and fat vacuoles were reduced. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly decreased (P < 0.05, 0.01, 0.001), while the activity of alcohol dehydrogenase (ADH) and reduced glutathione (GSH) level were significantly increased (P < 0.05, 0.01, 0.001). The levels of reactive oxygen species (ROS), malondialdehyde (MDA) and triglycerides (TG) were significantly decreased (P < 0.05, 0.01, 0.001), and the number of neutrophils was significantly reduced (P < 0.01, 0.001). The excitement state of zebrafish was decreased, and the rapid movement trajectory and total movement distance were decreased (P < 0.05, 0.001). The core targets associated with ILAE and ALD were protein kinase Bα (AKT1), nuclear factor-κB subunit 1 (NF-κB1), signal transducer and activator of transcription 3 (STAT3), key components were chrysin, eupatilin and rosmarinic acid, which exhibited strong binding affinity to the targets. Kyoto encyclopedia of genes and genomes (KEGG) analysis and gene ontology (GO) analysis showed that the process involved lipid metabolism, insulin resistance, bile acid secretion, and exogenous substance metabolism pathways. Western blotting results showed that ILAE significantly reduced the expression levels of NF-κB1, STAT3 proteins and their phosphorylated forms in ethanol-induced zebrafish (P < 0.05, 0.01, 0.001), and increased the expression levels of AKT1 protein and its phosphorylated forms (P < 0.05, 0.01). Conclusion ILAE may improve ALD by targeting AKT1, NF-κB1, STAT3 through rosmarinic acid, chrysin, caffeic acid, 7-hydroxycoumarin and other components. Its protective effect may be related to reducing inflammatory response, decreasing lipid accumulation and enhancing antioxidant activity.

R285.5

中山市中医药传承创新发展科研专项项目（2024B3065）；电子科技大学中山学院科研团队培育项目（424YT02）