Study On The Mechanism Of Xiao Chaihu Decoction Affecting Melanogenesis Based On Network Pharmacology

ObjectiveTo clarify the molecular mechanism of Xiao Chaihu decoction in inhibiting melanonegesis through AGEs-RAGE pathway and UVB-induced oxidative stress pathway.Methods1.In the network pharmacology part,the active components and action targets of Xiao Chaihu decoction were searched in TCMSP and TCMID,the melanogenesis related target genes were obtained from Gene Cards and NCBI Gene database,the core target information protein interaction analysis was carried out in STRING database,the network attributes were analyzed using Cytoscape.The results obtained from Metascape database were analyzed by GO enrichment analysis and KEGG enrichment analysis.Auto Dock Tools and Auto Dock Vina were used for molecular docking.The results were visualized by Py Mo L.2.In the experimental part,CCK-8 method was used to detect the effects of different concentrations of AGEs,different doses of UVB radiation and ethanolic extract of Xiao Chaihu decoction on cell viability;Fontana-Masson staining was used to observe the distribution of melanin;Na OH lysis was used to detect melanin content;visible spectrophotometry was used to detect tyrosinase activity;Western blot was used to detect the expression of MITF,ERK,p-ERK and other proteins;q PCR was used to detect the expression of melanin synthesis related genes MITF,TYR,TRP-1,TRP-2.The expression of gp100 and reactive oxygen species（ROS）was detected by immunofluorescence.Results1.A total of 282 effective targets,439 melanin synthesis related targets and 62intersection targets of Xiao Chaihu decoction were obtained.The main active components are praeruptorin,isorhamnetin and baicalein,and the core targets are AKT1,TP53,MAPK3 and so on.The core targets are significantly enriched in cancer-related pathways,AGEs-RAGE signaling pathways,PI3K-AKT pathways,cell senescence pathways and so on.2.There was no significant effect on cell viability when the radiation intensity of UVB was 300m J/cm~2.Compared with the blank control group,UVB andα-melanocyte-stimulating hormone（α-MSH）could promote melanin production in B16F10 cells（P<0.01,P<0.05）,but no obvious melanin production was observed in A375 cells.UVB promoted MITF expression（P<0.01,P<0.05）in both B16F10 cells and A375 cells,increased TYR activity（P<0.01）in B16F10cells;α-MSH could promote MITF expression（P<0.01）and TYR activity（P<0.01,P<0.05）in both B16F10 cells and A375 cells.Immunofluorescence results showed that there was no obvious expression of gp100 in A375 cells but with high expression in B16F10 cells.3.There were no significant effects on cell viability when the concentration of ethanolic extract was 10μg/m L as well as 10μg/m L of AGEs.Compared with the blank control group,AGEs could promote melanin synthesis,the expression of MITF,RAGE and TYR activity（P<0.01）.Compared with the AGEs group,the ethanolic extract had no obvious inhibitory effect on melanogenesis induced by AGEs,but the expression of RAGE can be inhibited（P<0.01）.q PCR results showed that AGEs and ethanolic extract of Xiao Chaihu decoction had no significant effect on MITF,TYR,TRP-1 and TRP-2 m RNA expression.4.Compared with the blank control group,the ROS content,melanin content,TYR activity and the expression of ERK,p-ERK and MITF in the UVB group increased significantly（P<0.01）.Compared with the UVB group,the ROS and melanin content,TYR activity decreased in the Xiao Chaihu decoction ethanolic extract group（P<0.01）,as well as the protein expression of ERK,p-ERK,MITF（P<0.01）.Conclusion1.Xiao Chaihu decoction can affect melanogenesis by multiple pathways.2.B16F10 cells are more suitable as cell models for melanogenesis related research than A375 cells,but more works are needed to prove this.3.The ethanolic extract of Xiao Chaihu decoction could reduce the expression of RAGE induced by AGEs,but had no significant effect on melanogenesis induced by AGEs-RAGE in B16F10 cells.4.The ethanolic extract of Xiao Chaihu decoction can reduce the oxidative stress induced by UVB,down-regulate the activity of TYR and the expression of MITF,decrease the level of phosphorylated ERK and inhibit melanin synthesis.