Abstract

This study aimed to explore the mechanism of Qingre Huoxue Fang (QRHXF) treatment on anti-angiogenesis in rheumatoid arthritis (RA) based on network pharmacology and in vitro experiments. We used the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Therapeutic Target (TTD) database to extract the active components of QRHXF and potential targets for regulating angiogenesis. First, we used Cytoscape bioinformatics software to construct the network of QRHXF-angiogenesis and screened the potential targets. Then, we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on the potential core targets. In addition, enzyme-linked immune assay and Western blot were used for in vitro validation and to verify the effects of different concentrations of QRHXF on the expression levels of the vascular endothelial growth factor receptor type 1 (VEGFR-1) and VEGFR-2 cytokines and phosphoinositide 3-kinase (PI3k) and Ak strain transforming (Akt) proteins in human umbilical vein endothelial cells (HUVECs). In results, we screened 179 core QRHXF antiangiogenic targets, including vascular endothelial growth factor (VEGF) cytokines. Enrichment analysis showed that the targets were enriched in 56 core signaling pathways, including PI3k and Akt. In vitro experiments showed that the migration distance and square, adhesion optical density (OD) values, and the number of branch points in tube formation significantly decreased in the QRHXF group compared with the induced group (P<0.01). Notably, the serum levels of VEGFR-1 and VEGFR-2 were lower compared with the induced group (P<0.05 or P<0.01). In addition, the expressions of PI3K and p-Akt proteins were decreased in the middle- and high doses groups (P<0.01). This study's results suggest that the downstream mechanism of QRHXF anti-angiogenesis might inhibit the PI3K-Akt signalling pathway and downregulate VEGF-1 and VEGF-2.

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