The Study About NF-?B Affecting On The Expression Of Jmjd3 And Odontogenic Differentiation In Dental Pulp Stem Cells

Objective: Dental pulp stem cells(DPSCs)are undifferentiated or poorly differentiated adult stem cells that can be found in the dental pulp tissue.They have a particular proliferation capacity and differentiation potential,and,when exposed to the appropriate conditions,can differentiate into numerous cell types.These cell types include osteoblasts,odontoblasts,adipocytes,and chondrocytes.Chronic damages such as deep caries and abrasion attract DPSCs to migrate to the injured site.Here,they can differentiate into odontoblast-like cells and produce reparative dentin to protect the pulp.Epigenetic modifications regulated by histone methylation play an important role in regulating osteogenic differentiation.Jumonji domain-containing3(Jmjd3),is a highly specific histone demethylase.Jmjd3 functions primarily to demethylate lysine27 of histone H3(H3K27)to activate gene transcription.Many studies have shown that Jmjd3 regulates the osteogenic differentiation of various cells,but whether Jmjd3 participates in the odontogenic differentiation of DPSCs is poorly understood.Nuclear factor(NF)-?B is a ubiquitous transcription factor that is widely involved in both physiological and pathological processes,including immunity,inflammation,and the differentiation of cells and tumors.Previous studies have identified that NF-?B can induce the expression of Jmjd3 in inflammation,tumors,and trauma,but whether it can regulate the expression of Jmjd3 during differentiation has not been clearly reported.In this study,we utilized DPSCs to detect changes in the expression of Jmjd3 during odontogenic differentiation.Here,we explored the role of Jmjd3 and NF-?B signaling in the process of odontogenic differentiation in DPSCs.Our study may lay the experimental foundation for vital pulp therapy.Methods:1.Experimental mothods1.1 Primary DPSCs' isolation and culture: After obtaining the Ethics Committee's approval and the informed consent of the patients,we collectd healthy and intactpermanent teeth which were extracted due to impaction or orthodontic reduction.And DPSCs were isolated from human dental pulp tissue by enzymatic digestion.1.2 Flow Cytometry(FCM)was used to detect the expressions of cell surface markers.1.3 DPSCs were induced to differentiate into osteoblasts in vitro for 21 days,and the mineralization level of the cells was measured by Alizarin red staining.1.4 DPSCs were induced to differentiate into neurons in vitro for 14 days,and immunofluorescence staining was used to detect the expression of ?-tubulin ?protein.1.5 DPSCs were induced to differentiate into odontoblast-like cells in vitro for 7 days,and ALP staining was used to detect the expression of ALP.1.6 DPSCs were induced to differentiate into odontoblast-like cells for different days(0,3,5,7,14 days).Quantitative polymerase chain reaction(qPCR)was used to detect the mRNA expressions of Jmjd3,DSPP,and DMP1,and Western Blot was used to detect the protein expression of Jmjd3.1.7 Differing concentrations(0,1,10 ?mol/L)of GSK-J4 were added to the odontogenic differentiation medium that was used to treat cells for 7 or 14 days.qPCR was used to detect the mRNA expressions of DSPP and DMP1.The cells were treated with GSK-J4 for 21 d and alizarin red staining was used to detect the mineralization level of the cells.1.8 DPSCs were induced to differentiate into odontoblast-like cells for 0,5,15,30,60,120,240 minutes.The protein expressions of NF-?B phospho-p65 and total NF-?B p65 was detected by Western Blot.DPSCs were cultured in odontogenic differentiation medium containing 10?mol/L BAY 11-7082 for 30 minutes.The protein expressions of NF-?B phospho-p65 and total NF-?B p65 was detected by Western Blot.1.9 DPSCs were cultured in odontogenic differentiation medium containing10?mol/L BAY 11-7082 for 7 days.qPCR was used to detect the mRNA expressions of Jmjd3,DSPP,and DMP1.1.10 DPSCs were transfected with non-specific(NC)or si RNA targeting NF-?B p65(si NF-?B p65)and cultured in odontogenic differention medium for 7 days.qPCRwas used to detect the mRNA expressions of Jmjd3,DMP1,and ALP.2.Statistical analysis Each experiment was performed at least 3 times.The experimental data was analysed by one-way analysis of variance(ANOVA)with SPSS 18.0 software package.Dunnett t test was used for comparison between multiple groups.p <0.05 was considered statistically significant.Results:1.The cells cultured in this study were spindle-shaped,like fibroblasts.These cells positively expressed the mesenchymal stem cell surface markers CD29,CD44,CD105,and CD146,but did not express hematopoietic stem cell surface markers CD34 and CD45.Under the appropriate conditions,DPSCs could differentiate into osteoblasts and neurons.2.DPSCs were cultured in odontogenic differentiation medium for 0,3,5,7,or 14 days.As time progressed,the mRNA expressions of Jmjd3,DSPP,and DMP1 significantly increased.Notably,the protein expression of Jmjd3 increased(p?0.05)and staining for ALP was positive.3.Differing concentrations(0,1,10?mol/L)of GSK-J4 were added to the odontogenic differentiation medium that was used to treat cells for 7 or 14 days.The mRNA expression of DSPP decreased when cells were treated with 1?mol/L GSK-J4 for 14 days(p ? 0.05).The mRNA expressions of DSPP and DMP1 significantly decreased when cells were treated with 10?mol/L GSK-J4 for 7 or 14 days(p?0.05).Moreover,Alizarin red staining also became significantly lighter compared to the control group after the cells were treated with 10?mol/L GSK-J4 for 21 days.4.DPSCs were cultured in odontogenic differentiation medium for 0,5,15,30,60,120,or 240 minutes.The protein expression of NF-?B phospho-p65 increased at 5minutes and reached maximal expression at 30 minutes.The protein expression of NF-?B phospho-p65 decreased when cells were treated with 10?mol/L GSK-J4 for 30 minutes.However,the protein expression of total NF-?B p65 was not changed.The expressions of Jmjd3,DSPP,and DMP1 increased in the cells which were treated with10?mol/L BAY 11-7082(p ?0.05).DPSCs were transfected with small interfering RNA(siRNA)to knock down p65 and the expressions of Jmjd3,DMP1,and ALPwere upregulated(p?0.05).Conclusions: Jmjd3 may play a positive role in the odontogenic differentiation of DPSCs,which is related to its demethylase activity.Furthermore,suppression of NF-?B signaling pathway can upregulate the expression of Jmjd3 and promote DPSCs odontogenic differentiation.