The Role Of Metformin-induced Autophagy On Differentiation Of Osteoblastic MC3T3-E1 Cells

Objective: Osteoporosis(OP)is a metabolic osteopathy characterized by reduced bone mass and strength,which is caused by abnormal osteoblast osteogenesis and/or osteoclast bone resorption.It is seriously harmful to human health.Current studies have shown that autophagy is involved in the pathogenesis of osteoporosis.Metformin(MF)is a drug for type 2 diabetes mellitus(T2DM).Some studies have found that metformin can also promote osteoblast differentiation.However,whether metformin is associated with autophagy in the process of promoting osteoblasts differentiation,there is still a gap in this field.In this paper,we take mouse osteoblasts precursor MC3T3-E1 cell line as the research object to explore the role of autophagy in metformin promoting the osteogenic differentiation of MC3T3-E1 cell line.Methods: 1.MC3T3-E1 cells were treated with different concentrations of MF(0,200,400,800 micromol/l).Alkaline phosphatase(ALP)staining was used to detect the changes of osteogenic differentiation ability of MC3T3-E1 cells.Western blot was used to detect the expression of LC3 and P62 protein and immunofluorescence was used to detect the fluorescence intensity of LC3 protein to evaluate the changes of autophagy activity.The best intervention concentration was selected from the above four concentrations.2.Experimental grouping: control group(CON group),MF group(optimal intervention concentration 400 micromol/l),MF(optimal intervention concentration 400 micromol/l)+autophagy inhibitor 3-methyladenine(3-MA,5mmol/l)group,3-MA(5mmol/l)group.Western blot was used to detect the expression of LC3 and P62 protein,immunofluorescence was used to detect the fluorescence intensity of LC3 protein,transmission electron microscopy(TEM)was used to detect the number of autophages to evaluate the changes of autophagic activity,and ALP staining,semi-quantitative RT-PCR was used to detect the expression of osteocalcin(OCN)and collagen I(COL1)gene,Western blot was used to detect the expression of OCN and COL1 protein.To detect changes in osteogenic capacity.Results: 1.The ALP activity of MC3TE-E1 cells treated with MF in the concentration range of 0-400 umol/l gradually increased with the increase of MF concentration;the ratio of LC3 II/I gradually increased,the ratio of MF0 to MF400 increased,P < 0.05,MF200 to MF400,P < 0.05;the expression of P62 protein gradually decreased,compared with MF0 and MF400,P < 0.001,MF200 to MF400,P < 0.05;the immunofluorescence intensity of LC3 gradually increased,and the immunofluorescence intensity of MC3TE-E1 cells increased.The autophagy intensity and osteogenesis ability of MC3TE-E1 cell line were dose-dependent with the intervention concentration of MF.The autophagy intensity and osteogenesis ability of MC3T3-E1 were positively correlated.In 400 micromol/l MF group,the autophagy intensity of MC3T3-E1 was the strongest,and the osteogenic differentiation ability was the strongest.MC3T3-E1 was the best intervention concentration.Compared with 400 micromol/l MF group,the ALP activity of cells in 800 micromol/l MF group began to decrease,the ratio of LC3II/I decreased,P < 0.05,the expression of P62 protein increased,P < 0.001,and the immunofluorescence intensity of LC3 decreased.2.After the intervention of CON,MF,MF + 3-MA and 3-MA,the autophagy index LC3II/I ratio increased,the expression of P62 protein decreased,the fluorescence intensity of LC3 increased,the number of autophages increased and the autophagy activity increased in MF group;the activity of ALP,the expression of osteogenesis related genes and protein OCN,COL1 increased,and the osteogenesis ability increased.After adding 3-MA to MF,the ratio of LC3 II/I decreased,P < 0.05;the expression of P62 protein increased,P < 0.05;the fluorescence intensity of LC3 decreased,the number of autophages decreased,the autophagic activity of MF + 3-MA group decreased;the activity of ALP decreased,the expression of OCN gene decreased,P < 0.01;the expression of COL1 gene decreased,P < 0.05.The expression of OCN decreased,P < 0.05,COL1 decreased,P < 0.05,and the ability of MF + 3-MA to form bone decreased.Autophagy inhibitor 3-MA attenuated the osteogenic effect of metformin on MC3T3-E1 cell line.Conclusion: Within the concentration range of 0-400 umol/l,the autophagic intensity and osteogenic ability of MC3T3-E1 cell line were dose-dependent with the intervention concentration of metformin;the autophagic intensity and osteogenic differentiation ability of MC3T3-E1 cell line were positively correlated;metformin could promote osteogenic differentiation by activating moderate autophagy of MC3T3-E1 cell line.