Amoebicidal action of isoliquiritigenin and glabridin from Glycyrrhiza species: Mechanisms and effects against Acanthamoeba castellanii.

BACKGROUND

Acanthamoeba is a widely distributed genus, causing keratitis, granulomatous amoebic encephalitis and chronic infectious ulcers. At present, there are no effective drugs available for treating Acanthamoeba infections, highlighting the urgent need for the development of novel anti-Acanthamoeba therapies.

PURPOSE

This study aims to investigate the inhibitory effect of isoliquiritigenin (ISL), a chalcone, and glabridin (GLA), a prenylated isoflavonoid, on Acanthamoeba castellanii trophozoites growth and elucidate their mechanisms of action.

METHODS

The effect of ISL and GLA on trophozoite viability was assessed employing the CellTiter-Glo assay, while the viability of cells was evaluated via cell counting kit-8. Apoptotic cells were identified through Hoechst 33,342/ propidium iodide (PI) double staining and caspase-3 detection. Reactive oxygen species (ROS) level was measured via flow cytometry utilising 2',7'-dichlorofluorescin diacetate (DCFH-DA). Mitochondrial dysfunction was analysed with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine (JC-1) and mitochondrial superoxide (mtSOX) Deep Red staining. Ribonucleic acid (RNA) sequencing and quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis were conducted to investigate the potential anti-Acanthamoeba mechanism.

RESULTS

Our results showed that ISL and GLA in vitro effectively inhibited A. castellanii trophozoite growth in a dose and time-dependent manner. ISL and GLA induced several apoptosis features, including Hoechst/PI-positive staining and increased caspase-3 expression. ISL and GLA increased intracellular ROS production, decreased superoxide dismutase (SOD) expression and mitochondrial membrane potential and enhanced mitochondrial ROS generation. ISL and GLA effectively prevent host cells from A. castellanii trophozoites invasion. RNA sequencing indicated that ISL may modulate the NAD metabolic process, while GLA may influence the sterol metabolic process. ISL reduced the NAD/NADH ratio, while GLA lowered the levels of 7-dehydrocholesterol in A. castellanii trophozoites.

CONCLUSION

These findings suggest that ISL and GLA, two components of Glycyrrhiza species, may possess activity against A. castellanii trophozoites. Our findings suggest that ISL suppresses trophozoites by regulating NAD metabolism, while GLA inhibits trophozoites through the regulation of sterol metabolism.