Lu Jin,1,2 Yunshuang Guan,3 Xue Li,3,4 Mingyue Wang,2 Ying Shen,1,2 Nianxue Wang,3 Zhixu He1,2,5 

1Department of Pediatrics, School of Clinical Medicine, Guizhou Medical University, Guiyang, Guizhou, People’s Republic of China; 2Department of Pediatric Hematology, Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou, People’s Republic of China; 3Department of Immunology, College of Basic Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, People’s Republic of China; 4Department of Central Laboratory, Guizhou Provincial People’s Hospital, Guiyang, Guizhou, People’s Republic of China; 5Tissue Engineering and Stem Cell Experiment Center, Guizhou Medical University, Guiyang, Guizhou, People’s Republic of China

Correspondence: Nianxue Wang, Email wangnianxue@gmc.edu.cn; Zhixu He, Email hzx@gmc.edu.cn

Purpose: To investigate the effects and underlying mechanisms of indirubin in treating ALL using network pharmacology and experimental validation.
Methods: Potential targets of indirubin- and ALL-related genes were identified using public databases. Core genes were filtered through protein-protein interaction analysis in Cytoscape. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted to explore the potential mechanisms of indirubin against ALL. Drug-disease-functional annotation-signaling pathway network maps were constructed. Molecular docking between indirubin and core proteins was performed using AutoDock Vina software. Finally, both in vitro and in vivo experiments were performed to validate these findings.
Results: PPI network analysis identified eight potential core targets of indirubin in ALL: AKT1, CASP3, and the mammalian target of rapamycin. GO and KEGG enrichment analyses suggested that the mechanism of action of indirubin against ALL involves multiple biological functions and signaling pathways, with the PI3K-AKT pathway likely playing a central role. Molecular docking findings further confirmed the strong binding affinity of indirubin for the core targets. Both in vitro and in vivo experiments demonstrated that indirubin inhibited ALL cell proliferation and induced cell cycle arrest and apoptosis; the underlying mechanism may involve the PI3K-AKT signaling pathway.
Conclusion: The action and mechanism of indirubin in ALL through network pharmacology, as well as in vivo and in vitro experimental validation were elucidated, offering new insights and potential therapeutic avenues for the treatment of ALL.

Keywords: indirubin, acute lymphoblastic leukemia, network pharmacology, molecular docking, experimental validation