| IntroductionDiabetes mellitus is a metabolic disorder characterized by hyperglycemia.With the change of life style,the incidence rate of type 2 diabetes shows a rapid increase.Moreover,with the significantly increased incidence of adolescent diabetes,and the improved sense of anesthetics,there have been burning needs for orthodontic treatment among people with type 2 diabetes.Therefore,the demand of providing better orthodontic treatment for patients with type 2 diabetes is becoming urgent.Orthodontic tooth movement is achieved by alveolar bone remodeling in response to a prolonged application of pressure to the tooth,leading to osteoclast-induced bone resorption on the compression side and osteoblast-induced new bone formation on the tension side.An alteration in the metabolic state that interferes with bone remodeling can result in different rates of tooth movement.The diabetic state up-regulates osteoclast migration and activity,resulting in abnormal orthodontic tooth movement.These findings suggest that patients with diabetes may respond to orthodontic treatment differently from those without diabetes in terms of how the periodontium and surrounding visceral cranium react to therapies.Osteocytes are the most numerous cells in bone tissue,which account for about 95%of the total number of cells.Osteocytes are able to control bone resorption and bone formation by secreting Receptor activator of nuclear factor kappa-B ligand(RANKL),Osteoprotegrin(OPG),Sclerostin(Sost),dentin matrix protein-1(DMP-1),and thus regulate bone metabolism.The previous results showed that the necrosis and apoptosis of osteocytes were related to bone resorption,which may be an important starting factor for orthodontic tooth movement.One previous study investigated the roles of osteocytes in osteoclastic bone resorption during orthodontic tooth movement by using the transgenic mice,in which osteocytes can be specifically ablated.These results provide in vivo demonstration for osteocyte involvement in osteoclastic bone resorption during orthodontic tooth movement.Osteocytes play important roles in proliferation and activity of osteoblasts.Approximately 75%of osteocytes from alveolar bones become apoptotic after DT administration,and most osteocytic lacunae become empty.Osteoblastic numbers and alkaline phosphatase(ALP)activity were markedly reduced at the endosteum of alveolar bone after DT administration compared with the control.Metformin(1-(diaminomethylidene)-3,3-dimethylguanidine)is the first-line therapy for patients with type 2 diabetes mellitus(T2DM)according to the American Diabetes Association/European Association for Study of Diabetes guidelines.During metformin’s worldwide spread for over 50 years,numerous studies concerning other potential indications have emerged.What’s more,recent studies have found that metformin has important effects on bone.Vestergaard et al.reported that patients with type 2 diabetes who were treated with metformin showed decreased risk for bone fractures.Gao et al.reported that metformin promotes osteoblastic differentiation and inhibits adipogenic differentiation in cultured marrow mesenchymal stem cells of rats through inhibition of peroxisome proliferator-activated receptor gamma,a nuclear receptor that regulates lipid and glucose metabolism.Zhen demonstrated that Metformin reverses the deleterious effects of high glucose on osteoblast function,including decreasing intracellular ROS and apoptosis and promoting osteogenic differentiation of osteoblasts.However,the previous studies about the adverse effects of diabetes on orthodontic tooth movement and the effects of metformin on bone metabolism are mostly focused on osteoblasts and osteoclasts,but the effects of both cells on osteocytes are rarely reported.In view of the important role of osteocytes in the regulation of bone remodeling,in this experiment,we investigate the damage of high glucose on osteocytes under fluid shear stress,the effect of metformin on high glucose-injured osteocytes,and the inductive effect of different osteocyte-conditioned media on osteoblasts and osteoclasts.Therefore,in this study,a fluid shear stress model was established in vitro to investigate the effect of different concentrations of high glucose on osteocyte-like cell MLO-Y4 under fluid shear stress and the effect of metformin on high glucose-injured osteocytes.The rats model of orthodontic patients with type 2 diabetes was constructed in vivo to find out the histological evidence that metformin affects alveolar osteocytes.In summary,this study investigated the effects and mechanisms of metformin