基于转录组学、网络药理学及体外实验探讨菟丝子-枸杞子治疗卵巢储备功能减退的主要有效成分及其作用机制OA

Objective To screen the key bioactive ingredients of TSZ-GQZ herbal pair by network pharmacology,molecular docking technology and in vitro experiments,and explore its mechanism for diminished ovarian reserve(DOR).Methods Active ingredients and potential targets of TSZ-GQZ were predicted by network pharmacology,and functional enrichment analysis was carried out based on DAVID database.UPLC-HRMS was used to identify chemical constituents of the two herbs,and AutoDock Vina 1.2.7 was adopted for molecular docking.A hydrogen peroxide induced injury model of human ovarian granulosa cells was established.Cell viability was detected by the CCK8 assay.Transcriptome sequencing was performed to screen differentially expressed genes(DEGs).Flow cytometric apoptosis analysis,ROS detection,SOD activity assay,RT-qPCR and Western blot(WB)were further applied to verify the intervention effect of the drug.Results Network pharmacology analysis screened quercetin as the active ingredient with the highest correlation degree in TSZ-GQZ,and UPLC-HRMS confirmed the existence of quercetin in both Cuscuta chinensis and Lycium barbarum.Enrichment analysis combining quercetin targets and transcriptomic DEGs of injured granulosa cells jointly identified MAPK signaling pathway as the core regulatory pathway.Molecular docking verified favorable binding affinity between quercetin and key proteins of MAPK pathway as well as apoptosis-related proteins.In vitro cell experiments showed that quercetin significantly improved the viability of damaged granulosa cells and inhibited cell apoptosis.It markedly upregulated EGFR and AKT1,while downregulating pro-apoptotic and pro-inflammatory genes including FOS,GADD45B,IL-1A,IL-1B,TP53 and BAX.WB results further proved that quercetin reduced the phosphorylation levels of JNK,p38 and ERK,as well as the expression of Cleaved Caspase-3.It suppressed excessive activation of the MAPK signaling pathway and thereby alleviated cell apoptosis.Conclusions Quercetin is the key active ingredient of TSZ-GQZ herbal pair against DOR.It can reduce ovarian granulosa cell apoptosis via inhibiting the activation of MAPK signaling pathway.