Neuroprotective effects of total phenolics from Hemerocallis citrina Baroni leaves through the PI3K/AKT pathway

Abstract

Neurological injury, as a major pathogenic mechanism in depression, holds significant importance in the research and development of antidepressant drugs. Hemerocallis citrina Baroni (H. citrina), referred to as “Forgetting Sadness Grass,” has been confirmed to possess remarkable neuroprotective effects. Studies have identified that the total phenolics in H. citrina Baroni leaves (HLTP) consist of flavonoids and phenolic acids and numerous studies have substantiated the neuroprotective effects of them. Based on this, we propose that HLTP may possess neuroprotective properties. To confirm this hypothesis, we initially employed network pharmacology techniques to predict potential targets for the neuroprotective effects of HLTP based on the Swiss Target Prediction database. GO and KEGG analyses were conducted to predict potential pathways, and a component-target-pathway network was constructed. Molecular docking experiments were then performed to analyze the binding abilities of the selected active components with the main targets. Furthermore, we validated the neuroprotective effects of HLTP and key targets selected through network pharmacology using a corticosterone-induced PC12 neuronal cell damage model. Network pharmacology research has identified that in the HLTP, Quercetin, Rutin, Apigenin, and Isoquercitrin are potential active components that may exert neuroprotective effects by modulating key targets such as AKT1, TNF, TP53, and CASP3 through crucial pathways including PI3K/AKT and apoptosis. Molecular docking revealed that 4-O-Caffeoylquinic acid, 5-O-Caffeoylshikimic acid, 4-p-Coumaroylquinic acid, and 5-O-Feruloylquinic acid exhibit low binding energies with key targets. Particularly, 4-O-Caffeoylquinic acid forms stable binding through hydrogen bonding with residues such as LYS389, GLU49, GLN47, LYS30, ASP44, and GLU40 in AKT1. PC12 cells were stimulated with 200 μmol/L Corticosterone (Cort) for 24 h, and then treated with 50, 100 and 200 μg/mL of HLTP for 24 h. The cell viability of damaged cells were significantly increased in a dose-dependent manner by 9.50%, 10.42% and 21.25%, respectively (P < 0.01). Western blot analysis confirmed that HLTP significantly (P < 0.01) increased the protein expression of PI3K and AKT by 15.24%, 30.44%, 41.03%, and 21.78%, 43.63%, 12.86%, respectively. In addition, through biochemical method, flow cytometry and WB analysis, we found that different concentrations of HLTP can all improve cell damage by reducing ROS, MDA, Ca2+, Cyt-C, Caspase-3, TNF-α and IL-1β, and increasing SOD, CAT, MMP, Bcl-2/Bax and IL-10. In particular, the HLTP at 200 μg/mL, compared with the Model group, decreased by 140.2%, 54.66%, 51.34%, 65.26%, 40.32%, 63.87%, and 55.38%, and increased by 39.65%, 35.45%, 38.38%, 28.54%, and 39.98%, respectively. Through the above experiments, we verified that HLTP may exert neuroprotective effects by mediating the PI3K/AKT signaling pathway to counteract oxidative stress damage, improve mitochondrial dysfunction, and alleviate inflammatory injury.