Effects Of Asperosaponin ? On The Proliferation And Osteogenic Differentiation Of Human Periodontal Stem Cells And Related Mechanism

Generally speaking,the duration of orthodontic treatment is 24 to 48 months.Some complicated cases may require an even longer time to achieve the desired results.Prolonged treatment time may increase the risk of side effects such as dental decalcification,root resorption,gingivitis and periodontitis.Therefore,how to accelerate tooth movement is of great concern for orthodontists.Periodontal tissue remodeling is the biological basis of orthodontic tooth movement.Periodontal tissue including periodontal ligament,alveolar bone and cementum.Orthodontic tooth movement is achieved through bone resorption mediated by osteoclasts on the pressure side and new bone formation mediated by osteoblasts on the tension side.In the process of tooth movement,the force applied onto the orthodontic tooth is transmitted by the periodontal ligament to the alveolar bone,causing periodontal tissue remodeling.Periodontal ligament cells play an important role in this process,transforming mechanical force into biological signal,induces the secretion,synthesis and activation of effector molecules,causing cells proliferation,differentiation and migration and then promote periodontal tissue remodeling.Periodontal ligament cells contain periodontal ligament fibroblasts,osteoblasts,undifferentiated mesenchymal stem cells and cementoblasts.The undifferentiated mesenchymal stem cells are also known as human periodontal ligament stem cells(hPDLSCs).hPDLSCs is a kind of cell with multi-directional differentiation potential and self-renewal,which can form alveolar bone,cementum and periodontal membrane like tissue under specific conditions.hPDLSCs can differentiate into osteoblasts under the mechanical force,which leads to alveolar bone remodeling and tooth movement.Therefore promote the osteogenic differentiation of hPDLSCs,so as to provide basic research for accelerating orthodontic tooth movement and shortening the orthodontic treatment courses become the focus of orthodontists.Asperosaponin VI(ASA VI)is the main active component of dipsacus asper wall.ASA ? has the effects of increasing bone mineral density and anti-osteoporosis in vivo.ASA ? can promote the osteogenic differentiation of MC3T3-E1,rat osteoblasts,rat bone marrow stromal cells and adipose-derived stem cells in vitro.Whether ASA VI can promote the proliferation and osteogenic differentiation of hPDLSCs has not been reported at domestic and foreign.The results of our previous study showed that local injection of ASA VI could increase the number and activity of osteoclasts.Further study the effect and mechanism of ASA VI on the proliferation and osteogenic differentiation of hPDLSCs can clarify the mechanism of ASA VI promoting orthodontic tooth movement and provide a safe and effective way to accelerate orthodontic tooth movement.Hippo signaling pathway is a highly conserved signaling pathway,which is mainly responsible for regulating stem cell self-renewal,organ size and tissue regeneration.It has been reported that Hippo signaling pathway plays an important role in the osteogenic differentiation of stem cells in recent years.In vitro,it was found that TAZ could promote the osteogenic differentiation of stem cells and inhibit lipogenic differentiation by interacting with TGF?,PPAR-y and RUNX2.G protein coupled receptor 30(GPR30)is a membrane estrogen receptor with high affinity for estrogen,which belongs to the G protein coupled receptor(GPCRs)family.GPCRs are a superfamily of membrane-bound receptors and also very important drug target.About 40%-60%of drugs serve GPCRs as a target to play a therapeutic role.Many studies have reported that GPR30 related to bone metabolism.In ovariectomized female rats,the activation of GPR30 significantly promoted bone formation and increased bone density;while GPR30 was inhibited,the bone formation was inhibited and bone density decreased.GPR30 has been detected in osteoblasts,osteocytes and periodontal ligament cells.When GPR30 was inhibited,the osteogenic differentiation of these cells was inhibited.The cell proliferation,differentiation,migration and other biological activities usually need the regulation of multiple signal pathways,single signal pathway could not complete complex cell regulatory functions.Previous studies reported that Hippo/TAZ signaling pathway is a key signal transducer downstream of GPR30 transduction in estrogen induced breast cancer cell proliferation,migration and breast cancer occurrence.Therefore,we propose the following hypothesis:ASA VI may bind to the membrane estrogen receptor(GPR30)on the membrane surface of hPDLSCs,subsequently the intracellular signaling pathway(Hippo/TAZ)is activated,then the related nuclear genes changed until the osteogenic differentiation of hPDLSCs is