IL-35 prevent bone loss through promotion of bone formation and angiogenesis in rheumatoid arthritis

Angiogenesis in bone and osteogenesis appear to be closely linked, suggesting the existence of molecular crosstalk between pro-angiogenic molecules and osteoblasts. The pro-angiogenic molecules vascular endothelial growth factor (VEGF) with its receptors Flt-1, Flk-1 and fibroblast growth factor (FGF)-2 play a crucial role in born formation, an early and critical event in the pathogenesis of rheumatoid arthritis (RA). Interleukin (IL)-35 is demonstrated to be an anti-inflammatory cytokine in RA. However, the mechanisms involved are not fully understood. This study aims to investigate whether IL-35 has an impact on angiogenesis in osteoblasts and its related signalling pathway in RA.

The effects of IL-35 on osteoblasts proliferation, apoptosis and pro-angiogenic molecules mRNA and protein were examined using osteoblast-like MC3T3E1 cells in vitro. The effects of IL-35 on proliferation and apoptosis were examined using cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Pro-angiogenic molecules expression were assessed by real time PCR and ELISA, respectively. The signalling pathway between IL-35, bone formation, angiogenesis and signalling pathway was also investigated.

IL-35 promoted osteoblasts proliferation and inhibited apoptosis in a dose-dependent manner in vitro. IL-35 increased basal and TNF-α induced pro-angiogenic molecules expression by osteoblasts. Blocking the Th17/IL-17 signalling pathway with plumbagin inhibited the pro-angiogenic effects of IL-35 in osteoblasts.

These results suggested that IL-35 promotes bone formation and angiogenesis by fostering osteoblasts proliferation, inhibiting apoptosis and upregulating pro-angiogenic molecules through Th17/IL-17 related-signalling pathway. Our findings extend the current understanding of mechanisms modulating bone formation and angiogenesis in RA.