The Effects And Mechanisms Of Chlorogenic Acid On Osteogenic Differentiation Of Dental Pulp Stem Cells In Vitro

Background:Periodontal disease(PD)is one of the main stomatology disease that lead to periodontal bone resorption and tooth loss in adults,and the incidence of which is as high as 80%?90%.The ratio of population with severe periodontal disease is 5%?20%.The therapy of periodontal disease is limited to remove the infection completely,but the repair of alveolar bone is very limited.How to repair alveolar bone effectively and preserve teeth as much as possible has always been a challenge in clinical treatment.The development of tissue engineering provides a new approach for the repair of alveolar bone defect.Seed cells are one of the three main components of tissue engineering.Dental pulp stem cells(DPSCs)are ideal seed cells.They have the advantages of wide sources,convenient collection,rapid proliferation,strong ability to clone in vitro.They also have the ability of osteogenic differentiation with low immunogenicity.They were no obvious tumorigenicity at present.Chlorogenic acid(CGA)is one of the main active components of traditional Chinese medicine Lonicerae japonica and Eucommia ulmoides.It is widely used in medical industry,food industry,cosmetics industry and health care industry.It has the antibacterial,cholagogic,hemostatic functions and other pharmacological functions.It also can induce osteogenic differentiation of bone marrow mesenchymal stem cells.This study was designed to clarify the effects and the underlying molecular mechanisms of CGA on osteogenic differentiation of DPSCs.This study expected to find a new method to serve as a foundation for further studies on how to repair defective alveolar bone for the patients with PD.Methods:DPSCs were isolated,expanded,and identified at first.CGA at 0.1?g/m L,1?g/m L,10?g/m L,100?g/m L and 1 mg/m L was used to treat DPSCs respectively.The effect of CGA on the proliferation of DPSCs was detected by MTT assay.DPSCs were randomly divided into six groups.For OSI group,DPSCs were treated with osteogenic medium.Four CGA groups,DPSCs were treated with osteogenic medium plus CGA at either 0.1,1,10 or 100?g/m L.For Control group,DPSCs were cultured in growth medium containing a low-sugar DMEM with 5%fetal bovine serum.The effect of CGA on osteogenic differentiation of DPSCs was detected by alizarin red staining after 21 days of osteogenic induction.RNA sequencing was used to detect the differentially expressed genes following osteogenic treatment.DPSCs were randomly divided into three groups:CGA group receiving osteogenic medium plus CGA(10?g/m L),OSI group receiving osteogenic medium,and negative Control group,3 replicates each.After osteogenic induction for 21 days,all cells were detected for RNA sequencing.The differentially expressed genes were screen out based on the result of RNA sequencing.Gene Ontology(GO)enrichment and the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways enrichment were further carried out using the default settings for the differentially expressed genes.The involved functional categories and signaling pathways of those genes were analyzed through bioinformatics analysis.Real-time fluorescent quantitative PCR(q RT-PCR)was used to verify the expression of the osteogenic-related genes including frizzled-related protein(FRZB)?pyruvate dehydrogenase kinase 4(PDK4)?asporin(ASPN)and cytokine like 1(CYTL1)in DPSCs.The influence of CGA on Wnt signaling was verified by western blot analysis.The protein level of FRZB was detected.The protein levels of active?-catenin and total?-catenin,which concerned with canonical Wnt/?-catenin signaling were detected.The protein levels of c AMP-reactive element-binding protein(CREB),phosphorylated CREB(p CREB),calmodulin-dependent kinase II(Ca MKII)and phosphorylated Ca MKII(p Ca MKII),which concerned with non-canonical Wnt/Ca²⁺signaling were detected too.Results:MTT assay showed that CGA had a dose-dependent effect on the proliferation of DPSCs at low and medium concentrations.However,when the concentration of CGA was 1 mg/m L and the treated time was more than 48 hours,CGA inhibited the proliferation of DPSCs.Alizarin red staining showed that CGA can promote DPSCs osteogenic differentiation,and with the increase of the concentration of CGA,its ability was enhanced.However,when the concentration of CGA increased to 100?g/m L,the effect was inhibited.The results of RNA sequencing,bioinformatics analysis and q RT-PCR showed that CGA treatment enhanced the expression of the osteogenesis genes for frizzled-related protein(FRZB)and pyruvate dehydrogenase kinase 4(PDK4)and inhibit the expression of the osteoclastogenesis genes such as those for asporin(ASPN)and cytokine like 1(CYTL1).Western blot analysis showed that besides FRZB,CGA treatment also caused reduction of both active and total?-catenin,while increased the total amount calcium-calmodulin-dependent kinase II(Cam KII)and the phosphorylated Cam KII(p Cam KII)and the phosphorylated c AMP-response element binding protein(p CREB).However,the total amount of the c AMP-reactive element-binding protein(CREB)did not change.Conclusions:CGA at low and medium concentrations can promote the proliferation and osteogenic differentiation of DPSCs.CGA can promote the expression of osteogenic genes FRZB and PDK4 and inhibit the expression of osteoclastic genes ASPN and CYTL1.Likely,the increased osteogenesis was associated with reduced canonical Wnt/?-catenin signaling but increased non-canonical Wnt/Ca²⁺signaling.The results suggested that CGA can promote the osteogenic differentiation of DPSCs by regulating osteogenesis-related genes and regulating Wnt signaling.