| Background and ObjectiveType 2 diabetes mellitus(T2DM)can aggravate the progression of periodontitis,resulting in alveolar bone resorption,tooth loss,and jaw atrophy.T2DM may also increase the risk of infection and failure after bone graft and affect the outcome of bone augmentation.The poor osseointegration of dental implants caused by T2DM may decrease the long-term survival rate of implants,which brings great challenge to dental implant treatment.Bone marrow mesenchymal stem cells(BMMSCs)are key factors in maintaining bone homeostasis.Bone augmentation technology based on BMMSCs combined with bone substitute materials is also expected to be an ideal method for bone defect reconstruction.However,the hyperglycemic microenvironment of T2DM may cause mitochondrial dysfunction,resulting in excessive production of reactive oxygen species(ROS),which inhibits the proliferation and osteogenic differentiation of BMMSCs.Glutathione peroxidase 7(GPX7)is an important oxidoreductase.The deficiency of GPX7 leads to the increase of ROS level in the cells and inhibit the osteogenic differentiation of BMMSCs.Nuclear factor erythroid 2 related factor 2(NRF2)is an important antioxidant transcription factor closely related to GPX7.The deficiency of NRF2 will lead to increased bone resorption.In the context of T2DM,persistent hyperglycemia significantly inhibits the expression of GPX7 and NRF2 in cells,resulting in excessive accumulation of ROS,and thus inhibit the osteogenic differentiation of BMMSCs.In addition,NRF2 can bind to the antioxidant response elements in the GPX7 gene promoter,promote GPX7 expression and improve the activity of stem cells.Therefore,NRF2-GPX7 pathway may be an important target for T2DM inducing oxidative stress and inhibiting osteogenesis.At present,there are few studies on NRF2-GPX7 pathway,and whether it is involved in the regulation of osteogenic differentiation of BMMSCs has not been reported.Therefore,the investigation of the mechanism of NRF2-GPX7 pathway is expected to provide a new target for the treatment of abnormal bone metabolism in T2DM environment.Metformin(MF)is a first-line drug in the treatment of T2DM.In addition to its hypoglycemic effect,MF is also able to promote the osteogenic differentiation in a high-glycemic environment,and improve the regeneration of bone defect and the formation of new bone around the implant in diabetic rats.However,its specific mechanism has not been elucidated.Studies have shown that MF can inhibit the production of endogenous ROS in cells,regulate the redox metabolism of mitochondria and delay stem cell senescence.At present,it is not clear whether MF can affect osteogenic differentiation of BMMSCs by regulating oxidative stress in T2DM environment,and there are few studies on whether NRF2-GPX7 signaling pathway is involved.The purpose of this study was to investigate whether MF inhibited oxidative stress and promoted osteogenic differentiation of BMMSCs in T2DM environment through NRF2-GPX7 pathway.Materials and Methods1 Effects of MF on osteogenic differentiation of Tr-BMMSCsFirstly,Sprague Dawley(SD)male rats of 200-250 g(6 weeks)were selected and fed with high-fat and high-sugar diet combined with peritoneal injection of streptozotocin(streptozotocin,STZ)to construct T2DM rat model.Tr-BMMSCs were isolated and cultured by whole bone marrow adherent method.The clonal formation was detected by crystal violet staining.The expressions of CD29,CD90,CD34 and CD45 were detected by flow cytometry.The differentiation ability of bone,lipid and chondrogenic was detected by alizarin red,oil red O and alcian blue staining.Secondly,cell counting kit-8(CCK-8),alkaline phosphatase(ALP)activity and western blot were performed to detect the effects of different concentrations of MF(0,50,100,200 and 500 μM)on cell activity,ALP activity and collagen-I(Col-I),runt related transcription factor 2(Runx2)and osteopontin(OPN)expression of Tr-BMMSCs.The optimal concentration of MF was 200 μM.Thirdly,real time quantitative polymerase chain reaction(RT-qPCR),ALP staining and alizarin red staining were further investigated to evaluate the effect of 200 μM MF on the expression of Col-I,Rux2 and OPN mRNA,ALP expression and calcium nodule formation of Tr-BMMSCs.2 The role of oxidative stress in regulating osteogenic differentiation of TrBMMSCs by MFFirstly,the effect of MF on oxidative stress of Tr-BMMSCs was detected,and the cells were divided into 5 group:1)Normal rats BMMSCs(Nr)group,2)Tr-BMMSCs(Tr)group,3)Tr+10 μM mitochondrial oxidative stress inhibitor(Mito-tempo)group,4)Tr+200 μM MF group,5)Tr+200 μM MF+10 μM oxidative stress activator(hydrogen peroxide,H2O2)group.The fluorescence expression of ROS in cells was detected after 24 hours of cell culture in stem cell medium or stem cell high-glucose medium.After 48 h,the