Adiponectin Inhibits Osteoclastogenesis By Regulating NF-?B And MAPK Signaling Pathway In Vitro Study

Background and objective:It is reported that adiponectin(APN)plays an important role in regulating bone mineral density(BMD)and is essential for bone metabolism regulation.However,the mechanism of its effect on osteoclasts remains unclear.Studies have shown that nuclear factor Kappa B(NF-kB)protein activation is an important regulatory factor for the survival and functioning of mature osteoclasts.In addition,the mitogenactivated protein kinase(MAPK)signaling pathway is closely related to the occurrence and development of osteoporosis.We has found that APN is expressed in the cartilage and subchondral bone in lumbar facet joint osteoarthritis(FJOA)in our previous studies,but the specific mechanism is still not clear.Accordingly,in our study,to investigate the effects of RAW264.7 cells after intervening by the APN in vitro experiment,we observe the osteoclasts mediated the changes of NF-k B and MAPK signaling pathways,to influence of bone dissolution,and study the possible mechanism of action.Methods:1.Cell viability assay and IC50 evaluation(1)RAW264.7 cell culture: RAW264.7 cells were cultured with 10% fetal bovine serum in the ?-MEM,and were used for subsequent experiments after 3generations.(2)RAW264.7 cells were cultured with different concentrations of adiponectin for 12 h,24h,48 h and 72 h,respectively.The absorbance value of the cells was determined by Enzyme Standard Instrument at the wavelength of 450 nm.2.In vitro osteoclastogenesis assay(1)APN induced by different concentrations: with different concentrations of APN(within the scope of IC50)and RANKL stimulating RAW264.7 cells,then the RWA264.7 cells were continuous cultured for 5 days,until it was observated osteoclast formation in the TRAP staining by the microscope.(2)Culture periodic induction experiment: with a certain concentration of APNand RANKL,RAW264.7 cells were cultured 1,3,5 days,respectively.The cells were stained at 1,3,5 days for TRAP.TRAP-positive multinucleated cells(MNCs)were counted under a microscope,and the percentage of TRAP stain-positive MNCs per area(/mm2)was measured using software.3.Measurements of related protein expression(1)The protein works for a short time: the experiment divided into the RANKL group and RANKL + APN group.RAW264.7 cells were cultured 0,5,10,20,30,60 min respectively.It was extrected cell total protein and detected related proteins: the IkB ?,p-IkB ?,p-p65,p65,p-p38,p38,p-JNK,JNK,p-ERK,ERK,?-actin.(2)The protein works for a long time: the experiment divided into the RANKL group and RANKL + APN group.RAW264.7 cells were cultured 0,1,3days,respectively.It was extrected cell total protein and detected related proteins:NFATc1,c-fos,?-actin.4.Detection of osteoclastic marker genes expressionThe experiment divided into the RANKL group and RANKL + APN group.RAW264.7 cells were cultured 1,3,5 days respectively.It was extrected cell total RNA and detected related genes: TRAP,CK,CTR,V-ATPase a3,V-ATPase d2,NFATc1,c-fos,GAPDH.Results:1.The proliferation effect of APN on RAW264.7 cells and IC50(1)According to the data analysis,when the concentration of adiponectin was10,20 and 30 ?g/ml,the proliferation of RAW264.7 was obviously inhibited.There was a significant difference among the groups,which was statistical significance(P <0.01).(2)Based on the data,we found that the IC50 of APN in RAW264.7 cell at 24 h and 48 h was 10.21 ?g/ml and 9.50 ?g/ml,respectively.2.APN effects RANKL-induced osteoclastogenesis in RAW264.7 cells(1)When the concentration of adiponectin was 0 ?g/ml,the number and area of mature osteoclast of the control group were significantly increased in the APN group after TRAP staining,which was great statistical significance(P < 0.01).(2)RAW264.7 cells were induced by 3,5 days.Compared with control group,the number and area of mature osteoclasts were significantly decreased the TRAP staining obviously at APN group.It has obvious statistical significance(P < 0.01).3.Expression of NF-kB signaling pathway and MAPK signaling pathway(1)The change of protein expression at a short timeBy Western blot,whether there was adiponectin intervention in cells or not,p-JNK,JNK,p-ERK,ERK did not change significantly in different time periods(P >0.05).While the expression of the IkB ? in the APN group was more than the control group when at 20,30 min(P < 0.01).P-IkB ? in the APN group relatively lower than the control when at 10,20,30 min(P < 0.01).P-p65 / p65 ratio at 5,10,20,30 and 60 min in the APN group significantly decreased,compared with the control group(P < 0.01).P-p38/p38 ratio in 5,10,20,30 min in the APN group was lower than the control group significantly(P < 0.01).(2)The changes of protein expression for a long timeAfter Western blot detecting,the expression of NFATc1 and c-fos in the APN group was lower than the control group obviously processing for 1,3 days,respectively(P < 0.01).3.The role of APN in the expression of osteoclastsBy PCR technique detecting,the RAW264.7 cells in the APN group were cultured for 1,3 days,significantly downregulate the expression of osteoclast related marker genes(P < 0.01),including genes: DC-STAMP,the TRAP,CK,CTR,V-ATPase a3,V-ATPase d2,NFATc1,c-fos.Conclusion:1.APN can inhibit the proliferation and growth in RAW264.7 cells,and this effect is present in a concentration-dependent manner.2.It was suggested that APN had a significant inhibitory effect,and this effect was dependent on the concentration-dependent manner.3.APN may alleviate the formation of osteoclasts by inactivating of I k B?/NF-?B signaling pathway and p38/MAPK signaling pathway.