CP-25 Exerts Anti-angiogenic Effects on a Rat Model of Adjuvant-induced Arthritis by Promoting GRK2-induced Downregulation of CXCR4-ERK1/2 Signaling in Endothelial Cells

Angiogenesis can produce an invasive and destructive front, also named a pannus, comprised of inflammatory vascular tissue that covers and erodes articular cartilage, subchondral bone and peri‑articular soft tissues in rheumatoid arthritis (RA). Paeoniflorin‑6'‑O‑benzene sulfonate (CP‑25) is a novel ester derivative of paeoniflorin. We previously demonstrated that CP‑25 exerts anti‑inflammatory and immunoregulatory effects. CP‑25 also exhibits a marked therapeutic effect on adjuvant‑induced arthritis (AA), and is able to inhibit synovial and immune cell function, according to our previous study. However, the effect of CP‑25 on angiogenesis remains unclear. In the present study, AA was initiated in Sprague‑Dawley rats via intradermal immunization in the right hind metatarsal footpad with heat‑killed Mycobacterium butyricum in liquid paraffin, and rats were divided into four groups: Normal, AA rat model, CP‑25 (50 mg/kg) and methotrexate (0.5 mg/kg) groups (n=10 rats/group). Subsequently, joint synovium in AA rats was pathologically evaluated by hematoxylin and eosin staining, synovial vascular proliferation was evaluated by immunofluorescence, the synovial expression levels of C‑X‑C motif chemokine ligand 12 (CXCL12) were detected by immunohistochemistry and ELISA, and synovial C‑X‑C chemokine receptor type 4 (CXCR4) was detected by western blotting. The results demonstrated that CP‑25 ameliorated clinical signs and pannus formation in the ankle joint in rats with AA. Furthermore, there was a positive correlation between pannus score and CXCL12 and CXCR4 expression. In addition, the effects of CP‑25 on endothelial cell function and CXCL12/CXCR4 signaling were studied in vitro using human umbilical vein endothelial cells (HUVECs). The results demonstrated that CXCL12 significantly promoted HUVEC proliferation, migration and tube formation, and that CP‑25 could reverse these abnormalities by inhibiting plasma membrane localization of G protein‑coupled receptor kinase 2 (GRK2) in HUVECs. These findings suggested that CP‑25 may markedly inhibit pannus formation in AA. This effect may be associated with a reduction in the plasma membrane localization of GRK2 in endothelial cells, an enhancement of the inhibitory effect of GRK2 on ERK1/2 in the cytoplasm, a reduction in the phosphorylation of ERK1/2 and in the function of HUVECs.