OBJECTIVE To explore the mechanism of action of Alocasia cucullata alcohol extract in the treatment of rheumatoid arthritis(RA) based on network pharmacology, molecular docking and animal experiments.
METHODS Potential active ingredients and targets of Alocasia cucullata alcohol extract were collected through PubChem, SwissTargetPrediction and related literatures. Genecards and DisGeNET databases were used to search for RA disease targets. The intersection targets between the Alocasia cucullata alcohol extract and RA diseases were obtained by Venn diagram. The STRING database was used for PPI protein interaction analysis of intersecting targets. Cytoscape 3.9.1 software was used to draw a “target-component-drug-disease” network diagram. DAVID database was used for gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis. AutoDock Tools 1.5.6 software was used to perform molecular simulation docking between key compounds and targets. The rats were randomly assigned into normal group, RA group, low dose group, medium dose group and high dose group of Alocasia cucullata alcohol extract, and the RA model was induced by the mixed emulsion of complete Freund's adjuvant and bovine type II collagen. The foot volume and arthritis index of rats in each group were measured after the intervention of different doses of Alocasia cucullata alcohol extract. HE staining was used to detect the morphology of rat ankle joint tissue. ELISA was used to detect the levels of inflammatory factors in rat serum. Western blotting was used to detect the protein expression of core targets in rat ankle joint tissue.
RESULTS A total of 17 active ingredients were screened out from the Alocasia cucullata alcohol extract, which shared 132 intersection targets with RA disease, mainly regulating core targets such as AKT1, EGFR, SRC, BCL2, TLR4 and involve plasma membrane, protein binding, signal transduction and other signaling pathways. Molecular docking results showed that luteolin and kaempferol, the main active components of Alocasia cucullata alcohol extract, had good binding activity with AKT1, EGFR, SRC, BCL2 and TLR4. Animal experiment results showed that the Alocasia cucullata alcohol extract could alleviate ankle joint tissue lesions in RA rats, reduce foot volume, arthritis index, ankle joint pathological score, down-regulate serum TNF-α, IL-17 and IL-6 levels as well as the expression of p-AKT1/AKT1, p-SRC/SRC, EGFR, BCL2 and TLR4 proteins in ankle joint tissue, in a dose-dependent manner(P<0.05).
CONCLUSION The main active ingredients of Alocasia cucullata alcohol extract in the treatment of RA are luteolin and kaempferol, which can reduce inflammatory response and improve RA ankle tissue lesions by regulating the expression of key target proteins AKT1, EGFR, SRC, BCL2 and TLR4.
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