The Effect And Mechanism Of Metformin On Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells And Osteoporosis In Ovariectomized Rats

Background:Diabetic osteoporosis(DOP)is a complication of diabetes complicated with osteoporosis,resulting in reduced bone mass.It is a serious complication of diabetes involving bone and seriously affects the quality of life of diabetic patients,the relationship between them are not definite mechanism,thus effectively control and treatment of diabetic osteoporosis is imminent.Metformin is a first-line drug for the treatment of type 2 diabetes patients,and its research in tumor,thyroid disease,osteoporosis and other fields has attracted more and more attention.Estrogen deficiency in menopausal women can lead to postmenopausal osteoporosis.Estrogen can inhibit the differentiation and maturation of osteoclasts,promote the growth and differentiation of osteoblasts,and thus play a protective role in bone formation.In recent years,it has been found that metformin is not only hypoglycemic,but also osteogenic.Metformin's role in osteogenesis involves a variety of molecular mechanisms.On the one hand,MF can enhance the expression and secretion of bone morphogenetic protein(bmp-2)and osteopontin(OPN)by activating the metabolic pathway of adenosine monophosphate-activated protein kinase(AMPK).Bmp-2 and OPN are important factors for osteoblast differentiation and maturation.In addition,metformin induces AMPK signaling pathway activation to enhance the expression of runt-related transcription factor-2(Runx2),and the formation of small Heterodimer Partner(SHP)and Runx2 complex in osteoblasts regulates the expression of osteoblasts Related target genes.The final result is to induce BMSCs to differentiate into osteoblasts,promote the proliferation,differentiation and maturation of osteoblasts and matrix mineralization,and enhance bone formation.On the other hand metformin can stimulate bone protection element(osteoprotegerin,OPG)the synthesis of secretion,reduce the NF kappa B receptor ligands predominate(ligand of receptor activate of nuclear factor kappa,RANKL)expression in osteoblast,OPG is the key factor of inhibiting bone resorption through competitive and RANKL thereby impede the combination of RANKL and RANK,inhibiting osteoclast activity and promote its apoptosis,reduce bone resorption function,Bone formation.Objective:The aim of this study is to explore the effects of metformin on bone mineral density and bone mineral content in ovariectomized(OVX)rats were observed with estradiol as positive control,at the same time from the level of cell cellular and molecular biology to explore metformin to ovary rat bone marrow mesenchymal stem cells to the osteoblast differentiation of cells,explore metformin play bone,the mechanism of protection may metformin for clinical prevention and treatment of diabetic osteoporosis provide scientific experimental basis for the rational use of drugs.Methods:Study 1:Effects of estrogen and MF on bone mineral density and bone mineral content in ovariectomized ratsSixty female SD(sprangue-dawley)rats aged three months were randomly dividedintofourgroups:shamoperation(SHAM)group,ovariectomized(OVX)group,ovariectomized +metformin(OVX+MF)group,ovariectomized+?-estradiol(OVX+E2)group.One week after ovariectomy or sham surgery,the OVX+MF group was given 100mg·kg-1·d-1by gavage of MF,the OVX+E2 group was given 0.1mg·kg-1·d-1 by gavage of17-estradiol,and the other groups were given the same volume of distilled water by gavage,and the dosage was adjusted according to the change of body weight.After sixty days of continuous administration,the rats were sacrificed by neck method,and the femur and tibia of the rats were separated.The right tibial bone density and bone mineral content of the rats in each group were determined by dual-energy X-ray absorptiometry.Study 2:Effects of estrogen and MF on osteoblast differentiation of BMSCs in ovariectomized rats2.1 Isolation and culture of BMSCs:bone marrow cell suspensions were taken from the bilateral femur and tibia of SD rats in each group,and BMSCs were isolated and cultured and induced to differentiate into osteoblasts.Cell activity and proliferation were determined by MTT method.2.2 Alkaline phosphatase(ALP)activity assay:cells in each group were inoculated with 5×10~5/well density in 6-well