Role And Mechanism Of Autophagy In Regulation Of Osteoblast Function By Thyroid Hormone

Objective:Objective to observe the effect of triiodothyronine(T3)on autophagy of MC3T3-E1 cells,and to explore the role and mechanism of autophagy in T3 mediated osteogenic differentiation and mineralization of MC3T3-E1 cells.Method:(1)MC3T3-E1 cells were treated with different concentrations of T3(0,1 n M,10n M,100 n M)and different intervention time(0,3 h,6 h,24 h),western blot was used to detect the expression of autophagy related proteins LC3-II / I and p62.The cells were divided into blank control group,T3 group,CQ group and T3 + CQ group,the expression of LC3-II / I and p62 protein was detected by WB.After MC3T3-E1 cells were treated with 10 n M T3 for 3 hours,the endogenous aggregation of LC3 was observed by immunofluorescence and the structure of autophagosome was observed by transmission electron microscope.(2)After the autophagy was blocked by bafilomycin A1,the osteogenic differentiation indexes Runx2,osterix,Col I and OCN of each group(blank control group,T3 group,Bafa1 group and T3 + Bafal group)were detected by q RT-PCR;ALP staining and ARS staining were applied to find the effect of autophagy on osteogenic differentiation and mineralization.(3)Mechanism exploration: The expression of AMPK and ULK1 in autophagy pathway was detected by WB;si RNA down regulated AMPK expression to observe the changes of downstream protein expression and autophagy,and its effect on osteogenic differentiation.Results:(1)After treated with different concentrations of T3(0,1 n M,10 n M 100 n M) for 3 hours,compared with the control group,the expression level of LC3-II protein in MC3T3-E1 cells in 10 n M and 100 n M treatment groups was markedly increased; the expression level of LC3-II protein in MC3T3-E1 cells treated with 10 n M T3 for 0,3,6 and 24 hours respectively was increased,while the expression level of p62 protein was gradually decreased.Compared with T3 alone,the expression of LC3-II protein was further increased after T3 combined with chloroquine treatment.Then, the aggregation of LC3 puncta was tested by immunofluorescence method,and the number of points in T3 group increased significantly.The results of transmission electron microscopy showed that the number of autophagosomes increased in T3 treated group,suggesting that T3 induced autophagy.(2)The cells were divided into blank control group,T3 group,bafal group and T3 + bafal group,Expression of markers of osteogenic differentiation Runx2, Osterix,Col I and OCN were detected 7 and 21 days after induction of osteogenesis. Compared with the cells treated with T3 alone,the expression of these genes was decreased after T3 combined with bafal,the intensity of ALP staining was significantly decreased,and the number of mineralized nodules was significantly reduced by ARS staining.(3)The expression of p-ampk(thr172)and p-ulk-1(ser555)increased in MC3T3-E1 cells treated with T3;After down regulating AMPK expression,the protein expressions of p-ampk(thr172),p-ulk-1(ser555),LC3-II and Runx2 were significantly decreased,suggesting that T3 may regulate autophagy through AMPK / ULK-1 signaling pathway,and then affects the osteogenic differentiation and mineralization of MC3T3-E1 cells.Conclusion:(1)Autophagy was induced by T3 in MC3T3-E1 cells;(2)T3 may promotes osteogenic differentiation and mineralization of MC3T3-E1 cells through autophagy mediated by AMPK / ULK-1 signaling pathway.