Study On NF-?B Pathway Mechanism During The Differentiation Of Human Stem Cells From Apical Papilla Induced By Yunnan Baiyao

Yunnan Baiyao,a traditional Chinese herbal medicine,has been widely used in clinical work for its ability of wound healing and bone regeneration.However,the effects of Yunnan Baiyao on odonto/osteogenic differentiation of SCAPs and the potential mechanisms remain unclear.This paper was designed to explore the questions from three aspects.Part ?.Preparation of Yunnan Baiyao conditioned medium and the screening of determined concentrationObjective:To obtain the Yunnan Baiyao conditioned medium and select the optimal concentration and investigate its effect on the proliferation ability.Methods:The optimal concentration was determined by ALP activity.CCK8 assay and FCM was performed to evaluate the proliferation activity.Results:ALP activity assay revealed that Yunnan Baiyao at the concentration of 50?g/mL was the optimal concentration during the differentiation of YNB-treated SCAPs.As compared with control group,the proliferation activity of SCAPs was not affected by YNB at 50 ?g/mL.Conclusion:50?g/mL Yunnan Baiyao conditioned medium can promote the mineralization ability of SCAPs without affecting their proliferation capacity in vitro.Part ?.Effects of YNB on the differentiation of SCAPsObjective:To observe the effects of YNB on the odontogenic/osteogenic differentiation of SCAPs.Methods:Alizarin red staining and quantitative calcium measurement at day 14 was respectively investigated.Odonto/osteoblastic genes and proteins(OCN/OCN,OPN/OPN,OSX/OSX,RUNX2/RUNX2,ALP/ALP,COL-I/COL-I,DMP1,DSP/DSPP)in SCAPs were respectively assessed by real-time RT-PCR and Western blot.Results:Calcium nodules formed in YNB group were significantly higher than those in the control group.The expressions of odonto/osteogenic markers(OCN/OCN,OPN/OPN OSX/OSX RUNX2/RUNX2,ALP/ALP,COL-I/COL-I DMP1,DSP/DSPP)in YNB-treated SCAPs were significantly higher than those in control group in vitro.Conclusion:YNB can promote the differentiation of SCAPs into odontogenic/osteogenic lineages in vitro.Part ?.NF-?B pathway mechanism embedding in the differentiation of YNB-treated SCAPsObjective:To explore the NF-?B pathway involvement in the differentiation of SCAPs.Methods:NF-?B pathway related proteins were measured by Western blot in cytoplasm or nucleus.Inhibitors of NF-?B pathway(BMS345541)were used to detect the expressions of I?B?,p-I?B?,P65,p-P65 by Western blot and immunofluorescent staining.The odonto/osteogenic potential of SCAPs treated by BMS345541 was measured by ALP activity assay,ALP staining,real-time RT-PCR and Western blot.Results:YNB increased the nuclear accumulation of P65 and induced a rapid degradation of I?B?.YNB increased the expression of p-I?B? and p-P65.The expression of P65 and I?B? was downregulated when NF-?B pathway was inhibited by BMS345541.The odonto/osteogenic differentiation was inhibited after BMS3345541 treatment.Conclusion:NF-?B pathway was activated instantly when SCAPs were treated with YNB.Therefore,we conclude that YNB-mediated odonto/osteoblastic differentiation of SCAPs may be determined by the activation of NF-?B pathway.