on osteocytes under fluid shear stress and high glucose environment in vitro and in vivo to provide treatment ideas for clinical diabetic orthodontic patients from the perspective of osteocyte,an important regulator of bone metabolism.Materials and methods1.The effects of different concentrations of high glucose on MLO-Y4 under fluid shear stress Under fluid shear stress,6 experimental groups were set according to different concentrations of glucose(5.5,11,22,33,44,66mM).Cell proliferation was detected by CCK8 assays,cell apoptotic rate was tested by flow cytometry assays,immunofluorescence was applied to analyze the effect of strains on cells,mineralization induction was used to observe the mineralization state of osteocytes under different concentrations of glucose,the changes of RANKL,OPG,SOST,DMP-1 in osteocytes by Western Blot and qRT-PCR.2.The effect of metformin on high glucose-damaged MLO-Y4 cells and the effect of the supernatants of MLO-Y4 cells collected after high glucose and metformin treatments on the biological characteristics of osteoblasts and osteoclasts2.1 Two different treatments were applied on MLO-Y4 cells under fluid shear stress:1)After using HG22mM high glucose medium for 2 weeks,different concentrations of metformin were added;2)After 14 days of culturing in HG22mM high glucose medium,the medium was changed into normal ones and different concentrations of metformin were added.The group with 5.5mmmol/L glucose and 0μM metformin was set as the control group.Cell proliferation was detected by CCK8 assays,cell apoptotic rate was tested by flow cytometry assays,mineralization induction was used to observe the mineralization state of osteocytes under different concentrations of glucose.Changes of the expression of the markers’ mRNA and protein were detected by qRT-PCR and Western Blot assays.2.2 Osteocytes culture medium under the above different conditions were collected to culture the mouse pre-osteoblasts MC3T3-E1.The apoptosis of osteoblasts was detected by flow cytometry;Mineralization ability of MC3T3-E1 was evaluated by mineralization induction;the changes of osteogenic markers’ mRNA and protein were detected by qRT-PCR and Western blot.Osteoclasts were induced using mouse RAW264.7 cells.The effects of different osteocytes’ medium on the number and function of osteoclasts were observed.3.The primary mechanisms of the effect of metformin on high glucose-damaged MLO-Y4 cells3.1 Analysis of bioinformatics technology DrugBank database was used to find the targets of metformin called PRKAB1,then String database was used to retrieve the ten genes that are closely related to the metformin targets,at last,KEGG database,GO database,and the genes above were applied to predict the biological processes and signaling pathways that metformin was involved in.3.2 Western Blot analysis After MLO-Y4 cells were cultured in high glucose medium(HG22mM)for 14 days under fluid shear stress,the medium was replaced by serum-free medium and cultured for another 12 hours.Then 1M metformin was added.Proteins were collected at Omin,5min,15min,30min,60min,90 min,2h-10h respectively and Western Blot analysis was applied to analyze the changes of different signaling pathways with the change of time.4.The effect of metformin on high glucose-damaged osteocytes under fluid shear stress in vivo Thirty Wistar rats were divided into three groups,the normal control group,the diabetes group,the metformin treatment group.Orthodontic tooth movement model was established on those three groups of rats,after 2 weeks,tooth movement distance was measured by mouth-cavity modulus,the animals were executed to take the maxillary,and the maxillary tissue section was made,and HE staining,the trap staining,the immunohistochemical staining were performed.Results1.The effects of high glucose on MLO-Y4 under fluid shear stress1)The results of immunofluorescence staining of F-actin showed that in the FSS group,the expression of F-actin was stronger than control.2)The results of CCK-8 assay and Flow cytometry showed that 11 and 22 mmol/L glucose groups promoted cell proliferation and decreased cell apoptosis rate in a dose-dependent manner(p<0.05),and 22 mmol/L glucose group showed more obvious promotion on proliferation(p<0.01),while 33,44,and 66 mmol/L glucose groups inhibited cell proliferation and promoted cell apoptosis(p<0.05);in the 66 mmol/L group,the apoptosis was most significant(p<0.01).3)Mineralization induction showed that the number of mineralized nodules and the mineralization ability decreased with the increase of glucose concentration.4)The results of