Abstract
ObjectiveOsteoporosis is a common metabolic bone disease mainly involving bone remodeling and blood vessels. The current study aimed to explore the suppressive role of interleukin (IL)-35 in nuclear factor kappa-B ligand receptor activator (RANKL) and macrophage colony stimulating factor (M-CSF)-induced osteoclastogenesis and angiogenesis in osteoclasts.MethodsOsteoclasts differentiation were induced by incubation of mouse leukemic monocyte/macrophage cell line RAW264.7 cells in the presence of RANKL and M-CSF and was assessed with tartrate-resistant acid phosphatase (TRAP) staining assay. The viability and apoptosis of RAW264.7 was measured using CCK-8 assay and flow cytometry, respectively. The expression of angiogenic genes and proteins were measured using RT-PCR, Western blots and ELISA. The inhibition of Th17/IL-17 axis was examined using plumbagin, which was demonstrated as an IL-17A related signaling pathway inhibitor.ResultsIL-35 inhibited the viability of RAW264.7 cells and promoted the apoptosis of RAW264.7 cells in a dose-dependent manner. Furthermore, IL-35 dose-dependently suppressed the expression of angiogenic markers including VEGF and its receptor. The suppressive effect of IL-35 was confirmed through the activation of Th17/IL-17 axis.ConclusionsWe demonstrated for the first time the immuno-suppressive function of IL-35 on RANKL and M-CSF-induced osteoclastogenesis and angiogenesis through Th17/IL-17 axis. Therapeutic approach involving augmentation of IL-35 regulatory response may serve as a novel treatment option for osteoporosis, especially by suppressing bone resorption and angiogenesis.
Highlights
Osteoporosis affects millions of people and is the most common bone metabolic disease worldwide
The RANKL and M-CSF induced osteoclasts were stained with TRAP, and the TRAP-positive cells with more than 3 nuclei were considered as osteoclasts (Figure 1)
To explore the effect of IL-35 on osteoclasts viability, we added different concentrations of IL-35 to osteoclasts which were successfully induced with RANKL and M-CSF
Summary
Osteoporosis affects millions of people and is the most common bone metabolic disease worldwide. Bone is a skeletal system and a vascularized tissue with a network of blood vessels and capillaries that provide nutrients and oxygen for bone development, which are regulated through the mediation of multiple signaling pathways between bone cells and endothelial cells [1, 2]. Blood vessels play a crucial role in the development of osteoporosis and are formed through two different biological processes. Most of the new blood vessels sprout by the process of angiogenesis, which is coupled with an expansion of the existing angiogenic networks through a series of steps including proliferation, migration of endothelial cell, sprouting vessel pruning, and anastomosis [4, 5]. Aberrant expression of VEGF in bone can cause an increase in bone resorption by osteoclasts [9]
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