contents of malondialdehyde(MDA)and glutathione(GSH)as well as the activities of superoxide dismutase(SOD)and catalase(CAT)were determined by extracting mitochondrial proteins.Secondly,the osteogenic differentiation ability of Tr-BMMSCs was detected.After the cells were cultured in osteogenic induction medium or osteogenic high-glucose induction medium for 10 days according to the above groups,ALP expression and the expression of Col-I,Runx2 and OPN protein were detected by ALP staining and western blot assay,respectively.After 14 days,the expression of osteocalcin(OCN)was detected by immunofluorescence assay.After 21 days,the formation of calcium nodules was detected by alizarin red staining.Thirdly,the role of oxidative stress in the regulating effect of MF on heterotopic osteogenesis of Tr-BMMSCs was detected.Thirty nude mice were randomly divided into 5 groups:1)Blank control group,2)Nr group,3)Tr group,4)Tr+MF group,and 5)Tr+MF+H2O2 group.After 21 days of osteogenic induction culture of Tr-BMMSCs,the cell-collagen scaffold complex was constructed and implanted under the skin of the back of nude mice.MF solution was separately injected locally or MF solution and H2O2 solution were alternately injected locally for 5 weeks according to the grouping.Tissue samples were obtained 6 weeks after surgery,the heterotopic bone formation in each group were evaluated by hematoxylin eosin(HE)staining and Masson staining.3 The regulatory effect of MF on the expression of NRF2 pathway related molecules and GPX7 in Tr-BMMSCsThe cells were divided into 5 groups:1)Nr group,2)Tr group,3)Tr+10 μM NRF2 pathway activator(TBHQ)group,4)Tr+200 μM MF group,5)Tr+MF+10μM NRF2 pathway inhibitor(ML385)group.After the cells were cultured in osteogenic induction medium or osteogenic high-glucose induction medium for 10 days according to the above groups,the expression of NRF2 in the nucleus and cytoplasm,as well as NADPH quinone oxidoreductasel(NQO1),heme oxygenase-1(HO-1)and GPX7 in cytoplasm were detected by western blot assay.4 Role of NRF2-GPX7 pathway in the regulating effect of MF on the oxidative stress and osteogenic differentiation of Tr-BMMSCsFirstly,GPX7 overexpressed plasmid(ovGPX7)and empty plasmid(ncGPX7)were constructed,and Tr-BMMSCs were transfected by transposon method.Secondly,the role of NRF2-GPX7 pathway in MF regulation of Tr-BMMSCs oxidative stress was examined.The cells were divided into 6 groups:1)Nr group,2)Tr group,3)Tr+200μM MF group,4)Tr+200 μM MF+10 μM ML385 group,5)Tr+200 μM MF+10μM ML385+ncGPX7 group,6)Tr+200 μM MF+10 μM ML385+ ovGPX7 group.The fluorescence expression of ROS in cells was detected after 24 hours of cell culture in stem cell medium or stem cell high-glucose medium.After 48 hours,MDA and GSH content and SOD and CAT activity in mitochondria were detected.Thirdly,the role of NRF2-GPX7 pathway in MF regulation of the osteogenic differentiation of TrBMMSCs was detected.After 10 days of cell culture in osteogenic induction medium or osteogenic high-sugar induction medium,the expression of ALP was detected by ALP staining.After 14 days,the expression of OCN protein was detected by immunofluorescence assay.After 21 days,the formation of calcium nodules was detected by alizarin red staining.5 Role of NRF2-GPX7 pathway in MF regulation of Tr-BMMSCs in the repair of mandibular bone defects in T2DM ratsTwenty-four T2DM rats were randomly divided into 6 groups:1)Blank control group,2)MF injection alone group,3)Tr group,4)Tr+MF group,5)Tr+MF+ML385 group,6)Tr+MF+ML385+ovGPX7 group.Firstly,Tr-BMMSCs and GPX7 overexpressed Tr-BMMSCs were cultured through osteogenic induction for 21 days,and the cell-collagen scaffold complex was constructed.Secondly,mandibular bone defect model(5 mm×4 mm×1 mm)of T2DM rats were constructed,the cell-collagen scaffold complexes were implanted into the bone defects,and MF solution was injected separately or MF solution and ML385 solution were injected alternately according to the group for 5 weeks.Thirdly,samples were collected 6 weeks after surgery.The new bone formation area of each group was evaluated by micro computed tomography(Micro CT),HE staining and Masson staining,and the expression of OCN protein was observed by immunohistochemistry.Results1 MF promoted osteogenic differentiation of Tr-BMMSCsThe T2DM rat model was successfully constructed in this study,and Tr-BMMSCs were obtained by whole bone marrow adherent method.The cells were long spindleshaped and showed clonal colony growth.Mesenchymal stem cell derived surface markers CD29 and CD90 were positively expressed,while hematopoietic stem cell derived surface markers CD34 and CD45 were negatively expressed.The multidifferentiation potential ability of osteogenic,lipogenic and chondrogenic multidirectional differentiation of cells were also exhibited.CCK-8 