qRT-PCR and Western Blot indicated that with the increase of high glucose concentration in a certain range(5.5-66mM),the expression of OPN mRNA,OCN mRNA and DMP-1 mRNA decreased,the expression of SOST mRNA and the ratio of RANKL/OPG increased in a dose-dependent manner.2.The effect of metformin on high glucose-damaged MLO-Y4 cells and the effect of the supernatants of MLO-Y4 cells collected after high glucose and metformin treatments on the biological characteristics of osteoblasts and osteoclasts2.1 The effect of metformin on high glucose-damaged osteocytes under fluid shear stress2.1.1 Under fluid shear stress and high glucose conditions,different concentrations of metformin were added1)The results of CCK-8 assay and flow cytometry showed that:the different concentrations of metformin(100-800μM)could all promote the proliferation and decrease the apoptosis of MLO-Y4 cells,and the peak appeared at the 400μM;2)Mineralization induction showed that different concentrations of metformin(100-800μM)could all promote the mineralization of MLO-Y4 cells,and metformin at the concentration of 100μM showed the most obvious effect.3)The ratio of RANKL/OPG mRNA decreased in a dose dependent manner;the expression of OCNmRNA increased with 100-200μM of metformin and reached the peak at 100μM;the expression of OCNmRNA decreased when 400-800μM metformin was added,the peak appeared at 800μM;the expression of DMP-1mRNA increased,the expression of SOSTmRNA decreased in a dose-dependent manner with 100-800μM of metformin.4)The results of Western Blot showed that metformin at low concentration(100-200μM)could alleviate the inhibitory effect of high glucose on OCN,OPG and DMP-1 to a certain extent,this effect was also obvious in groups with metformin at high concentration(400-800μM).At the same time,low concentration of metformin(100-200μM)could reduce the promotive effect of high glucose on OCN,OPG and DMP-1 to a certain extent,and this effect was more obvious at high concentration(400-800μM)of metformin.2.1.2 After using HG22mM high glucose medium for 2 weeks,the medium was replaced by normal medium,and different concentrations of metformin(0-800μM)were added1)The results of CCK-8 assay and flow cytometry showed that:metformin promoted cell proliferation and decreased the apoptosis rate in the range of 0-400 μM,and the peak appeared at the concentration of 100μM,while metformin at the concentration of 800μM suppressed cell proliferation and enhanced the apoptosis rate of MLO-Y4.2)Mineralization induction showed that different concentrations of metformin could all significantly promote the mineralization of MLO-Y4 cells,and the effect of metformin at the concentration of 200μM was the most obvious.3)The the ratio of RANKL/OPG mRNA dropped in a dose dependent manner;With 100-800μM metformin,the expression of OCN mRNA and OPN mRNA increased and reached the peak at the concentration of 200μM;the expression of DMP-1mRNA was increased and the expression of SOST mRNA was decreased.DMP-1 mRNA showed the highest while SOST mRNA showed the lowest expression when 400μM metformin was applied.4)The results of Western Blot showed that compared with the control group,groups added with metformin presented significantly promoted expression of OCN,OPG,DMP-1 protein,and decreased expression of RANKL and SOST protein.2.2 Effects of different supernatants of MLO-Y4 cells on pre-osteoblast MC3T3-E1 and osteoclasts2.2.1 Effect of the supernatants of MLO-Y4 cells collected after high glucose and metformin treatments on the biological characteristics of pre-osteoblast MC3T3-E1The results of flow cytometry showed that after normal MC3T3-E1 culture medium plus different conditions of osteocyte culture medium(1:1)were added,high glucose MLO-Y4-culture supernatants and high glucose plus 200μM metformin groups showed increased apoptotic rate of MC3T3-E1 and damaged the mineralization ability;while normal medium plus metformin groups showed decreased rates of apoptosis and the function of MC3T3-E1 was rescued.2.2.2 Effect of the supernatants of MLO-Y4 cells collected after high glucose and metformin treatments on the biological characteristics of osteoclasts High glucose MLO-Y4-culture supernatants(HG 22mM)could significantly promote the formation and function of osteoclasts.And high glucose plus 200μM metformin groups(HG 22mM+MF 200μM)could alleviate the effect of high glucose on osteoclasts.The groups with conditional media in which high glucose medium was replaced by normal medium with metformin inhibited the formation and function of osteoclasts in a dose-dependent manner.3.The mechanisms