| PurposeOrthodontic tooth movement(OTM)relies on mechanical force-induced alveolar bone remodeling.Alveolar bone marrow mesenchymal stem cells(ABMMSCs)are able to proliferate,migrate and differentiate into osteoblast under mechanical force,which are considered an important effector of OTM.All of the above cellular behaviors are often accompanied by active reprogramming of cellular energy metabolism to meet increased energy demands.It has been shown that the glycolysis was up-regulated during osteogenic differentiation.However,whether lactate as the end-product of glycolysis could respond to mechanical stimulus during OTM and whether lactate has an impact on the alveolar bone remodeling during OTM remain to be further elucidated.Therefore,the aim of this research is to investigate the role of lactate on the bone remodeling during OTM.Methods1.Rat ABMMSCs were harvested and cultured in vitro.The proliferation potential was proved by clone formation test.The osteogenic and adipogenic differentiation potential of ABMMSCs was identified by Alizarin Red staining and Oil O Red staining respectively.Flow cytometry was used to detect the surface antigen of ABMMSCs.Cells were subjected to cyclic mechanical stretch(10%elongation,0.5 Hz;sinusoidal waveforms)for 0,6,12,and 24 h.The expression of lactate dehydrogenase A(LDHA)at transcriptional and translational level were quantified by qRT-PCR and Western blot respectively.Intracellular and Extracellular lactate levels were detected using the Lactate Content Assay Kit.2.The rat OTM model was established for 0,3,7,and 14 d.The expression of LDHA in the alveolar bone was detected by Immunohistochemistry.And the lactate content in the alveolar bone around the moved teeth was also detected using the Lactate Content Assay Kit.3.GNE-140,a specific LDHA inhibitor,was applied to inhibit the lactate production during cyclic stretch in ABMMSCs to detect the effect of lactate on the cellular characteristics of ABMMSCs under mechanical stress.CCK-8 and cell cycle test were applied to detect cellular proliferation.The apoptosis was analyzed by Annexin V-FITC/PI staining.The migration was evaluated by scratch test.The osteogenic differentiation was detected by alkaline phosphatase(ALP)activity test and ALP staining as well as the expression of ALP and RUNX2.4.The level of histone lactylation in the alveolar bone during OTM was evaluated by immunohistochemistry staining.Immunofluorescence and Western blot were applied to detect the level of histone lactylation in ABMMSCs under mechanical stress or under GNE-140 treatment.The enrichment level of histone lactylation at the promotor region of bone remodeling-related genes was detected by chromatin immunoprecipitation sequence.The alteration of mRNA expression of these genes was further evaluated by qRT-PCR.Results1.Rat ABMMSCs were successfully isolated and culture in vitro,they were in spindle or triangle shape.The cellular clone was observed,and the osteogenic and adipogenic differentiation potential were confirmed by enhanced mineralized nodules and formation of lipid droplets respectively.The ABMMSCs expressed MSC-specific positive markers(CD90 and CD44)and failed to express MSC negative markers(CD31 and CD45).Besides,the intracellular and extracellular lactate level increased under cyclic mechanical stretch.2.The expression level of LDHA in the alveolar bone was elevated during OTM.Besides,the lactate level in the alveolar bone surrounding the moved teeth was also increased during OTM.3.GNE-140 significantly down-regulated the expression of ALP and RUNX2.The results of ALP activity and ALP staining also proved attenuated osteogenic differentiation in GNE-140 group.Furthermore,the proliferation,cell cycle progression,and migration were suppressed,while the apoptosis were increased by GNE-140 treatment.4.The level of histone lactylation in the alveolar bone was increased during OTM.And level of histone lactylation was also elevated in ABMMSCs under mechanical stress,while it was decreased by GNE-140.The enrichment level of lactylated histone at the promotor region of bone remodeling-related genes was decreased by GNE-140.Meanwhile,the expression of above genes at the mRNA level was suppressed by GNE-140.ConclusionTaken together,our results suggest that force-induced lactate is involved in controlling bone remodeling-related cellular activities in ABMMSCs and plays a vital role in the alveolar bone remodeling during OTM.Our findings indicate that lactate might be a critical modulator for alveolar bone remodeling during OTM,providing a novel therapeutic target for controlling tooth movement more effectively and improving the stability of orthodontic results. |
