The Role And Mechanism Of Apelin-13 Mediated Dentin Formation In High Glucose Environment

Background and ObjectiveEndodontic medicine studies the bidirectional relationship between endodontic infection and systemic diseases.Diabetes is one of the most common metabolic disorders,which can cause or aggravate a variety of oral diseases,including dental pulp diseases,which is a hot and difficult point in the field of endodontic medicine.The ideal treatment of dental pulp disease is based on the principle of regenerative medicine to produce new tissue through dental pulp regeneration to replace necrotic pulp tissue.Apelin-13 is an endogenous bioactive peptide involved in many biological processes,which can regulate bone tissue growth,promote bone regeneration,improve insulin sensitivity and reduce blood glucose.We speculated that Apelin13 might be involved in dentin formation and regeneration in high glucose environment.The purpose of this study was to study the expression of Apelin-13 in tooth germ development and pulp-dentin complex,and the role and mechanism of Apelin-13 on the differentiation of human dental pulp stem cells(hDPSCs)into odontoblast-like cells in high glucose environment,so as to provide possible theoretical guidance and experimental basis for the repair of dental pulp injury in patients with diabetes mellitus.Methods1.Immunofluorescence technique was used to observe the expression of Apelin in different stages of mouse tooth germ development and human pulp-dentin complex.2.Human dental pulp cells were cultured by enzyme digestion-tissue block method.The hDPSCs were obtained by monoclonal method.Stem cell surface markers were detected by flow cytometry,and their multi-directional differentiation ability was observed by alizarin red and oil red O staining.3.The expression of Apelin-13 during the induction of hDPSCs mineralization was detected by quantitative real-time polymerase chain reaction(qRT-PCR).Cell counting kit-8(CCK-8)assay was used to detect the effect of Apelin-13 on hDPSCs activity,and qRT-PCR was used to screen the appropriate concentration of Apelin-13.The effect of Apelin-13 on the differentiation of hDPSCs into odontoblast-like cells was detected by qRT-PCR,Western blot,immunofluorescence technique,alkaline phosphatase(ALP)staining and activity assay,alizarin red staining and cetylpyridinium chloride(CPC)analysis.4.To detect the effect of Apelin-13 on the differentiation of hDPSCs into odontoblast-like cells in high glucose environment:the expression of genes and proteins related to odontoblast differentiation was detected by qRT-PCR and Western blot,and the expression of runt-related transcription factor 2(Runx2)was detected by immunofluorescence.ALP staining and activity assay,alizarin red staining and CPC analysis were carried out at the same time.5.To explore the mechanism of the effect of Apelin-13 on the differentiation of hDPSCs into odontoblast-like cells in high glucose environment:the expression of Apelin and putative receptor protein related to the angiotensin Ⅱ type 1 receptor(APJ)in hDPSCs was detected by double immunofluorescence staining.The effect of Apelin-13 on the expression of proteins related to Wnt/β-catenin pathway was detected by Western blot and immunofluorescence.After adding the pathway inhibitor Dickkopf-related protein 1(DKK1),the expression of odontoblast differentiation-related factors was detected by qRT-PCR,Western blot and immunofluorescence.Results1.The results of tissue immunofluorescence showed that Apelin expressed spatiotemporal ly during the development of mouse teeth germ and specifically expressed in human pulp-dentin complex.2.The dental pulp stem cells cultured by enzyme digestion-tissue block method were long fusiform and had the ability of colony formation,the characteristics of mesenchymal stem cells and the potential of multi-directional differentiation.3.The results of qRT-PCR showed that the messenger ribonucleic acid(mRNA)expression level of Apelin-13 increased in a time-dependent manner during the induction of hDPSCs mineralization.CCK-8 results showed that the activity of hDPSCs did not change significantly with the increase of Apelin-13 concentration(0-10μM).The results of qRT-PCR showed that 10 nM Apelin-13 could significantly promote the gene expression level of Runx2,and it was selected as the appropriate effective concentration.The addition of Apelin-13 in vitro significantly promoted the differentiation of hDPSCs into odontoblast-like cells.4.The results of qRT-PCR,Western blot and cellular immunofluorescence showed that 10 nM Apelin-13 significantly promoted the expression of odontoblast differentiation-related factors in high glucose environment,which was consistent with ALP staining and activity assay,alizarin red staining and CPC analysis.5.Double immunofluorescence staining results showed that Apelin and its receptor APJ were co-expressed in hDPSCs under high glucose environment.The results of Western blot and cellular immunofluorescence showed that the expression of β-catenin and Wnt10a related to Wnt/β-catenin signaling pathway increased.After the addition of inhibitor DKK1,the activation of Wnt/β-catenin pathway was inhibited and the expression of related factors decreased.Conclusion1.Apelin expressed spatiotemporally during the development of mouse teeth germ and specifically expressed in human pulp-dentin complex.2.Apelin-13 promoted the differentiation of hDPSCs into odontoblast-like cells under normal and high glucose conditions.3.Apelin-13 promoted the differentiation of hDPSCs into odontoblast-like cells partly through Wnt/β-catenin signaling pathway.