Inhibitory Effect Of Carbon Monoxide Releasing Molecule-3 On Osteoclastogenic Differentiation Of Mouse RAW264.7 Cells And Its Mechanism

Background and ObjectivesPeriodontitis is a chronic infectious disease of periodontal tissues,which is a common disease of oral cavity.The incidence of periodontitis is as high as 80%.Periodontitis may lead to the destruction of periodontal tissue structure,the loss of periodontal attachment,the formation of periodontal pocket and even the loss of teeth,which is the main cause of tooth loss in adults.At present,the clinical treatment of periodontitis is still based on basic periodontal treatment,combined with systemic or local use of antibiotics.When various bone resorption inducing factors activate a large number of osteoclasts,significant alveolar bone resorption occurs.It is difficult to reverse the destruction trend of alveolar bone by simple physical and chemical therapy.Therefore,it is important to inhibit osteoclastogenic differentiation and osteoclast maturation in the early stage of periodontitis,which is particularly crucial for the treatment of periodontitis,the maintenance of teeth function and even the preservation of affected teeth.The purpose of this experiment was to study whether CORM-3 can inhibit the osteoclastogenic differentiation and maturation of osteoclasts,and whether CORM-3 can suppress the alveolar bone resorption in experimental periodontitis in mouse.RAW264.7 cells are mouse-derived osteoclast precursor cells.The cell strain was originally derived from tumor induced by Abelson mouse leukemia virus.It is a better way to obtain osteoclast through RANKL induction of RAW264.7 cells.There have been a variety of osteoclast cell lines(such as FDCP,HL-60 cells,C7 cells and FLG29.1)used in the study of osteoclast.In the present,the onlv recognized osteoclast precursor cell is RAW264.7.RAW264.7 cells can express genes that represent phenotypic markers of osteoclasts,which are very similar to the gene expression profiles of osteoclasts.Furthermore,RAW264.7 cells were able to form bone resorption fossa.RANKL stimulated the up-regulation of the expression of osteoclast specific factors in RAW264.7 cells,such as TRAP,ca-thepsin K,et al,which indicates that RAW264.7 cells can be used as a good osteoclast model.There are a series of signature genes produced in the process of differentiation from pre-osteoclasts to mature osteoclasts.RANKL/RANK/OPG is the key system for regulation of osteoclast differentiation,maturation and bone resorption.When osteogenic/stromal cells are stimulated by various bone resorption factors,RANKL expressed on the surface of osteoblasts/stromal cells can specifically recognize the osteoclast precursors,combined with RANK,the functional receptor on the membrane,stimulating osteoclast precursors to differentiate into mature osteoclasts.At the same time RANKL can also make mature osteoclasts form actin ring rapidly through rank pathway and activate mature osteoclasts in bone resorption.Tartaric acid phosphatase(TRAP)is a key enzyme highly expressed in osteoclasts and is involved in osteoclast-mediated bone transformation.Cathepsin K(Cts-K)is a cysteine proteinase expressed on osteoclasts in the form of sol-proto-form.Both Cts-K and TRAP are important genes for the identification of osteoclasts.Matrix metalloproteinases(MMP)belong to the zinc binding endopeptidase family,which plays an important role in extracellular matrix degradation for various tissues including bone tissue.MMP,especially the highly expressed MMP-9 in osteoclasts,is an important protease for the degradation of extracellular matrix,and bone remodeling.It plays an important role in bone resorption with enhanced osteoclast activity.Carbon monoxide releasing molecules(CORMs)are a kind of newly synthesized compounds in recent years,which can be used as a new source of exogenous CO.CORMs are able to release CO after dissolving in proper solvent.CORM-3 is