assay,ALP activity assay and western blot assay showed that the cell activity and osteogenic differentiation ability of Tr-BMMSCs gradually increased with the increase of MF concentration,reaching a peak value of 200 μM,and significantly decreased when the MF concentration was further increased to 500 μM.Thirdly,ALP staining and alizarin red staining further showed that 200 μM MF significantly promoted the ALP expression and calcium nodule formation of Tr-BMMSCs.Therefore,200 μM MF was used for subsequent experiments.RT-qPCR detection,ALP staining and alizarin red staining further showed that 200 μM MF significantly promoted the expression of Col-I,Runx2,OPN mRNA,ALP expression and calcium nodule formation of Tr-BMMSCs.Therefore,in this study,MF with a concentration of 200 μM was used for subsequent experiments.2 MF inhibited oxidative stress and promoted osteogenic differentiation of TrBMMSCsROS fluorescence detection and mitochondrial MDA,GSH content and SOD,CAT activity detection showed that MF significantly inhibited the oxidative stress level of Tr-BMMSCs,decreased the ROS expression and MDA content,and promoted the GSH content and SOD,CAT activity of Tr-BMMSCs.The effect was similar to Mitotempo,a mitochondrial oxidative stress inhibitor.After addition of H2O2,the ROS expression and MDA content of Tr-BMMSCs were significantly increased,while the GSH content,SOD and CAT activities were significantly decreased,indicating that the inhibitory effect of MF on oxidative stress was reversed by H2O2.The results of ALP staining,alizarin red staining,western blot detection and immunofluorescence detection showed that MF significantly promoted the ALP expression,calcium nodule formation and osteoblast protein expression of Tr-BMMSCs,while H2O2 significantly inhibited the promoting effect of MF on osteogenic differentiation of Tr-BMMSCs.In the heterotopic osteogenesis experiment of nude mice,the results of HE and Masson staining also showed that MF significantly promoted the new bone formation of TrBMMSCs under the skin of nude mice,while H2O2 completely inhibited the promotion effect of MF.3 MF promoted the expression of NRF2 pathway related molecules and GPX7 in Tr-BMMSCsWestern blot experiments showed that MF significantly promoted the expression of NRF2 in the nucleus,and the expressions of NRF2,NQO1,HO-1 and GPX7 in the cytoplasm,similar to that of NRF2 pathway activator TBHQ.After adding NRF2 inhibitor ML385,the promoting effect of MF was significantly inhibited,and the expression of NRF2 in the nucleus and NRF2,NQO1,HO-1 and GPX7 in the cytoplasm were significantly decreased.4 MF inhibited oxidative stress and promoted osteogenic differentiation of TrBMMSCs through NRF2-GPX7 pathwayROS fluorescence expression detection and mitochondrial MDA and GSH content detection as well as SOD and CAT activity detection showed that ML385 significantly inhibited the antioxidant effect of MF on Tr-BMMSCs,while GPX7 overexpression effectively reversed the inhibitory effect of ML385.ALP staining,alizarin red staining and immunofluorescence detection showed that ML385 significantly inhibited the promoting effect of MF on ALP expression,calcium nodule formation and OCN expression of Tr-BMMSCs,while GPX7 overexpression effectively reversed the inhibitory effect of ML385 on osteogenic differentiation.5 MF promoted Tr-BMMSCs to repair mandibular bone defects in T2DM rats through NRF2-GPX7 pathwayThe results of Micro CT,HE staining,Masson staining and immunohistochemical staining showed that the combined application of MF and Tr-BMMSCs could effectively repair mandibular defects in T2DM rats,and the effect was better than that of MF or Tr-BMMSCs alone.NRF2 pathway inhibitor ML385 completely inhibited the combined effect of MF and Tr-BMMSCs.When MF was used in combination with GPX7 overexpressed Tr-BMMSCs,both new bone mass and OCN expression were significantly increased,indicating that GPX7 overexpression effectively reversed the osteogenesis inhibition of ML385 on Tr-BMMSCs.It is further suggested that NRF2GPX7 pathway plays an important role in the process of MF promoting Tr-BMMSCs to repair mandibular defects in T2DM rats.Conclusion1.The oxidative stress level of Tr-BMMSCs was significantly higher than that of normal rat BMMSCs,and the osteogenic differentiation ability was significantly reduced.2.MF inhibited ROS expression of Tr-BMMSCs and increased their antioxidant levels,thus promoting osteogenic differentiation of Tr-BMMSCs in vitro and heterotopic osteogenesis under the skin of nude mice.3.MF activated NRF2-GPx7 pathway and promoted the expression of NRF2 and GPX7 in Tr-BMMSCs.4.MF inhibited oxidative stress level and promoted osteogenic differentiation of TrBMMSCs and the repair of mandible bone defects of T2DM rats through NRF2GPX7 pathway. |
PDF Full Text Request