Metformin Improves Osteogenesis To Alleviate Diabetic Osteoporosis Via The MiR-21/Mef2c/SOST Pathway

Objective:Diabetic Osteoporosis(DOP)is one of the most common skeletal complication of diabetes and it greatly increases the risk of fracture.The decreased bone formation caused by the imbalance of osteoclast/osteogenesis is the physiological basis of DOP.How to promote bone formation to improve diabetic osteoporosis has always been one of the main focuses in clinic.There are many kinds of hypoglycemic drugs.It is necessary to pay attention to the effect of hypoglycemic drugs on bone metabolism when formulating hypoglycemic plan.Metformin can reduce the incidence of diabetic osteoporosis and fractures,but the mechanism of it is not entirely clear.The pathogenesis of diabetic osteoporosis is complex.In addition to the possible mechanisms such as RANKL/OPG and oxidative stress,the role of sclerostin in inhibiting bone formation in DOP has also attracted scholar’s attention.MicroRNA-21(hereinafter referred to as,miR-21)promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells,and may participate in the regulation of sclerostin,thus becomes an intervention target for DOP.In this study,elderly female with diabetes,C57BL/6J and miR-21-/-mice and MLO-Y4 osteocyte lines were respectively taken as research subjects.By adopting population study,animal experiment and cell experiment,the research is aimed at exploring issues as follows:1.The levels of miR-21 in bone tissue of elderly female with diabetes and the bone mineral density and bone turnover of elderly female with diabetes;2.The role and mechanism of miR-21 on bone metabolism;3.The mechanism of how high glucose promotes diabetic osteoporosis by regulating miR-21 and the intervention effect of metformin.Method:1.Population study:According to the inclusion and exclusion criteria,elderly women were selected as the research subjects and divided into diabetes Group(DM Group)and non-diabetes Group(non-DM Group)according to the WHO diagnostic criteria for type 2 diabetes.The expression level of miR-21 in bone tissue of the subjects was detected,together with serum biochemical indicators such as blood glucose,liver function and renal function,biomarkers of bone turnover and bone mineral density.2.Animal research:C57 BL/6J and miR-21-/-mice are taken as research objects,and diabetic models were induced by using high-fat diet and low-dose STZ induction.The mice are divided into normal control Group(NC Group),a miR-21 knockout Group(miR-21-/-Group),type 2 diabetes Group(DM Group)and metformin intervention Group(DM+Met Group).After modeling of diabetes,mice were intervened with metformin for 10 weeks,and then body weight and blood glucose were measured.Three-point bending test was used to evaluate bone strength.Micro-CT was used to detect the bone microstructure and evaluate the bone quality of mice.The morphological change of bone tissue was observed.The expression level of miR-21 in bone tissue of mice was detected by RT-PCR.The expression levels of miR-21 target gene and its downstream proteins(Mef2c and sclerostin)as well as the expression of target proteins of Wnt/β-catenin pathway(Cyclin D1 and Runx2)was detected by Western blot.3.Cell research:The MLO-Y4 osteocyte line was used as the research object.The miR-21 mimics and inhibitor were used to transfect osteocytes for the add-back rescue experiment.The RT-PCR method and Western blot method were used to detect the expression levels of genes and proteins of each component in the pathway.Elisa method was used to detect the level of sclerostin in the supernatant.CCK-8 method was used to detect the cell proliferation.The role and mechanism of miR-21 in osteocyte function were explored from the gene level.To investigate the possible role and mechanism of miR-21 in DOP,high glucose(33.3mmol/L)and HG+miR-21 mimics were administered to intervene with osteocytes to detect the changes of gene and protein levels of each component of signaling pathway,cell proliferation changes by CCK-8 method and the microfilaments of osteocytes by F-actin immunofluorescence staining.Under high glucose conditions,high dose and low dose of metformin were respectively given to interfere with the osteocytes,and miR-21 inhibitor was given to transfect the osteocytes to detect the changes of gene and protein levels of each component of the signaling pathway,cell proliferation changes were detected by CCK-8 method,and the microfilaments of osteocytes were detected by F-actin immunofluorescence