| Pulpitis is the one of the most prevalent disease,which can lead to severe pain.And then,if left untreated,It can cause periapical periodontitis and has an serious influence on the life of people.DPSCs were isolated from dental pulp firstly in 2000.It is reported that DPSCs isolated from teeth with irreversible pulpitis can exist and be isolated and still retain tissue regenerative potential,but the odonto/osteogenic potential of DPSCs from inflammatory pulp is lower than DPSCs from healthy pulp.Baicalin,a major active component,was isolated from traditional Chinese medicine Scutellaria radix.This compound has anti-oxidant,anti-bacterial,anti-tumor,anti-viral,and anti-inflammatory activity,which clearly implies a potential value for medicinal use.Baicalin downregulates Porphyromonas gingivalis lipopolysaccharide-upregulated IL-6 and IL-8 expression in human oral keratinocytes.Baicalin can decrease the expression of TLR4 and the secretion of TNF-α in dental pulp cells induced by lipopolysaccharide.And baicalin can reduce the resorption of the bone and destruction of the collagen in periodontitis.In conclusion,baicalin has a significate application prospect in the treatment of inflammation and restoration of the injuries.But its mechanism of anti-inflammatory and the restoration of pulpitis is still not clear.The aim of this study is to provide theoretic and experimental basis for clinical application by exploring the effect of baicalin on the proliferation and odonto/osteogenic differatiation of i DPSCs.Part I The biology behavior of the inflammatory dental pulp stem cells(i DPSCs)Aim: To analyze the change of biology behavior of i DPSCsMethod: 1)The stem cells were isolated from normal dental pulp and the inflammatory dental pulp and purified by limited dilution technique.2)The expression of mesenchymal stem cell markers of n DPSCs and i DPSCs were measured by immunofluorescence assay.3)The real time RT-PCR was used by measured the expression of inflammatory cytokin including IL-1β and TNF-α of n DPSCs,i DPSCs and LPS-treated n DPSCs.4)ALP activity at day 3,5,7,was measured.5)We evaluated the odonto/osteogenic differentiative potential of three groups by real time RT-PCR and Western blot.Result: n DPSCs and i DPSCs were immunopositive against vimentin and,while negative against CK.The expression of IL-1β and TNF-α of i DPSCs and LPS-treated n DPSCs were both significantly up-regulated as compared with n DPSCs.The odonto/osteogenic differentiative potential of i DPSCs and LPS-treated n DPSCs was lower than that of n DPSCs.Conclusion:In our study,we can isolated DPSCs from the inflammatory dental pulp successfully,but DPSCs have lower odonto/osteogenic differentiative potential.Part II Effects of baicalin on the proliferation and odonto/osteogenic potential of i DPSCsAim: To explore the effects of baicalin on the proliferation and odonto/osteogenic potential of i DPSCs.Methods: Methods: 1)We test the proliferation of n DPSCs,i DPSCs and baicalin-treated i DPSCs at the concentrations of 0,10,20,50,100μM by MTT assay.2)Cell cycle fractions(G0/G1,S and M phase)were determined by flow cytometry.3)ALP activity at day 3,5,7,alizarin red staining and quantitative calcium measurement at day 21 in n DPSCs,i DPSCs and baicalin-treated i DPSCs in mineralization media was respectively measured.4)Furthermore,the expression of cytokines(IL-1β,TNF-α)and odonto/osteoblastic markers(DMP1/DMP1,RUNX2/RUNX2,DSPP/DSP,OSX/OSX,OPN/OPN,OCN/OCN)in n DPSCs,i DPSCs and baicalin-treated i DPSCs was investigated by real time RT-PCR and Western blot.Results: MTT assay showed that Baicalin exposure did not affect i DPSCs proliferation at the concentrations of 0-20μM as campared with the untreated controls,and there appeared to be a decrease in i DPSCs growth in the presence of50μM baicalin.The expression of IL-1β and TNF-α in baicalin-treated i DPSCs were significantly lower those in i DPSCs(P<0.05).MTT assay and Cell-cycle analysis revealed that the proliferation index was similar in baicalin-treated i DPSCs and untreated,and that of i DPSCs was increased as compared with n DPSCs.ALP activity,calcified nodules formed in baicalin-treated group were higher than untreated group(P<0.05).The expression of odonto/osteoblastic markers(RUNX2/RUNX2,DSPP/DSP,OSX/OSX,OPN/OPN,OCN/OCN)in baicalin-treated group were significantly up-regulated compared with the untreated group.Conclusion: The proliferation of i DPSCs was higher than n DPSCs.20 μM baicalin did not affect on the proliferation of i DPSCs,but could decrease the expression of IL-1β and TNF-α and enhance the capacity of donto/osteoblastic differentiation of i DPSCs.Part III Regulation of baicalin on odonto/osteogenic potential of i DPSCs via NF-κB and Wnt signaling pathwayAim: To explore regulation of baicalin on odonto/osteogenic potential of i DPSCs via NF-κB and Wnt signaling pathwayMethods: NF-κB pathway-related proteins(IκBα,p-IκBα,P65,p-P65)and Wnt pathway-related proteins(β-catenin,GSK3β,Ca MKII,NLK)in n DPSCs and i DPSCs were respectively measured by western blot in cytoplasm or nucleus.Real-time RT PCR and western blot measured the odonto/osteogenic differentiative potential of i DPSCs treated by baicalin,BMS345541(one NF-κB pathway inhibitor)and DKK1(one Wnt pathway inhibitor).Results: Western blot showed that the expression of NF-κB pathway proteins(p-P65 and p-IκBα)and Wnt pathway-related protein(β-catenin)in i DPSCs was significantly enhanced as compared with those in n DPSCs,however,the proteins in baicalin-treated group were lower than i DPSCs;the expression of GSK3β,Ca MKII and NLK in i DPSCs was decreased as compared with those in n DPSCs,and the proteins in baicalin-treated group were enhanced.The expression of odonto/osteogenic markers(DMP1/DMP1,RUNX2/RUNX2,DSPP/DSP,OSX/OSX,OPN/OPN,OCN/OCN)were upregulated by baicalin,BMS345541 and DKK1 in i DPSCs.Conclusion: Baicalin can regulate the NF-κB and β-catenin/Wnt pathway to rescue the decreased differentiation potential of i DPSCs. |
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