Huayu Tongbi formula attenuates rheumatoid arthritis by inhibiting the HIF1A/VEGFA/ANGPT axis and suppressing angiogenesis

BACKGROUND: Angiogenesis, integral to the pathogenesis of rheumatoid arthritis (RA), contributes to the advancement of synovial hyperplasia, pannus formation, and bone destruction. The precise effect of the Huayu Tongbi formula (HT), a proven traditional Chinese medicine for RA treatment, on angiogenesis remains uncertain. PURPOSE: To investigate the effects of HT on RA angiogenesis and the potential underlying molecular mechanisms. METHODS: to simulate hypoxic conditions. Human umbilical vein endothelial cells (HUVECs) were cultured with conditioned medium (CM) from RA-FLS supplemented with HT or the HIF1A inhibitor LW-6 to investigate the impact of synovial cell secretions on angiogenesis, encompassing proliferation, migration, adhesion, and lumen formation. Western blot was employed to assess protein levels of hypoxia-inducible factor 1 subunit alpha (HIF1A), vascular endothelial growth factor receptor 2 (VEGFR2), and phosphorylated VEGFR2 (p-VEGFR2). Enzyme-linked immunosorbent assay (ELISA) was utilized to quantify VEGFA secretion, while quantitative real-time PCR (qRT‒PCR) measured mRNA expression of VEGFA, Angiopoietin 1 (ANGPT1), and ANGPT2. In vivo, a collagen-induced arthritis (CIA) model was established. Pathological changes and vessel counts were observed via hematoxylin-eosin (HE) staining, bone structure was evaluated using X-ray, mature and immature vessels were classified via immunofluorescence, and the mechanism was further verified using immunohistochemistry and ELISA. Liquid chromatography‒mass spectrometry (LC‒MS) was employed to identify the active components of HT drug-containing serum, and the potential metabolites were identified by bioinformatics analysis. RESULTS: Network pharmacology analysis indicated potential targets of HT in angiogenesis, correlating with HIF1A and VEGF pathways. In vitro, HT reversed proliferation, migration, and adhesion of the cell lines and suppressed luminal formation by HUVECs. HT (10 % and 15 %) effectively reduced HIF1A and VEGFA expression, attenuated VEGFR2 levels and its phosphorylation, and downregulated VEGFA, ANGPT1, and ANGPT2 mRNA levels. In vivo, HT mitigated inflammation and bone destruction, reduced immature (CD31+/α-SMA-) and mature (CD31+/α-SMA+) vessel counts in the synovium, and inhibited the HIF1A-VEGFA-ANGPT axis. Finally, 19 active components from HT drug-containing serum were successfully identified, and we also unexpectedly discovered that HT might affect metabolic pathways such as proline and tyrosine metabolism, which have been reported to be involved in angiogenesis. CONCLUSION: HT has a promising effect on alleviating RA angiogenesis, at least in part through HIF1A-VEGFA-ANGPT axis inhibition, which reduces the cross-talk between RA-FLS and HUVECs. Our results suggest that HT is a drug candidate for the treatment of RA angiogenesis.