P046 Effects of CB2 and TRPV1 stimulation on osteoclast overactivity induced by iron in pediatric inflammatory bowel disease

Abstract

Background The reduction of bone mineral density and osteoporosis (OP) has an high impact on inflammatory bowel disease (IBD) patients’ health. We have previously shown that a dysregulated iron metabolism occurs in IBD and leads to a decrease in circulating iron concentration and an excessive intracellular sequestration of iron. Studies suggest that iron overload affects the bone, accelerating osteoclasts (OCs) differentiation and activation with consequent bone resorption. The aim of this study was to evaluate the role of Cannabinoid Receptor type 2 (CB2) and transient receptor potential vanilloid type-1 (TRPV1) and of the iron in the development of osteoporosis in pediatric IBD. Methods We enrolled 21 young subjects aged less than 18 years stratified into 3 groups on the basis of the clinical diagnosis: 7 with Ulcerative Colitis (UC) 7 with Crohn’s disease (CD) and 7 were non-IBD controls (CTR). We evaluated the role of CB2 and TRPV1 receptors and of iron in the development of OP in pediatric IBD at diagnosis and analyzed the effects of the pharmacological modulation of CB2 and TRPV1 receptors on osteoclast activity and on iron metabolism by Western Blotting, Tartrate-resistant acid phosphatase assay (TRAP assay) and Iron Assay Kit. We evaluated bone resorption through a commercially available kit visualizing and counting the resorption pits with the optical microscope. Results We observed higher levels of TRPV1 and lower levels of CB2 in the OCs from UC and CD patients compared to CTR. In IBD patients we found a significant up-regulation of osteoclast markers TRAP and Cathepsin K and a consequent osteoclast hyper-activation. The treatments of OCs from IBD patients with the CB2 agonist JWH-133 and the TRPV1 agonist Resinferatoxin, RTX, led to a significant reduction of osteoclastic hyper-activation affecting bone markers expression, number and size of active OCs, and bone resorption area. We also analyzed the [Fe3+] concentration and the expression of iron metabolism modulators, ferroportin1(FPN-1) and divalent metal transporter1(DMT1), in OCs derived from IBD patients. We demonstrated an increase of [Fe3+] and accordingly a decrease of FPN-1 in both groups of patients and an increase of DMT1 levels. After 48h of exposure to JWH-133 and RTX we observed a significant reduction of DMT1 expression in OCs from IBD patients together with a strong increase of FPN-1. Conclusion Our data confirm the well-known role of CB2 and TRPV1 receptors in bone metabolism and suggest that their stimulation can reduce the osteoclast overactivity induced by iron, thus providing new insights into the pathogenesis and in the treatment of pediatric IBD-related bone resorption.