plates.The cells were washed with PBS and dissolved in 0.5ml 0.1%Triton x-100 with the ALP kit after 7 days of osteogenic induction.2.3 Mineralized nodule formation and the determination of calcium content:groups of cells with 5 x 10~5/hole density vaccination in 6 orifice,osteogenesis induction training 28 days using alizarin red S staining,petri dish washing twice,with PBS in situ fixed by 95%alcohol,1%alizarin red(2%alcohol preparation,PH 8.3)staining,the microscopic observation,with 10 mm the coordinates of the mesh count the number of calcified nodule formation to the naked eye.In the cell culture layer,0.6n hydrochloric acid was decalcified for 24 hours,and the calcium content in the calcium containing supernatant was determined by o-cresolphthalein and ketone method.2.4 RNA extraction and real-time PCR detection of osteoblasts genes:cells in each group were inoculated with 5×10~5cells/well density in a 6-well plate.After 21days of osteogenic induction solution culture,RNA extraction and fluorescence quantitative real-time PCR were used to determine osteoblasts genes:collagen type I?OC?OPG?RANKL?IL-6 mRNA and GAPDH were used as watch genes.At the end of the reaction,the amplification curve and dissolution curve of real-time PCR were confirmed,and the relative quantitative calculation of PCR was carried out by double CT method.Real-time PCR was used to extract total RNA from tissues or cells.The mRNA was used as a template and the cDNA was reverse-transcribed using Oligo(dT)or random primers using reverse transcriptase.CDNA was used as the template for PCR amplification to obtain the target gene or detect gene expression.Results:1.To study the effect of metformin and estrogen on bone mineral density and bone mineral content in ovariectomized rats and determine the bone mineral density and bone mineral content in each group:compared with SHAM group,bone mineral density and bone mineral content in OVX group were significantly reduced(P<0.01).Metformin and estrogen significantly improved bone mineral density and bone mineral content in ovariectomized rats(P<0.01),but the bone mineral density and bone mineral content in the OVX+E2 group were higher than that in the OVX+MF group(P<0.05).The bone mineral density and bone mineral content of OVX+MF group and OVX+E2 group were close to that of the control group,with no statistical difference(P>0.05).2.The results of the MTT assay suggested that BMSCs were isolated from the bilateral femur and tibia of SD rats in each group and cultured.BMSCs were then cultured with mineralization induction solution to induce osteoblast differentiation.Cell growth was observed under an inverted microscope after inoculation.The cell growth and adaptation period was from 1 to 3 days after inoculation,and the cell proliferation was slow.On the 6th day,the cell proliferation rate reached a peak,then gradually slowed down,and the number of cells began to decline.Under the inverted phase contrast microscope,represented the rat BMSCs in the induction medium,after differential centrifugation to stick wall inside a small round ball is suspended in the culture medium,begin the sidewall within 2 to 3 h,three days after inoculation cells were fusiform,fusiform,polygon,triangle,and reaches outward growth,6-8 last single,borrow some cells swelled mutual connection,gradually to the osteoblast form transformation of cells in the areas of high density;Growth interconnected confluent,osteoblasts are grown in multilayer overlapping,and showed a trend of calcification,cell matrix secretion increased and package,10-12 days appear more scattered dense clumps,and gradually increase,mass surrounding area cell density is higher,and contour is fuzzy,mass center gradually dim,pervious to light sex difference,formed distinct nodules.The morphological changes of BMSCs cells in each group were observed under fluorescence microscope,and it was found that after 3 days of cell culture,the typical morphology of osteoblasts appeared in each group:polygon,triangle and spindle.In the OVX group,there were significantly increased dead cells with red fluorescence,while in the SHAM group,there were almost no dead cells.Compared with the OVX group,the number of dead cells in the OVX+E2 group and the OVX+MF group decreased,and the cells showed a trend of fusion.It indicated that MF could promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts,promote the activity of osteoblasts,and reverse the cell death.The cell activity of osteoblasts in ovariectomized rats was significantly decreased,and both MF and estrogen significantly improved the inhibitory effect on the activity of osteoblasts in ovariectomized rats,and the effect of both was stronger than that of estrogen.3.Changes of cell proliferation,alkaline phosphatase activity and calcium deposition after differentiation of rat bone marrow mesenchymal stem cells into osteoblasts in each group:compared with SHAM group,proliferation capacity and ALP activity of OVX bone cells were significantly inhibited,and calcium deposition was significantly reduced(P<0.05).Compared with the OVX group,the proliferation ability and ALP activity of oophotomized rat osteoblasts were significantly enhanced after MF and estradiol intervention,and the amount of calcium deposition was significantly increased(all P<0.05).The proliferation capacity,ALP activity and calcium deposition of bone cells composed of OVX+E2 were higher than those of the OVX+MF group,with statistically significant differences(all P<0.05).4.Bone marrow mesenchymal stem cells differentiate into osteoblast of each group of rat after the formation of mineralized nodules:in mineralized induced liquid culture after 21 days,the microscopic observation,SHAM group,osteoblast multilayer overlapping growth gradually formed nodules,as collagen accumulation and calcium salt deposition,eventually forming the opaque mineralized nodule cells,calcified nodule number,around the calcified nodules can be observed more calcium salt deposition;OVX group,osteoblastic calcification was significantly inhibited,and there was no significant cell fusion.No significant mineralized nodules were observed.However,OVX+E2 and OVX+MF group,osteoblasts began to overlap and gradually formed mineralized nodules,with more calcification nodules and more calcium salt deposition around calcification nodules.The inhibition effect of MF and estrogen on osteoblast mineralization in ovariectomized rats was significantly improved,but the effect of estrogen was stronger.5.Changes in gene expression of rat osteoblasts in each group:Compared with SHAM group,mRNA expression levels of collagen type I?OC and OPG were significantly decreased in the OVX group,while RANKL and IL-6 were significantly enhanced in the OVX group,and the difference was statistically significant(P<0.05).Compared with the OVX group,the OVX+E2 group and the OVX+MF group significantly increased the expression levels of collagen type I?OC and OPG mRNA,and inhibited the expression levels of RANKL and IL-6mRNA,with statistically significant differences(P<0.05).Further comparison with SHAM group showed that the expression levels of collagen type I?OC?OPG?RANKL and IL-6mRNA in OVX+MF group were all higher than SHAM group,with statistically significant differences(P<0.05).Compared with the OVX+E2 group,mRNA expression levels of collagen type I?OC and OPG in the OVX+MF group were lower than those in the OVX+E2 group,and the expression levels of RANKL and IL-6mRNA were higher than those in the OVX+E2 group,with statistically significant differences(P<0.05).Conclusions:1.Bone mineral density and bone mineral content of ovariectomized rats were significantly decreased,while estrogen and metformin could significantly increase the bone mineral density and bone mineral content of ovariectomized rats,but metformin was slightly weaker than estrogen.2.The proliferation activity,ALP activity and calcification ability of osteoblasts in oarian rats were significantly reduced,while estrogen and metformin significantly increased the proliferation activity,ALP activity and calcification ability of osteoblasts,but metformin was slightly weaker than estrogen.3.Differentiation of bone marrow mesenchymal stem cells into osteoblasts in ovariectomized rats was inhibited,the expression levels of collagen type I?OC and OPG were significantly inhibited,and the levels of RANKL and il-6mRNA were significantly enhanced.Both estrogen and metformin significantly increased the levels of collagen type I?OPG?RANKL and IL-6mRNA.MF may promote the differentiation of bone marrow stromal cells into osteoblasts and inhibit the activity of osteoclasts through the OPG/RANKL/RANK cell signaling pathway.