| Purpose: This thesis was performed with the aim of investigating the effect of FNDC5/Irisin and its homologue FNDC4 in bone homeostasis.To test if circulating Irisin level was associated with bone mineral density in human,a comprehensive meta-analysis was performed based on observational studies.According to previously published papers,we assume that circulating Irisin could be positively correlated with bone mineral density since many papers reported bone-anabolic effect of Irisin.To confirm the effect of elevated circulating Irisin level in bone metabolism,we used muscle creatinine kinase(MCK)driven FNDC5 over-expressing mice model.Finally,as the effect of Irisin in bone-regulating cells like osteoblasts,osteoclasts and osteocytes has been thoroughly studied,we attempted to evaluate the effect of FNDC4 in osteoclast,since FNDC4 is a secreted factor sharing high homology with the exercise-associated Irisin and the anti-inflammatory effect of FNDC4 has been reported.Methods: Four online databases including Pub Med,Embase,ISI Web of Science and CNKI were searched from their inception data up to December 2018.Observational studies featured to assess the association of circulating Irisin and bone mineral density was considered potentially eligible for inclusion.The meta-analysis was performed using Rev Man 5.2 software.Provided that Irisin was positively associated with bone mineral density in humans,we tried to evaluate the effect of elevated Irisin in bone modeling and remodeling.A FNDC5 over-expressing mice model,MCK_FNDC5,was employed here,in which FNDC5 expression was continuously activated by MCK.Histomorphometry was performed according to the latest ASBMR guideline,by using von Kossa staining,TRAP staining and Toluidine Blue staining,tibial trabecular bone microstructure was evaluated, along with dynamic and static indicators reflecting the activity of bone formation and resorption.The distribution and morphology of osteocytes in these trans-genetic mice were also evaluated using back scattered electron microscope(BSEM).Finally,in vitro experiments were performed using FNDC4,which is a homologue of FNDC4/Irisin,considering its anti-inflammatory effect which was proved in a mice colitis model.RANKL-treated BMMs were cultured with different concentrations of FNDC4 to evaluate the effect of FNDC4 on osteoclast differentiation.Luciferase assay and western blotting analysis were conducted to determine whether FNDC4 inhibited osteoclast formation via NF-KB signaling.Furthermore,to identify gene signatures in FNDC4 treated BMMs and used these to elucidate the underlying molecular mechanisms during osteoclast formation,we adopted a bioinformatics approach by downloading the GSE76172 gene expression profiling dataset from the Gene Expression Omnibus database.Differentially expressed genes(DEGs)were screened using RStudio and combined for a protein-protein interaction(PPI)network(visualized by Cytoscape software).According to the connectivity degree,the DEG with highest connectivity degree was selected by our study and included for further functional analysis.Results: A total of five studies were included in the meta-analysis.The concentration of circulating Irisin was compared between postmenopausal osteoporotic women and postmenopausal non-osteoporotic women.Based upon the results our meta-analysis,women with postmenopausal osteoporosis(PMOP)had significantly lower level of serum Irisin than non-PMOP control(SMD-0.64,95%CI-0.79,-0.50;P<0.0001).Furthermore,among these five included studies three studies reported the association of circulating Irisin level and bone mineral density.The combined Z values indicated that Irisin was positively associated with both lumbar spine bone mineral density(Z=0.21,95%CI 0.11,0.31;P<0.0001)and femoral neck bone mineral density(Z=0.27,95%CI 0.19,0.35;P<0.0001).A FNDC5 over-expressing mice model was used to evaluate the effect of elevated Irisin on bone metabolism.Male mice were sacrificed at 2 months and 13 months,right tibiae were collected for histomorphometry analysis and subsequent BSEM assessment.From the von Kossa staining sections,we observed less trabeculae in 2-month-old trans-genetic mice compared to wild type mice,and all the four bone microstructure indicators differed significantly between two groups.To further investigate the bone homeostasis in wild type and trans-genetic mice,dynamic parameters of bone formation,mineral apposition rate(MAR),mineralizing surface/bone surface(MS/BS)and bone formation rate(BFR)were assessed.MAR and BFR/BS were significantly decreased in trans-genetic mice compared to wild type mice.Osteoid surface/bone surface(OS/BS)showed an around 20% decrease in tran-genetic mice compared to wild type mice and Osteoid thickness(O.Th)showed a statistically significant reduction in trans-genetic mice,confirming the dynamic parameters and the decrease in bone formation.According to our Two-way Anova analysis,we concluded that the large differences in bone formation activity at 2-month-old were abolished and reached steady state at 13-month-old because of aging,thus similar level of bone mass were observed.Additionally,continuously elevated Irisin changed the morphology and distribution of osteocytes residing in cortical bone,rendering them an aged like phenotype.In our in vitro study,FNDC4 inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner,as shown by the significantly reduced number of TRAP-positive osteoclast.Administration of 1000ng/m L FNDC4 led to a sharp decrease in osteoclast number;the expression of osteoclast-related genes(CK,TRAP,NFATc1)that are upregulated in response to RANKL,decreased significantly with the treatment of 1000ng/m L FNDC4 for 24 hours;the mature osteoclast resorptive function was also suppressed in the presence of FNDC4 and the resorption was almost absent in the 1000ng/m L group.Additionally,we examined NF-KB transcriptional activity using a luciferase assay,the results suggested that 1000ng/m L FNDC4 could significantly suppress the RANKL-induced NF-KB transcriptional activity.Based on the GSE76172 gene expression profiling dataset and PPI network construction,CXCL10 was identified as the DEG with highest connectivity degree(degree=23).CXCL10,which has been proved to regulate osteoclast formation,was drastically down-regulated in the presence of 1000ng/m L FNDC4.Furthermore,treatment with CXCL10(10ng/m L)partially ameliorated the FNDC4-induced inhibition of osteoclast formation.Conclusion: Based on the findings of our current meta-analysis we found a significant association between elevated Irisin and high bone mineral density.However,in the FNDC5 over-expressing mice model,continuously activated FNDC5 expression led to significantly decreased bone volume,which was attributed to impaired bone formation activity.The normal osteocytes morphology and distribution were also disrupted by elevated Irisin.Finally,in the in vitro study,FNDC4 could inhibit RANKL induced osteoclast differentiation and function via inhibiting NF-KB pathway and down-regulation of CXCL10. |