fully water soluble,which might therefore be of therapeutically interest by delivering CO in a controllable fashion.It has been proved that CORM can improve arthritis mice clinical manifestations effectively by inhibiting the expression of inflammatory cytokines in pathological region.Through its antioxidant,anti-inflammatory and anti-apoptosis effects,CORM can reduce the damage of multiple organ function induced by lipopolysaccharide in rats.In our previous studies,we found that CORM,as a donor of exogenous CO,can inhibit the expression of adhesion molecules in human gingival fibroblasts induced by inflammatory factors,and reduce the infiltration and adhesion of immunoreactive cells.It was also found that CORM could effectively inhibit the expression of TNF-a and IL-1? in serum,the absorption of alveolar bone and the infiltration of inflammatory cells in diseased areas of experimental periodontitis rats.Heme oxygenase-1(HO-1)is a rate-limiting enzyme for heme degradation.HO system is a microsomal enzyme system widely exists in human and mammalian bodies.The expression of HO-1 can be induced by many factors,such as heme,cytokine,CO et al.Studies have shown that HO-1 plays a direct regulatory role in the process of bone remodeling.In this study,we used mouse RAW264.7 cells as in vitro model to investigate the effect of CORM-3 on osteoclastogenic differentiation of the cells,and explore the possible mechanism underlying the regulation.We implemented C57 mouse as in vivo model to study the influence of CORM-3 on alveolar bone resorption in experimental periodontitis mouse.We found that,firstly,CORM-3 up-regulated the mRNA and protein expression of the osteoclast-specific markers TRAP,RANK,MMP-9 and Cathepsin K.Notably,the regulatory effect of CORM-3 was mediated by the release of CO.Secondly,the effect of CORM-3 on the expression of TRAP,RANK,MMP-9 and Cathepsin K was partly mediated by HO-1 pathway.Thirdly,CORM-3 suppressed the alveolar bone resorption in experimental periodontitis mice.The research provides a new strategy for the clinical treatment of periodontitis.Methods1.Establishment of osteoclast differentiation model of mouse RAW264.7 cellsMouse RAW264.7 cells were cultured on 6-well plate at the density of 5×104.The cells were divided into two groups:control group and osteociastogenic differentiation group(100 ?g/L RANKL+50 ?g/L M-CSF).After 5 days of culture,the osteoclast differentiation of the two groups was detected by TRAP staining.2.The effect of different concentrations of CORM-3 on the activity of RAW264.7 cellsRAW264.7 cells were cultured in 0,100,200,400 ?M and 800 ?M CORM-3 medium for 24h and 48h,respectively.The effects of different concentrations of CORM-3 on the activity of RAW264.7 cells were detected by CCK-8 assay.3.Identification of inhibitory effect of CORM-3 on osteoclast differentiation of RAW264.7 cells by TRAP stainingMouse RAW264.7 cells were cultured on 6-well plate at the density of 5×104.The cells were divided into three groups:control group,osteociastogenic differentiation group(100 ?g/L RANKL+50 ?g/LM-CSF)and CORM-3-osteoclastogenic differenti-ation group(200 ?M CORM-3+100 ?g/L RANKL+50 ?g/L M-CSF).After 5 days of culture,the osteoclast differentiation between the three groups was detected by TRAP staining·4.Detection of mRNA and protein expression of four osteoclast related factors(RANK,TRAP,MMP-9,Cts-K)in mouse RAW264.7 cells suppressed by CORM-3Mouse RAW264.7 cells were cultured on 6-well plate at the density of 5×104.The cells were divided into four groups:control group,osteociastogenic differentiation group(100 ?g/L RANKL+50 ?g/L M-CSF),degassed CORM-3-osteoclastogenic differentiation group(200 ?M degassed CORM-3+100 ?g/L RANKL+50 ?g/L M-CSF),and CORM-3-osteoclastogenic differentiation group(200 ?M CORM-3?100?g/L RANKL+50 ?g/L M-CSF).On day 5,7 9,fluorescence real-time quantitative PCR was used to detect the mRNA expression of RANK,TRAP,MMP-9 and Cathepsin