Bioinformatics Analysis Of Osteoclast-associated Genes And Effect Of NF-?B Inhibitor JSH-23 On Osteoclasts

Background and purpose of the study: Osteoporosis is common in the older people and postmenopausal women.The main reason is that the bone resorption rate is greater than the bone formation rate,which causes the bone density to decrease,leading to fractures.Osteoclasts are the only cells which can digest bone.Osteoporosis may benefit from treatments that inhibit osteoclast formation and function.In this study,we used gene chips to screen differential genes in different stages of osteoclasts,and analyzed them by information biology to find possible gene protein targets.The corresponding inhibitors were then used to verify their effects on osteoclasts,providing new drug options for osteoporosis treatment.Methods: 1.Two sets of gene chip data containing different stages of osteoclasts were downloaded in the GEO database.Differential genes during osteoclast formation and differentiation were screened using the R language Limma package.DAVID online analysis software was used to analyze the gene ontology function and KEEG pathway of differential genes,and the gene protein interaction network was constructed by STRING online analysis software.The Core Molecular Module was finally selected using Cytoscape's MCODE plugin to identify the core genes.2.Bone marrow macrophages were obtained from C57B/L6 mice and induced to form osteoclast precursor cells with 30 ng/mL M-CSF.The NF?B inhibitor JSH-23 was used to intervene with different doses of NF?B inhibitor.After 5 days,the effect of JSH-23 on the proliferation of osteoclast precursor cells was evaluated by MTS assay.Bone marrow mononuclear macrophages were induced to differentiate into mature osteoclasts by using 30 ng/mL M-CSF and 100 ng/mL RANKL.Different concentrations of JSH-23 were added during RANKL-induced osteoclast differentiation,and after 5 days,tartrate-resistant acid phosphatase was stained and the size and number of osteoclasts were observed by microscopy.Bone marrow macrophages cells were cultured in hydroxyapatite plates and spiked with RANKL,and different concentrations of JSH-23 were added.Finally,the absorption area of the hydroxyapatite coating was observed under a microscope and analyzed by ImageJ software.Then different concentrations of JSH-23 were added during the process of RANKL-induced osteoclast differentiation.After 5 days,the RNA was extracted and transcribed into cDNA.Finally,the corresponding mRNA expression was determined by Quantitative Real-time PCR.At last,the osteoclast precursor cells were induced with RANKL alone or with RANKL and JSH-23 for 1 hour,and the total protein was extracted at different time points,and then the protein expression was detected by Western blotting.Results: 1.By analyzing the gene chip of the two groups of osteoclast differentiation process,30 up-regulated genes and 19 down-regulated genes in the early stage of osteoclast differentiation were screened,and 4 up-regulated genes were screened in the middle stage of osteoclast differentiation.At the same time,the protein interaction network was constructed and two key molecular networks in the differential gene were selected,that was,Mmp9,Src,Plau,Vegfc,Met,Mmp14,as well as Traf1,Nfkbie,Nfkb2,Fos,Ctsk and Acp5.2.NF-?B inhibition JSH-23 has no toxic effect on osteoclast precursor cells under a concentration of 10 ?M.JSH-23 can inhibit RANKL-induced osteoclast differentiation and osteoclast hydroxyapatite dissolution.JSH-23 inhibited the expression of TRAP,CTSK,c-Fos and NFATc1 mRNA in osteoclasts induced by RANKL,and inhibited the phosphorylation of NF?B p65 during RANKL-induced osteoclast differentiation.Conclusion: 1.We screened 53 differential genes during the two stages of osteoclast differentiation,providing a theoretical basis for further research.2.NF?B inhibitor JSH-23 can inhibit osteoclast differentiation and bone resorption,and can affect the expression of related genes and protein.