Latexin Deficiency In Mice Exacerbating Bone Destruction In Rheumatoid Arthritis Via Enhancing Osteoclast Differentiation

Bone is a dynamic organ of the body,and bone homeostasis is maintained mainly by the action of bone formation by osteoblasts and bone resorption by osteoclasts.When the rate of bone resorption is greater than the rate of bone formation,bone homeostasis is disrupted,and causing many bone diseases,such as osteoporosis and rheumatoid arthritis.Rheumatoid arthritis is a common autoimmune disease with typical pathological manifestations of joint inflammation and bone destruction.Latexin（LXN）is a carboxypeptidase inhibitor in mammals,and some studies have found that LXN plays an important role in regulating stem cell differentiation and maintaining functional stability of the body,and LXN expression is upregulated in osteoarthritis.Therefore,we speculated whether LXN also has a regulatory role in the differentiation of bone marrow-derived macrophages to osteoclasts;and and whether LXN may have an impact in arthritic diseases in which osteoclast overactivation leads to bone destruction.Here,we investigated the regulatory mechanisms of LXN on osteoclast differentiation and arthritic bone destruction by studying the changes of LXN during the development of arthritis and the effects of LXN on osteoclast differentiation using collagen-induced arthritis mice as a model.In the second chapter,we examined the expression of LXN in two different strains of mice,C57BL/6 and DBA/1,by Western Blot and Q-PCR,and found that the expression level of LXN was lower in DBA/1 mice;while DBA/1 was used as a common strain of mice for collagen-induced arthritis（CIA）modeling,we speculated whether LXN would be changed during the CIA process.Then,we performed CIA model using DBA/1 mice,and examined the protein expression of LXN in control and CIA mice by Western Blot,and found that LXN expression was upregulated in CIA model;to further investigate the effect of LXN on collagen-induced arthritis model,we constructed collagen-induced arthritis model in wild-type（WT）mice and LXN knockout（KO）mice.The incidence and severity of arthritis were significantly higher in KO mice compared with WT mice,and Micro-CT bone scan,HE staining and TRAP（anti-tartrate phosphatase）staining revealed that the degree of inflammation,number of osteoclasts,and bone loss were more severe in KO mice than in WT mice after modeling.These experimental results suggest our that LXN is involved in the development of CIA and has an effect on the bone destruction of arthritis.In the third chapter,We conducted a vitro experimental study.Bone marrow mononuclear cells were extracted from WT group mice and KO mice,and osteoclasts were induced using RANKL（nuclear factor-κB receptor-activating factor ligand）.The results showed that BMMs（bone marrow-derived macrophages）in the LXN-KO group were more capable of inducing osteoclast differentiation;then we inoculated the cells onto bovine bone sections,and detect the bone resorption ability of osteoclasts.The results showed that the bone resorption pits formed by osteoclasts in the LXN-KO group were significantly more and larger than those in the WT group;meanwhile,we examined the protein expression changes related to osteoclast differentiation in WT and KO BMMs under RANKL stimulation and found that the expression of NFATC1（activated T-cell nuclear factor 1）,TRAF6（tumor necrosis factor receptor-associated molecule 6）,and C-FOS（cellular proto-oncogene）were significantly higher in the KO group than in the WT group.Finally,we examined the expression changes of LXN in the presence of RANKL induction and found that LXN expression was upregulated after RANKL stimulation.The above results suggest that LXN deficiency promotes osteoclast differentiation,enhances ability of osteoclast bone resorption,and upregulates the expression of genes related to osteoclast differentiation.In Chapter 4,we investigated the relationship between LXN and osteoblast differentiation and its regulatory mechanisms.We first examined the activation status of NF-κB（nuclear factorκB）signaling pathway after differentiation induction in BMMs from WT and KO mice,and found that P-IKBαexpression was significantly up-regulated and IKBα（nuclear factorκB inhibitor proteinα）expression was down-regulated in WT cells after RANKL stimulation,indicating that the NF-κB signaling pathway was activated at this time,while there was no significant change in the KO group of cells compared with the WT group.In order to investigate the reason for the enhanced differentiation of osteoblasts in KO group BMMs under RANKL stimulation,we extracted BMMs from WT and KO mice,extracted RNA for transcriptome sequencing after differentiation induction,and found no significant difference in NF-κB signaling pathway between WT and KO groups in both uninduced and induced conditions.Notably,we found differences in the ITAM-mediated differentiation pathway,and the expression of SIRPβ1a,SIRPβ1b,SIRPβ1c（signal regulatory proteinβ1）,DAP12（natural killer-activated receptor protein 12）and TREM2（trigger receptor expressed on myeloid-2）were significantly higher in the KO group than in the WT group after induction,and Q-PCR and Western Blot experiments were performed to verify the data of some transcriptome.Meanwhile,we examined the intracellular Ca²⁺signal intensity using Fluo-3 AM reagent and found that the intracellular Ca²⁺signal was significantly higher under RANKL stimulation,and the Ca²⁺signal was higher in the LXN-KO group than in the WT group.To further clarify the regulatory role of LXN in this pathway,we examined the phosphorylation level of Syk（splenic tyrosine kinase）and found that the phosphorylation level of Syk was significantly upregulated in KO group BMMs under RANKL stimulation within 30 min.When we treated cells with the Syk inhibitor R406 followed by induction,the upregulation of osteoclast-specific gene expression was also inhibited,and osteoclast differentiation was inhibited after knocking down Syk by si RNA,while the inhibition was more pronounced in the KO group,indicating that LXN deletion promotes osteoclast differentiation by activating Syk kinase activity.It has been shown that Syk kinase activation recruits to SIRPβ1 and activates downstream molecules to exert relevant effects.We examined the interaction of SIRPβ1 with Syk by immunoprecipitation assay and found that the interaction of SIRPβ1 with Syk was enhanced after LXN knockdown.Immunofluorescence experiments confirmed that LXN deficiency promoted the interaction of SIRPβ1 with Syk.Finally,we performed CIA modeling on LXN-KO mice and then treated CIA mice with Syk inhibitor R406,and found that R406 treatment effectively reduced joint inflammation in mice,HE staining showed that synovial inflammation was effectively relieved after R406 treatment,and TRAP staining revealed that the number and activity of osteoclasts were significantly reduced after treatment,and the results of staining with saffron O showed that CIA-induced cartilage damage was effectively relieved.In summary:this thesis found that（1）LXN expression is upregulated in collagen-induced arthritis;（2）LXN deficiency promotes collagen-induced arthritis;（3）LXN deficiency promotes osteoclast differentiation;（4）LXN deficiency activates Syk kinase activity by promoting the interaction between SIRPβ1 and Syk,thereby promoting osteoclast differentiation;（5）using Syk inhibitor R406 treatment of BMMs effectively inhibited the upregulation of osteoclast differentiation-related protein expression,and osteoclast differentiation was inhibited after knockdown of Syk by si RNA of Syk;R406 reduced the symptoms of joint inflammation,cartilage destruction,and bone loss caused by CIA.It is concluded that under normal conditions,RANKL stimulation mainly regulates osteoclast differentiation through classical signaling pathways such as NF-κB,while LXN deficiency enhances Syk kinase activity and promotes the expression of molecules related to this signaling pathway,thus promoting osteoclast differentiation and enhancing bone resorption function.