regulated.ObjectivesThe purpose of this study was to investigate the effect of ASA VI on the proliferation and osteogenic differentiation of human periodontal ligament stem cells,and to explore the potential role of GPR30 and hippo/TAZ signaling pathway in the process of ASA VI promoting the osteogenic differentiation of hPDLSCs.Furthermore,through application of ASA VI on the orthodontic tooth movement model of rats to explore the effects of ASA VI on the osteoclastic activity of the pressure side and osteogenic activity of the tension side,which provided a theoretical basis for the clinical application of ASA VI in orthodontics.Methods(1)hPDLSCs were isolated and cultured and the potential of multi-directional differentiation was verified by osteogenic and lipogenic differentiation.The expression of CD34,CD44,CD45,CD90 and CD105 were detected by flow cytometry.(2)Cell-counting kit-8(CCK-8)kits were used to detect the effect of ASA VI on the proliferation of hPDLSCs.The effects of ASA VI on the expression of ALP and Runx2 genes and proteins were detected by RT-PCR and Western blot.Alkaline phosphatase activity,alkaline phosphatase staining and alizarin red staining were used to verify the effect of ASA VI on the osteogenic differentiation of hPDLSCs.(3)The protein levels of GPR30,p-TAZ,TAZ was detected by Western blot to explore the effect of ASA VI on GPR30 and Hippo signaling pathway.(4)The effect of TAZ on the osteogenic differentiation of hPDLSCs was verified by knocking down TAZ using a small interfering RNA targeting TAZ(siTAZ).(5)GPR30 was knocked down by a small interfering RNA targeting GPR30(siGPR30)to verify the role of GPR30 in the process of ASA VI promoting the osteogenic differentiation of hPDLSCs and its regulation of Hippo signaling pathway.(6)In order to investigate the effect of ASA VI on the orthodontic tooth movement in rats.ASA VI solution was injected into buccal sub-mucoperiosteal of maxillary molars.Tooth movement measurements and alveolar bone volumetric changes were analyzed using a micro-computed tomography(microCT)scan.Molecular changes were evaluated by quantitative real-time RT-PCR and immunohistochemical staining(IHC).Results(1)The primary human periodontal ligament stem cells were successfully isolated,purified and cultured.Flow cytometry verified that it derived from mesenchymal.Osteogenic and lipogenic induction experiments showed that it had the multi-directional differentiation potential.Flow cytometry results showed that the expression of CD44,CD90 and CD 105 was positive,and the expression of CD45 and CD34 were negative.The cells grew well after passage.(2)ASA ? can significantly promoted the proliferation,ALP activity,ALP staining,mineralization nodule formation and the expression of ALP,Runx2 mRNA and protein of hPDLSCs in vitro(P<0.05).And the optimal concentration is 10-5M(P<0.05)(3)PCR and Western blot analysis showed that ASA VI could significantly promoted the mRNA and protein expression of GPR30 and TAZ in a time-dependent manner(P<0.05).(4)The promotion of osteogenic differentiation of hPDLSCs inducted by ASA VI was inhibited after TAZ was knocked down,indicating that TAZ involved in ASA VI induced osteogenic differentiation of hPDLSCs.(5)Depletion of GPR30 offset the facilitation effect of ASA VI on osteogenic differentiation of hPDLSCs and thus demonstrated an essential role of GPR30 in ASA?-induced osteogenic differentiation.Moreover,the deactivation of LATS1/2 and the activation of TAZ induced by ASA ? were significantly reversed by the application of knockdown GPR30.(6)Local injection of ASA VI could accelerate the orthodontic tooth movement in rats.ASA VI induced a significant decrease in bone volume and density and an increase in trabecular spacing and RANKL expression at the compression side.Furthermore,ASA VI stimulated bone formation on the tension side by enhancing OCN and RUNX2 expression,increasing bone volume and density and decreasing in trabecular spacing.ASA VI promoted the expression of GPR30 and TAZ in orthodontic periodontal tissues,indicating that GPR30 and TAZ were involved in the process of ASA VI promoting periodontal tissue remodeling in rats.Conclusions(1)ASA VI can significantly promote the proliferation and osteogenic differentiation of hPDLSCs,and the optimal concentration is 10-5M.(2)GPR30 and Hippo/TAZ signaling pathway were involved in the osteogenic differentiation of hPDLSCs promoted by ASA VI.(3)GPR30 mediated Hippo/TAZ signaling pathway plays an important role in the osteogenic differentiation of hPDLSCs promoted by ASA VI(4)ASA VI may accelerate tooth movement via increasing the activity of osteoclasts,stimulating bone resorption at the compression side.Furthermore,ASA VI has a positive effect on bone formation at the tension side.(5)GPR30 and TAZ were involved in the improvement of periodontal tissue remodeling promoted by ASA VI.