K in cultured cells.The protein expression of RANK,TRAP,MMP-9 and Cathepsin K was investigated by Western Blot analysis.5.HO-1 Gene silencing lentivirus transfer experimentMouse RAW264.7 cells were cultured on 12-well plate at the density of 5×105.The cells were divided into five groups:blank group,NC control group,SH-1 virus group,SH-2 virus group and SH-3 virus group.Each group was divided into three MOI value(MOI=40,50,60).After 5 days of infection,the best MOI value was selected using fluorescence microscope;The cells in the best MOI value group were detected by PT-PCR and Westen Blot,and the lentivirus with the highest infection efficiency was screened out for the follow-up formal infection experiment.6.Detection of mRNA and protein expression of four osteoclast related factors(RANK,TRAP,MMP-9,Cts-K)in HO-1 gene silencing mouse RAW264.7 cells suppressed by CORM-3The HO-1 gene silencing mouse RAW264.7 cells were cultured on 6-well plate at the density of 5×104.The cells were divided into four groups:control group,osteoclastogenic differentiation group(100 ?g/L RANKL+50 ?g/L M-CSF),degassed CORM-3-osteoclastogenic differentiation group(200 ?M degassed CORM-3+100 ?g/L RANKL+50 ?g/L M-CSF),and CORM-3-osteoclastogenic differentiation group(200 ?M CORM-3+100 ?g/L RANKL+50 ?g/L M-CSF).On day 5,7 9,fluorescence real-time quantitative PCR was used to detect the mRNA expression of RANK,TRAP,MMP-9 and Cathepsin K in cultured cells.The protein expression of RANK,TRAP,MMP-9 and Cathepsin K was investigated by Western Blot analysis.7.The inhibitory effect of carbon monoxide releasing molecule-3 on the differentiation and maturation of osteoclasts and the alveolar bone resorption in experimental periodontitis mice36 male 8-week-old C57 mice(23g-25g)were randomly divided into three groups(n=12):control group,chronic periodontitis group(CP group),and CORM-3 intervention group(CORM-3 group).The experimental periodontitis was established by ligating maxillary second molars with silk thread and local injection of endotoxin in CP group and CORM-3 group.CORM-3 solution(CORM-3,10 mg/kg/d)and aseptic saline were injected intraperitoneally in CORM-3 group and CP group from the day of ligation.On day 7 and 14,6 mice in each group were sacrificed,respectively.Bilateral maxilla specimens were made.The alveolar bone resorption of one side of maxilla was observed under stereoscopic microscope by toluene blue staining.The other side of the maxilla specimen was fixed,decalcified,transparent and embedded,and then the conventional paraffin section was made.After TRAP staining,the differentiation and maturation of osteoclasts in alveolar bone were observedResults1.Establishment of osteoclast differentiation model of mouse RAW264.7 cellsAfter RAW264.7 cells were induced by osteoclastogenic induction solution for 5 days,TRAP staining showed obvious osteoclast formation,while in the control group,there had no obvious osteoclast formation.2.The effect of different concentrations of CORM-3 on the activity of RAW264.7 cellsThe results of 24h and 48h CCK-8 showed that 100 ?M and 200 ?M CORM-3 did not affect the activity of RAW264.7 cells,200 ?M CORM-3 significantly promoted the proliferation of RAW264.7 cells,while 800 ?M CORM-3 significantly inhibited the proliferation of RAW264.7 cells,(p<0.05).Therefore,the concentration of 200 ?M CORM-3 was selected as the experimental concentration.3.Identification of inhibitory effect of CORM-3 on osteoclast differentiation of RAW264.7 cells by TRAP stainingRAW264.7 cells were induced by osteoclastogenic induction solution in the presence or absence of CORM-3.After 5 days,TRAP staining showed that no obvious osteoclasts were found in the CORM-3 group and control group.In osteoclastogenic differentiation group,there were significantly increased osteoclast formation.4.Detection of mRNA and protein expression of four osteoclast related factors(RANK,TRAP,MMP-9,Cts-K)in mouse RAW264.7 cells