Exploration of the Effect and Mechanism of Fructus Lycii, Rehmanniae Radix Praeparata, and Paeonia lactiflora in the Treatment of AMD Based on Network Pharmacology and in vitro Experimental Verification

PURPOSE: The aim of this study was to observe the mechanism of Fructus Lycii (FL), Rehmanniae Radix Praeparata (RRP) and Paeonia lactiflora (PL) in treating age-related macular degeneration (AMD) based on network pharmacology and biological experiments. METHODS: Bioactive compounds, potential targets of FL, RRP and PL, and genes related to AMD, were acquired from public databases. Functional and pathway enrichment analyses of the core targets were conducted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Subsequently, the finding was further verified with cell experiments. The MTT assay and flow cytometric analysis were used to assess cell viability and apoptosis. The production of reactive oxygen species (ROS) was analyzed by DCFH-DA staining; the activity of antioxidant enzymes was chemically measured with assay kits. The expression of key proteins was evaluated by Western blot analysis. RESULTS: Fifty-nine active compounds, 182 potential targets, and 2536 AMD-related human genes were identified. A total of 103 key targets of the three herbs on AMD were identified by protein–protein interaction (PPI) analysis. The abovementioned targets were correlated with nuclear receptor activity, oxidative stress, and apoptosis pathways according to the GO and KEGG analyses. MTT assay and flow cytometry demonstrated that pretreatment of ARPE-19 cells with the three herbs significantly increased cell viability and decreased apoptosis induced by H(2)O(2). The three herbs might reduce the intracellular ROS levels and increase the SOD and CAT activities after H(2)O(2). Furthermore, the three herbs significantly inhibited oxidative stress via increasing the expression of Nrf2, HO-1 and NQO1. CONCLUSION: The combined results of network pharmacology and validation experiments showed that FL, RRP and PL reduce oxidative stress and apoptosis in RPE cells to exert its effect in the treatment of AMD.