Network pharmacology, molecular docking and experimental verification of the mechanism of huangqi-jixuecao herb pair in treatment of peritoneal fibrosis

Ethnopharmacological relevance: The Huangqi-Jixuecao herb pair (HQJXCHP) is a traditional herbal formula composed of two widely applied TCM prescriptions, Huangqi (Astragalus membranaceus (Fisch.) Bunge) and Jixuecao (Centella asiatica (L.) Urb.), used for hundreds of years to replenish qi and clear away heat. However, the therapeutic effects of HQJXCHP against peritoneal fibrosis (PF) and potential targets are currently unclear. Aims of the study: The main objective of this study was preliminary prediction and validation of the effects and molecular mechanisms of action of HQJXCHP against PF based on network pharmacology analysis and experimental verification. Materials and methods: The ingredients of HQJXCHP were analyzed via HPLC-Q-TOF/MS. Bioactive compounds of HQJXCHP used for network pharmacology analysis were obtained from the TCMSP database. HQJXCHP-related therapeutic targets in PF were obtained from the GeneCards, OMIM, Therapeutic Targets and PharmGkb databases. Therapeutic target-related signaling pathways were predicted via GO and KEGG pathway enrichment analyses. The targets of HQJXCHO were further validated in a PDS-induced PF mouse model in vivo and PMCs MMT model in vitro. Results: A total of 23 bioactive compounds of HQJXCHP related 188 target genes were retrieved. The HQJXCHP compound-target and PF-related target networks identified 131 common target genes. Subsequent protein-protein interaction (PPI) network analysis results disclosed Akt1, TP53, TNF, VEGFA and CASP3 as the top five key targets of HQJXCHP. Further molecular docking data revealed strong affinity of the two key compounds of HQJXCHP, quercetin and kaempferol, for these key targets. GO and KEGG pathway enrichment analyses further showed that PI3K/Akt, IL-17, TNF and TLR pathways contribute to the therapeutic effects of HQJXCHP on PF. An in vivo PDS-induced PF mouse model and in vitro PMCs mesothelial-to-mesenchymal transition (MMT) model with or without HQJXCHP intervention were used to confirm the effects and mechanisms of action of HQJXCHP. Western blot and qRT-PCR results showed that HQ, JXC and HQJXCHP reduced PDS-induced inflammatory cell aggregation and peritoneal thickening through suppressing the MMT process, among which HQJXCHP exerted the greatest therapeutic effect. Moreover, HQJXCHP inhibited activation of the PI3K/Akt, IL-17, TNF and TLR signaling pathways induced by PDS. Conclusions: This is the first study to employ network pharmacology and molecular docking analyses to predict the targets of HQJXCHP with therapeutic effects on PDS-related PF. Data from in vivo and in vitro validation experiments collectively showed that HQJXCHP delays the PF process through inhibiting PI3K/Akt, IL-17, TNF and TLR signaling pathways. Overall, our findings highlight the successful application of network pharmacology theory to provide a scientific basis for clinical utility of HQJXCHP against PF.