staining,in order to explore the mechanism of miR-21 in the improvement of diabetic osteoporosis by metformin.Result:1.Effect of DM on BMD and miR-21 level in bone tissue of elderly female with diabetes1.1 Compared with the non-DM Group,the FBG of patients in the DM Group was significantly increased,and the BMDs of femoral neck,whole hip and vertebral were all significantly decreased,together with the significant reduction in the level of bone formation marker PINP.1.2 Compared with the non-DM Group,the expression of miR-21 in bone tissue in DM Group was significantly decreased,and it was negatively correlated with blood glucose and HbAlc(r=-0.459,p=0.036;r=-0.449,P=0.041)。2.Role and mechanism of miR-21 in bone metabolism2.1 The results of three-point bending test showed that Stiffness and Maximum load of mice in the miR-21-/-Group were significantly decreased(P<0.05),and the Failure load showed a downward trend(P>0.05).2.2 Analysis of microCT showed that in the miR-21-/-Group,BV/TV was decreased,BSA/BV and TPF were increased,Tb.Th was thinner,and Tb.Sp was increased(P<0.05).Tb.N tended to decrease(P>0.05).2.3 HE staining showed that in the miR-21-/-Group,the cortical bone became thinner,the number of trabeculae decreased,and the thickness of trabeculae became thinner.2.4 The expression levels of Mef2c and Sclerostin in the bone tissue of the miR-21-/-Group were up-regulated,while the expression levels of Cyclin D1 and Runx2 were down-regulated.2.5 Transfecting osteocytes with miR-21 mimics,remarkably reduced gene and protein expression of Mef2c and SOST,and remarkably increased proliferation of osteocytes.Transfecting osteocytes with miR-21 inhibitor,significantly increased gene and protein expression of Mef2c and SOST,and significantly reduced proliferation of osteocytes.2.6 After transfection of osteocytes with Mef2c-siRNA,the expression of SOST detected by RT-PCR was significantly decreased.3.Role and mechanism of miR-21 in DOP3.1 The results of the three-point bending test showed that the Stiffness,Maximum load and Failure load of the diabetic mice were significantly decreased(P<0.05).3.2 Analysis of microCT showed that in the DM Group,BV/TV was decreased,while BSA/BV and TPF were increased.Tb.Th became thinner and Tb.Sp was increased(P<0.05).Tb.N tended to decrease(P>0.05).3.3 HE staining showed that the number of trabecular in DM Group was decreased,and thickness became thinner.3.4 In DM Group,the expression of miR-21 in bone tissue of mice was significantly decreased.The expressions of Mef2c and sclerostin in bone tissue were increased,while the expressions of Cyclin D1 and RUNX2 were decreased.3.5 Osteocytes were cultured with 22.2mmol/L glucose.The levels of miR-21 were detected at 0h,24h,48h and 72h,respectively.The results showed that the expression of miR-21 decreased gradually with the prolongation of culture time,and it was the lowest at 72h.Therefore,72 h was selected as the intervention time point.Taking 72h as the treatment time detection point,glucose with the concentrations of 5.5mmol/L,22.2 mmol/L,33.3 mmol/L and mannitol were respectively administered as the osmotic pressure control Group.The results showed that the expression of miR-21 decreased gradually with the increase of glucose concentration,and the expression in the 33.3 mmol/L glucose Group was the lowest,without significant change in each osmotic pressure control Group.Therefore,33.3 mmol/L glucose was selected as the intervention glucose concentration.3.6 The normal control Group(NC Group)was incubated with 5.5mmol/L glucose and the high glucose Group(HG Group)was incubated with 33.3mmol/L glucose.After 72h,we found that the expression of miR-21 in osteocytes of HG Group was decreased significantly,together with the mRNA and protein expressions of Mef2c and SOST.The proliferation of osteocytes in HG Group was decreased by CCK-8 detection,while the optical density value of osteocytes in HG Group was decreased by F-actin immunofluorescence staining.3.7 The osteocytes in HG Group were transfected with miR-21 mimics.The expression of miR-21 in the HG+miR-21 mimics Group was significantly higher than that in the HG Group.The mRNA and protein expression levels of Mef2c and SOST were decreased.The proliferation of osteocytes was increased,and the OD value was increased.4.Role and mechanism of miR-21 in the improvement of DOP by metformin4.1 The Stiffness and Maximum load of mice in the metformin intervention Group(DM+Met Group)were significantly increased compared with those in