Study On The Mechanisms Of Lonafarnib Inhibiting Macro-phage Activation In Inflammatory Osteolysis

Objective Inflammatory osteolysis disease is a group of diseases triggered by inflammatory diseases,characterized by macrophage activation,increasing production of pro-inflammatory mediators and excessive activation of osteoclasts,leading to bone matrix degradation,bone calcium loss,focal bone erosion and increase the risk of fragility fractures.Currently,there are no drugs that can effectively inhibit inflammatory osteolysis.Repurposing from known drugs may be another way to develop anti-inflammatory and targeted osteoclast-inhibited drugs.This study aimed to explore the effects and mechanisms of Farnesyltransferase(FTase)inhibitor Lonafarnib(Lon)on the production of pro-inflammatory mediators,macrophage polarization and osteoclast activation in vivo and in vitro,so as to provide a theoretical basis for clinical prevention and treatment of inflammatory osteolytic diseases.Methods 1.We searched database to find out the targets of Lon and predict the targets of inflammation and osteolysis using a network pharmacology method.The GO and KEGG pathway enrichment analysis was performed on the common targets,and the protein interaction network was constructed to find out the core targets.2.RAW264.7 cells and BMMs were treated with different concentrations of Lon,and cell viability was detected by CCK8 assay.RAW264.7cells were stimulated with LPS,and RT-PCR and ELISA were used to detect the effects of Lon on the m RNA and protein expressions of pro-inflammatory mediators,respectively.Immunofluorescence and flow cytometry were used to track the expression of macrophage markers and analyze the effect of Lon on macrophage polarization.BMMs were induced by RANKL,the effect of Lon on cell apoptosis was detected by flow cytometry,and tartrate-resistant acid phosphatase staining was used to observe the effect of Lon on the formation of osteoclasts.The effect of Lon on the bone resorption function of osteoclasts was observed by podosome actin belt formation assay,osteoclast acidification assay and bone pits resorption assay.The osteoclast-relevant genes expression was explored by RT-PCR.Immunofluorescence was used to detect NFATc1.Si RNA silence assay was applied to examine the influence of FTase on osteoclasts activation.Related signaling pathways were identified by western blot assay.3.A mouse calvarial local osteolysis model induced by titanium particles and LPS-induced mouse systemic inflammatory bone loss model were established.Micro-CT and histopathology were used to detect the protective effects of Lon on inflammatory osteolysis.TRAP staining was used to observe the number of osteoclasts in bone tissue.Immunohistochemical staining and immunofluorescence staining were utilized to detect the production of inflammatory cytokines and macrophage makers,respectively.ELISA was used to detect bone catabolites in serum,and the protein in bone tissue was extracted to detect the relative expression of RANKL.Results 1.Twenty-six common targets of Lon on inflammatory osteolysis were predicted by network pharmacology.Pathway enrichment analysis of these targets indicated that Lon might affect TNF signaling pathway.And Lon influenced osteoclast differentiation and macrophage differentiation.TNF was the core target among these targets.2.Lon didn’t inhibit the proliferation of RAW264.7 cells and BMMs when the concentration was not more than 1μM.Lon down-regulated the expression of genes such as TNF-α,CD86,and i NOS in RAW264.7 cells,reduced LPS-induced secretion of pro-inflammatory mediators such as IL-1β,and inhibited M1 macrophage polarization.Lon suppressed RANKL-induced osteoclast formation in a dose-dependent manner in vitro,and promoted apoptosis of precursor osteoclasts.Lon inhibited the formation of osteoclast podosome actin belt and osteoclastic acidification.Lon affected osteoclastic resorption activity in a dosage-dependent manner.Lon down-regulated the expression of osteoclast-relevant genes,and suppressed NFATc1 nuclear translocation and auto-amplification.Lon decreased osteoclast-relevant protein expression.Mechanistically,Lon dampened FTase,and inhibition of FTase reduced osteoclast formation by suppressing ERK signaling.3.Lon reduced titanium particle-induced local osteolysis in mouse calvaria,inhibited osteoclast bone resorption,reduced the production of local pro-inflammatory cytokines,and inhibited macrophage M1 polarization in vivo.Meanwhile,Lon significantly ameliorated LPS-induced systemic bone loss,and reduced osteolysis of cancellous bone in the distal femur of mice,and decreased the serum levels of a bone catabolite,CTX-1.Lon reduced the formation of osteoclasts by inhibiting the production of RANKL.Conclusions Lon reduces the production of pro-inflammatory mediators,inhibits macrophage M1 polarization,and decreases the activation of osteoclasts by targeting FTase through affecting the phosphorylation of ERK in vitro.Lon protects the local osteolysis of mouse calvaria induced by titanium particles and systemic bone loss induced by LPS in vivo.Lon is a promising treatment option for inflammatory osteolytic diseases.