Exploring the Modulatory Mechanism of Eleng Capsule in the Treatment of Endometriosis Based on Integrated Strategy of Transcriptomics Combined With Network Pharmacology

Abstract

BackgroundEndometriosis causes severe chronic pelvic pain and infertility. Chinese medicine plays an active role in the treatment of endometriosis. ELeng Capsule(ELC) is a Chinese medicine formula used for the treatment of endometriosis for several years. The previous studies have shown that ELC inhibits endometriosis. However, the mechanisms of action of ELC have not been characterized. In this study, network pharmacology and mRNA transcriptome analysis were used to study various therapeutic targets in ELC.Methods Ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry analysis(UPLC-Q-TOF/MS) was used to identify the compounds in ELC.And network pharmacology was used to analyze the network of targets and identified compounds of ELC. Apoptosis was assessed using the terminal deoxynucleotidyl transferase (TdT) deoxyuridine 5'-triphosphate (dUTP) nick-end labeling (TUNEL) assay.The protein expression of VEGFA,VEGFB,VEGFC and α-SMA in the ectopic endometrium were identified by immunohistochemistry(IHC).The level of VEGFA,VEGFB and IL1-β in serum were used by ELISA. Further, RNA-sequencing was used to identify differentially expressed genes (DEGs) in ELC. Biological functions and pathways were determined through the Gene Ontology(GO) and Kyoto encyclopedia of genes and genomes pathway(KEGG) analyses. In addition, Gene Set Enrichment Analysis (GSEA) analysis was used to further analyze the genetic network and modular genetics. ResultsWe had identified 26 new bioactive compounds in ELeng Capsule by UPLC-Q-TOF/MS analysis. The KEGG pathways of ELC associtaed targets related to neuroactive ligand-receptor interaction, the toll-like receptor signaling pathway, and the vascular endothelial growth factor signaling pathway by network pharmacology analysis.Further, ELC could induce cell apoptosis, inhibit angiogenesis through reduce the expression of VEGFA and VEGFC, and inhibit the fibrosis through reduce the expression of α-SMA in ectopic lesions(P＜0.05). In addition, the development of endometriosis in the rat model may be related to mechanisms of inflammation,epithelial-mesenchymal transition(EMT) by RNA-sequencing. And the targets in the treatment of ELC were predominantly associated with actin and cytoskeleton,EMT,focal adhesion,and inflammatory immunity based on the DEGs analysis. In additon, the GSEA analysis showed that the treatment of ELC was associated with the Notch signaling pathway, Hippo signaling pathway, etc.ConclusionELeng Capsules exerted beneficial effects against endometriosis, potentially by induce apoptosis,modulating the angiogenesis,cytoskeleton, epithelial-mesenchymal transition(EMT), and cell junction-associated pathways,etc. These findings could provide evidence for an innovative treatment strategy and novel therapeutic targets for endometriosis.