suppressed by CORM-3 Real-time fluorescence quantitative PCR was used to detect the mRNA expression of four osteoclast associated genes RANK?TRAP?MMP-9 and Cathepsin-K on the 5th,7th and 9th day.The expression of TRAP,RANK,MMP-9 and Cts-K mRNA of the cells in osteoclastogenic differentiation group was significantly increased compared with the control group on day 5,7 and 9(P<0.05).In CORM-3-osteoclastogenic differentiation group four osteoclast associated genes were significantly lower than that in osteoclastogenic differentiation group(P<0·05).In degassed CORM-3-osteoclastogenic differentiation group,the level of TRAP,RANK,MMP-9 and Cts-K mRNA was parallel with that in osteoclastogenic differentiation group(P>0.05).The result that degassed CORM-3 failed to regulate the TRAP,RANK,MMP-9 and Cts-K mRNA expression suggested that the effect of CORM-3 was mediated by the release of CO.In consistent with the regulatory effect of CORM-3 on the mRNA level of the osteoclast-specific genes,the protein expression TRAP,RANK,MMP-9 and Cts-K in CORM-3-osteoclastogenic differentiation group significantly decreased compared with the cells without CORM-3 pretreatment(r<0.05).With degassed CORM-3 pretreatment,the protein expression of the cells maintained the same level as that in osteoclastogenic differentiation group at each indicated time point(P>0.05),which suggested again that the regulatory effect of CORM-3 was mediated by CO releasing.5.HO-1 Gene silencing lentivirus transfer experiment The result showed that,SH-3 virus had the highest silencing efficiency to HO-1 gene in mouse RAW264.7 cells when the MOI value was 60.6.Detection of mRNA and protein expression of four osteoclast related factors(RANK,TRAP,MMP-9,Cts-K)in HO-1 gene silencing mouse RAW264.7 cells suppressed by CORM-3RAW264.7 cells were transfected with HO-1-shRNA lentivirus.Transfected cells were subjected to osteoclastogenic differentiation with or without CORM-3 pretreatment.On day 5,7 and 9,the expression of TRAP,RANK,MMP-9 and Cts-K were detected by RT-qPCR.The mRNA expression of TRAP,RANK,MMP-9 andCts-K in osteoclastogenic differentiation group was significantly increased than control group on each time point(P<0.05).While the expression of these four osteoclast-specific genes in osteoclastogenic differentiation group was maintained the same level as that in CORM-3-osteoclastogenic differentiation group and degassed CORM-3-osteoclastogenic differentiation group(P>0.05).These results that CORM-3 was no more effective on the inhibition of the osteoclast-specific gene expression after HO-1-shRNA transfection suggested that the regulatory effect of CORM-3 was mediated by HO-1 pathway.7.The inhibitory effect of carbon monoxide releasing molecule-3 on the differentiation and maturation of osteoclasts and the alveolar bone resorption in experimental periodontitis miceAfter intraabdominal injection of CORM-3 in experimental periodontitis mice,the height of the alveolar bone in CP group was significantly lower than that in CORM-3 group(P<0.05),and the number of mature osteoclasts in alveolar bone in CORM-3 group was significantly less than that in CP group(P<0.05).Conclusions1.CORM-3 inhibits the osteoclastogenic differentiation of mouse RAW264.7 induced by RANKL and M-CSF.2.CORM-3 suppresses the mRNA and protein expression of four osteoclast associated genes RANK?TRAP?MMP-9 and Cathepsin-K in the osteoclastogenic differentiation process.The inhibitory effect of CORM-3 is mediated by the released CO.3.CORM-3 inhibits the osteoclastogenic differentiation of RAW264.7 cells through HO-1 pathway,which is evidenced by that CORM-3 is unable to inhibit the expression of the four osteoclast associated genes RANK?TRAP?MMP-9 and Cathepsin-K of the cells after HO-1 silence by lentivirus.4.CORM-3 inhibits the differentiation and naturation of osteoclasts in the alveolar bone,and decreases the alveolar bone resorption in experimental periodontitis mice.