To investigate the effects of Platycodin D (PD) on the proliferation, migration, and angiogenesis of EA.hy926 endothelial cells stimulated by supernatant from a rheumatoid arthritis (RA) synovial cell model (MH7A), this study aims to preliminarily explore its potential mechanisms. The findings may provide a novel therapeutic target for the management of RA.

The TNF-α-stimulated MH7A cells were utilized as a model for rheumatoid arthritis (RA) cells. The supernatant from these cells was collected and designated as conditioned medium (CM), which was then used to stimulate EA.hy926 cells, thereby establishing an RA endothelial cell model. The groups included: a normal control group, a model group (optimal CM stimulation group), and PD treatment groups (PD at concentrations of 1.25 mg/L, 2.5 mg/L, and 5 mg/L); CM group, CM+PD group, CD146-OE+CM group, and PD+CD146-OE+CM group. Cell proliferation was assessed by the CCK-8 assay; EA.hy926 cells were transduced with lentivirus to overexpress CD146; cell migration was evaluated through the wound-healing assay; angiogenic capability was determined via tube formation assays; and CD146 expression levels were measured by Western blotting. 

Compared to the normal group, CM stimulation significantly enhanced endothelial cell proliferation (p<0.01). However, after 24 hours of PD treatment, a notable decrease in cell proliferation was observed (p<0.05). Additionally, CM stimulation improved cell migration ability relative to the normal group; this enhancement was significantly diminished following PD treatment (p<0.01). Furthermore, tube formation capability was markedly increased with CM stimulation compared to the normal group but showed significant inhibition after 24 hours of PD treatment (p<0.0001). Moreover, CD146 expression levels were significantly elevated in the model group when compared to the normal group and subsequently decreased following PD treatment (p<0.01), and in a dose-dependent manner. In the rescue experiment, CD146 overexpression was performed. Compared with the PD treatment group (CM + PD 5 mg/L), the proliferation, migration, and tube formation abilities of cells in the CD146 overexpression + PD treatment group (CM + CD146-OE + PD 5 mg/L) were significantly increased (p < 0.0001, p < 0.0001, p < 0.0001). Interestingly, when compared with the overexpression group (CM + CD146-OE), the proliferation, migration, and tube formation abilities of cells in the overexpression + PD treatment group (CM + CD146-OE + PD 5 mg/L) remained significantly suppressed, but the levels were still higher than those in the PD treatment group alone (p < 0.0001, p < 0.0001, p < 0.001). 

Our team previously confirmed that CD146 is highly expressed in the synovium of rheumatoid arthritis (RA) patients, particularly in vascular regions, and demonstrated its pro-angiogenic and pro-proliferative effects in EA.hy926 cells, with transcriptome sequencing implicating the Wnt pathway. In this study, we found that PD significantly inhibits angiogenesis, proliferation, and migration of EA.hy926 cells in a dose-dependent manner and suppresses CD146 expression. CD146 overexpression partially attenuated PD's anti-angiogenic effect, but PD still exhibited strong anti-angiogenic activity in CD146-overexpressing cells, suggesting that PD acts partly through CD146 regulation and possibly other pathways, reflecting its multifaceted nature. This study is the first to link PD's anti-angiogenic effect to CD146, revealing a promising therapeutic direction for RA.