Leniolisib Relieves LPS-induced Skull Osteolysis By Inhibiting Osteoclast Differentiation

Research background and purpose: The main cause of inflammatory osteolysis is that the secretion of inflammatory cytokines such as IL-1β,IL-6,and TNFα.The inflammatory cytokines can stimulate the recruitment and activation of osteoclasts,resulting in enhanced bone resorption,leading to disorders of bone metabolism.Osteoclasts,as the only cells with bone resorption,play a crucial role in the pathogenesis of inflammatory osteolysis.Understanding the mechanism by which osteoclasts absorb bone and the cytokines that regulate their formation and activity can provide potential mechanism-based or cytokine-based therapeutic targets to prevent inflammatory bone loss.Although existing therapeutic agents are effective against osteoclast-mediated bone loss,harmful side effects preclude the long-term use of these agents.The PI3K-AKT signaling pathway plays a key role in the occurrence and development of osteoclasts.Leniolisib(CDZ173)is a highly effective and selective PI3Kδ small molecule inhibitor.This topic studies the inhibitory effect of CDZ173 on the differentiation and function of osteoclasts and alleviates partial osteolysis induced by LPS,and further investigates its molecular mechanism.Methods:(1)In vitro experiment: First,bone marrow derive macrophages(BMMs)were extracted from the femurs and tibias of C57BL/6J mice and cultured for 4 days.Then,the CCK-8 kit reagent was applied to detect the proliferation effect of CDZ173 on BMMs stimulated by 30 ng/m L M-CSF.And TRAP staining was further carried out to observe the effect of CDZ173 on osteoclast differentiation.Secondly,the podosomal actin skeleton fluorescence experiment and the hydroxyapatite cell plate bone resorption experiment were designed to detect the effect of CDZ173 on the function of osteoclast resorption of bone matrix.At the same time,RT-PCR was used to observe the effects of various concentrations of CDZ173 on the expression of osteoclast-related specific genes.Furthermore,Western blotting was applied to explore the roles of CDZ173 on osteoclast-related signaling pathways PI3K-AKT,NF-κB and MAPK.Finally,ALP staining and ARS staining were performed to observe the effect of CDZ173 on osteoblast differentiation and mineralization.(2)In vivo experiment: LPS mouse model was constructed: male C57BL/6J mice(6-8 weeks)were randomly divided into 4 groups(Sham,LPS,CDZ173(2.5 mg/kg)+ LPS and CDZ173(5 mg/kg)+ LPS groups).At the end of the experiment,the skulls of mice were taken for Micro-CT analysis.Results: Results:(1)In vitro experiment:(1)In this study,we firstly found that CDZ173 can effectively inhibit the formation of osteoclasts in a safe dose range in vitro.(2)Through bone resorption experiments,it was found that CDZ173 inhibited the fusion of podosomal actin belts of osteoclasts and reduced the area of hydroxyapatite plates absorbed by osteoclasts.(3)Through RT-PCR experiments,we discovered that CDZ173 can down-regulate the expression of osteoclast-related specific genes Dc-stamp,Ctsk,Mmp-9,Trap,c-Fos and NFATc1 at the gene level.(4)In terms of molecular mechanism,it was further found that CDZ173 inhibited the activation of PI3K-Akt,ERK and JNK signaling pathways,and suppressed the expression of downstream transcription factor proteins c-Fos and NFATc1.(5)Alkaline phosphatase kit and alizarin red S staining showed that CDZ173 had no effect on osteogenesis and mineralization.(2)In vivo experiment: The analysis of Micro-CT scan results showed that the bone mass of the skull in the LPS group was significantly missing compared to the Sham group,and it was statistically significant;however,it was significantly improved after treatment with 5 mg/kg CDZ173.Conclusion: CDZ173 down-regulates the expression of osteoclast-related genes by inhibiting the activation of PI3K-Akt,ERK and JNK signaling pathways stimulated by RANKL,and then suppresses osteoclast differentiation and function,thus alleviating the progress of local osteolysis induced by LPS.