the DM Group(P<0.05),and there was no statistical difference between DM+Met Group and the NC Group(P>0.05).There was no significant difference in the failure loads between the DM+Met Group,the DM Group,and the NC Group(P>0.05).4.2 micro CT analysis showed that the BV/TV of mice in the DM+Met Group was higher than that in the DM Group,but lower than that in the NC Group(P<0.05).The BSA/BV of mice in the DM+Met Group was lower than that in the DM Group(P<0.05),and there was no statistical difference with that in the NC Group(P>0.05).The TPF of mice in the DM+Met Group tended to decrease as compared with that in the DM Group(P>0.05).The Tb.Th of mice in the DM+Met Group was increased as compared with that in the DM Group(P>0.05).The Tb.Sp of mice in the DM+Met Group was lower than that in the DM Group(P<0.05),but it was not significantly changed from that in the NC Group(P>0.05).There was no statistical difference in Tb.N among the three Groups(P>0.05).4.3 HE staining showed that the number of trabecular bones in the DM+Met Group was more than that in the DM Group,and the trabecular bones showed increased thickness,connectivity and pore size.4.4 The expression of miR-21 in the bone tissue of mice in the DM+MET Group was significantly higher than that in the DM Group(P<0.05),and there was no statistical difference as compared with that in the NC Group(P>0.05).The expression levels of Mef2c and sclerostin in the bone tissue of mice in the DM+Met Group were significantly decreased(P<0.05),but they were still slightly higher than those in NC Group(P<0.05).4.5 The expression level of Cyclin D1 in the bone tissue of mice in the DM+Met Group was significantly up-regulated as compared with that in the DM Group(P<0.05),and there was no statistical difference as compared with that in the NC Group(P>0.05).The expression level of Runx2 was significantly increased compared with that in the DM Group(P<0.05),but it was still slightly lower than that in the NC Group(P<0.05).4.6 Under the condition of high glucose(33.3mmol/L glucose),treated with high dose metformin at 50μmol/L(μM)and low dose metformin at 10μmol/L(μM)for 24hours,the results showed that the miR-21 expression in HG+Met-10μM Group was higher than that in the HG Group(P<0.05),but it was still lower than that in the NC Group(P<0.05).The expression of miR-21 in HG+Met-50μM Group was significantly higher than that in HG Group(P<0.05),but there was no statistical difference between the Met-10μM Group and NC Group(P>0.05).4.7 The gene and protein expression levels of Mef2c and SOST in the HG+Met-10μm Group and the HG+Met-50μM Group were significantly lower than those in the HG Group and decreased in a dose-dependent manner of metformin.No statistical difference was found in the sclerostin between the HG+Met-50μM Group and the NC Group.4.8 The osteocyte proliferation rates in the HG+Met-10 μ m Group and the HG+Met-50μM Group were higher than that in the HG Group.The OD value was also higher than that in the HG Group and showed a dose-dependent increase after metformin intervention.The OD value of HG+Met-50μM Group was not significantly different from that of NC Group.4.9 Osteocytes transfected with high dose of metformin by miR-21 inhibitor showed that compared with the HG+Met-50μM Group,the expression of miR-21 of osteocytes in the HG+Met-50μM+miR-21 inhibitor Group was decreased significantly,together with the gene and protein levels of Mef2c and SOST.In addition,the proliferation of osteocytes was decreased,and OD value was decreased significantly.All of the above indicators except for the expression level of Mef2c protein were rescued to the level of HG Group.Conclusions1.The bone mineral density of elderly female with diabetes is reduced and bone formation is inhibited.The expression of miR-21 in bone tissues of elderly female with diabetes is down-regulated.2.miR-21-/-inhibits bone formation reduces bone quality and bone strength by targeting Mef2c/sclerostin pathway.3.High glucose inhibits bone formation via miR-21/Mef2c/bone sclerostin pathway,as well as by regulating proliferation and microfilament of osteocyte,and eventually reduces bone strength and bone quality in mice.4.Metformin reverses the high glucose-induced reduction in the expression of miR-21,then further inhibits the expression of Mef2c/bone sclerostin,meanwhile enhances the proliferation and microfilament of osteocytes,so that Metformin stimulates bone formation in diabetic mice and improves the bone strength and bone quality of diabetic mice.