of the effect of metformin on high glucose-damaged MLO-Y4 cells3.1 Analysis of bioinformatics technology1)Through the DrugBank database,the target of metformin 5’-AMP-activated protein kinase subunit beta-1 was found and its gene name was PRKAB1.And the ten genes discovered by string database that were closely related to PRKAB1 were PRKAG1m PRKAA2,PRKAA1,PRKAG2,PRKAG3,STK11,ULK1 ACACA,TP53 and MTOR.2)The signaling pathways retrieved by KEGG database which metformin was involved in were as follows:AMPK,mTOR,Adipocytokine,Insulin,Regulation of autophagy,Glucagon,Insulin resistance,Circadian rhythm,Non-alcoholic fatty liver disease(NAFLD)and Oxytocin signaling pathways.Among these signaling pathways,the AMPK pathway showed the closest relationship with metformin.3)Through GO database,the biological processes discovered that metformin were involved were as follows:the stagnation of cell cycle,the phosphorylation of proteins,macroautophagy,the signal transduction mediated by p53,the biological synthesis of fatty acid,the positive regulation of gene expression,cell reaction at nutrition level,the positive regulation of autophagy,the carnitine shuttle,and cell reaction activated by PGE.3.2 Western Blot analysisUnder fluid shear stress,after metformin was added in the high glucose-damaged osteocytes,the expression of p-AMPK,p-ERK,and p-JNK varied with time,while p-p38 and pp65 were not.4.The effect of metformin on high glucose-damaged osteocytes under fluid shear stress in vivo1)The fasting blood glucose levels of NG rats were 5.2±0.5 mmol/L,whereas the fasting blood glucose levels of DB rats were 18,1±0.8 mmol/L.The hyperglycemic state was maintained during the entire experimental course in the diabetes group.Metformin treatment significantly reversed the diabetic state(blood glucose,5.3 ±0.6mmol/L;p<0.01).The normal blood glucose level of metformin group was also maintained during the entire experimental course.2)The distance of tooth movement was measured on day 14 after the application of the tooth movement appliance in each experimental group.The results showed a greater amount of tooth movement in diabetes rats at 14d of mechanical loading compared with normal rats and metformin-administrated rats at the same time-point.3)ALP staining showed weaker ALP expression in the DB group compared with the control group,and the MA group showed stronger ALP expression than the DB group.Statistical analysis revealed significant differences in optical densities of ALP between the MA and DB groups.4)Compared with control group,the number of TRAP-positive osteoclasts was significantly increased in rats of DB group.However,the number of TRAP-positive osteoclasts was decreased in the rats of MA group.Osteoclasts in the control group were flat and slender with 1-2 nuclei,whereas osteoclasts in the DB group were round or polygonal with many nuclei.5)The ratio of SOST positive bone cells in diabetes mellitus group increased,and the ratio of SOST positive bone cells in MA group was decreased.The ratio of DMP-1 positive bone cells in the DB group decreased significantly,but increased in the MA group.Conclusions1.Rocking system is a convenient,effective and feasible method for fluid shear stress loading;Low concentration of high glucose MEM promoted cell proliferation,but high concentration of high glucose MEM inhibited cell proliferation;Under the fluid shear stress,and with the increase of high glucose concentration,the mineralization ability of MLO-Y4 decreased the expression of OPN,OCN and DMP-1 decreased and the expression of SOST increased in a dose-dependent manner.2.Metformin can promote proliferation and reduce the apoptosis rate of osteocytes under fluid shear stress and different glucose concentrations.Therefore,under fluid shear stress,Metformin could improve the function of MLO-Y4 cells damaged by high glucose to a certain extent under high glucose condition,and could completely reverse the function of MLO-Y4 cells damaged by high glucose and promote its osteogenic ability significantly under normal conditions.3.Under fluid shear stress,AMPK,ERK,JNK signaling pathways were involved in the regulation of metformin on high glucose-damaged MLO-Y4,while p38,NF-kB signaling pathways were not.4.Diabetes increased the number and activity of osteoclasts under orthodontic force resulting in a greater distance of tooth movement.Metformin administration decreased the number and activity of osteoclasts,improved the activity of osteoblasts,rescued the function of osteocytes in diabetes rats,and consequently leaded to normalized tooth movement. |
PDF Full Text Request