The Effects Of Adipo Ron On Proliferation,Differentiation And OPG/RANKL,Wnt/β-catenin Signaling Pathway In MC3T3-E1 Cells

Objective:To study the effect of adipo Ron on proliferation,differentiation and OPG/RANKL,Wnt/β-catenin signal pathway in mouse MC3T3-E1 cells,thus providing a experimental evidence for treatment of bone metabolic diseases.Methods: MC3T3-E1 cells were incubated with adipo Ron at different concentrations: 0(normal control group)3 mg/L,10 mg/L and 30 mg/L respectively for 48 h.The effects of adipo Ron on the proliferation activities of MC3T3-E1 cells were detected by MTT colorimetry.The activity of alkaline phosphatase(ALP)was detected by alkaline phosphatase kit.Alizarin red calcium staining was used to evaluate the mineralization nodules of MC3T3-E1 osteoblasts.The effect of adipo Ron on the cell cycle of MC3T3-E1 was evaluated by flow cytometry(FCM).The effect of adipo Ron on the apoptosis rate of MC3T3-E1 cells was detected by flow cytometry(Annexin V-FITC/PI double dyeing).The expression levels of RANKL,OPG and key molecules such as Wnt3 a,LRP6 and β-catenin in Wnt/ β-catenin signaling,and also Adipo R1,Adipo R2 in MC3T3-E1 cells were detected by Western blot respectively.Results: 1.The final concentrations of 3,10,30,90,and 270 mg/L of adipo Ron all inhibited proliferation of MC3T3-E1 cells when compared with cells in 0 mg/L adipo Ron and had statistical significance.In 0 to 30 mg/L concentration range,the proliferative effect decreased with the increment of adipo Ron dose,but 90 and 270 mg/L adipo Ron treatment no longer reduced the proliferation,that is to say,the largest inhibitory effect of adipo Ron on proliferation of MC3T3-E1 was at 30 mg/L.The results showed that adipo Ron in a certain concentration range had the dose-dependent inhibitory effect on proliferation of MC3T3-E1.2.When the final concentrations of 0,3,10,30 mg/L adipo Ron treated MC3T3-E1 cells for 48 h,the cells retarded at G0/G1 phase were increased as the concentration of adipo Ron increased,meanwhile the proportion of cells at G2/M phase was reduced.The result meaned that adipo Ron inhibited cell growth at G2/M phase.3.Compared with 9.2% of apoptosis ratio at 0 mg/L adiponectin treatment for 48 h,the cell apoptosis ratio at 3,10,30 mg/L adiponectin treatment groups increased significantly(for 13.35%,18.57% and 47.2% respectively).The result suggested that adipo Ron induced cell apoptosis in a certain concentration range.4.When adipo Ron at different concentrations(0,3,10,30mg/L)were incubated with MC3T3-E1 cells for 48 h,the activities of ALP of the cells were reduced,accompanied with the reduced formation of calcium nodules in cells.The results showed that adipo Ron intervention in a certain range restrained activities of ALP and suppressed mineralization of the cells,and exhibited dose-dependent effect.5.Adipo Ron,with a final concentration of 0-30 mg/L,promoted the expression of RANKL protein and inhibited the expression of OPG protein in MC3T3-El cells.6.The protein expressions of key molecules wnt3 a,β-catenin and LRP6 in Wnt/β-catenin signaling pathway were decreased with the increased concentrations of 0-30 mg/L adipo Ron treatment,which suggested that adiponectin may inhibit the physiological function of MC3T3-El cells through Wnt/β-catenin signaling pathway.7.Western Blot results showed that Adipo R1 and Adipo R2 were both expressed in MC3T3-El cells.Conclusion: 1.Adipo Ron(0-30mg/L)is a dose-dependent inhibitor of MC3T3-El cells proliferation.2.Adipo Ron(0-30mg/L)treatment showed a dose-dependent inhibition of MC3T3-El cells entering into G2/M phase.3.Adipo Ron(0-30mg/L)is a dose-dependent inducer of MC3T3-El cells apoptosis.4.Adipo Ron could inhibit the protein expression of OPG,meanwhile promoting the protein expression of RANKL,thus reduce the ratio of OPG/RANKL.5.Adipo Ron may inhibit the physiological function of MC3T3-El cells osteoblasts through inhibiting the Wnt/β-catenin signaling pathway.6.MC3T3-El cells express Adipo R1 and Adipo R2.