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.

Isodon lophanthoides (Buch.-Ham. ex D. Don) Hara var. gerandianus (Benth.) Hara  /  alcoholic liver disease  /  zebrafish  /  inflammatory response  /  lipid accumulation  /  oxidative stress  /  rosmarinic acid  /  chrysin  /  caffeic acid  /  7-hydroxycoumarin  /  AKT1  /  NF-κB1  /  STAT3