Autophagy Regulates Odontoblasts Defense In Inflammatory Enviroment

Part I:Autophagy in odontoblasts defensing inflammationObjectives:Odontoblasts derive from neural crest-derived odontogenic mesenchymal cells,and they are important barrier of defense for the host.Facing the invasion of carious bacteria,odontoblasts and cells from the subodontoblastic layer have the capacity to produce reactionary dentin.Autophagy can eliminate damaged organelles and recycle cellular components to facilitate cellular homeostasis.It can be activated with external stressors,such as starvation,hypoxia,and infection.In this study,the role of autophagy in inflamed odontoblasts was explored,and its possible mechanism was investigated.Methods:With LPS stimulation,odontoblast viability was assessed with CCK8,and autophagy related proteins were detected with or without LPS stimulation within 24h.CQ was used to block the autophagy process,then the cell viability and Akt/survivin signaling pathway were explored.LY294002 and YM155 were used to inhibit the activation of Aktand survivin respectively,then investigate the relationship between autophagy and Akt/survivin signaling pathway.Results:Cell viability showed no significant difference between the LPS-treated and control groups within 24h.With LPS stimulation,the protein levels of LC3,Atg5-Atgl2,p-ULK1S555,p-AMPKaT172,and Beclinl increased and peaked at 6 h,then gradually decreased at 12 and 24 h.Cleaved caspase3 slightly increased with time.Cell viability showed no significant change in the control,CQ,and LPS groups but decreased in the CQ+LPS groups.However,it increased once autophagy was inhibited with CQ in both control and LPS groups at 12 h.The expression of p-Akt showed no significant increase at 6 h,while it up-regulated in the CQ and LPS+CQ groups at 12h.That suggest the dual role of autophagy in pre-odontoblast with LPS stimulation,and autophagy was a rather early pro-survival event at early stage,The expression of survivin showed no change when p-Akt was inhibited with LY294002 at 6h.While p-Akt and survivin were decreased with LY294002,and the ratio of LC3II to LC3I increased at 12h.It suggested that increased Akt and survivin at 12 h might reduce autophagic cell death in mDPC6T.Conclusion:Our findings show autophagy induced by LPS may be an even early pro-survival mechanism than stellar pro-survival molecules Akt/survivin.The Akt/mTOR/survivin pathway may also play a vital role in protecting cells from autophagic cell death in the later stage of inflarmmation.Part ?:Autophagy regulates odontoblastic differentiation in an inflammatory environmentObjectives:In the event of carious invasion,odontoblasts induce immune inflammatory responses and reactionary dentin to defend against invading pathogens.When odontoblasts are damaged,the progenitors in the pulp can migrate and differentiate into odontoblast-like cells and form reparative dentin.Autophagy is an important survival mechanism,which is also proved in odontoblast resisting inflammation in above.This study explores the role of autophagy in odontoblast differentiation with lipopolysaccharide stimulationin vitro and the colocalization of p-NF-?B and LC3 in caries teeth.Methods:With LPS stimulation,odontoblast differentiation capacity was evaluated with Alizarin red staining and ALP activity.Autophagy expression level was detected with western blot,monodansylpentane(MDH),and autophagic flux was morphologically traced with an mRFP-GFP-LC3 plasmid.The odontoblast differentiation,autophagy and apoptosis related proteins were analyzed,after CQ,rapamycin and BAY11-7082 were used to block autophagy,active autophagy and inhibit NF-?B signal respectively.To investigate the relationship between autophagy and p-NF-?B,Atg5 siRNA was transiently transfected to cells to inhibit autophagy.The double immunofluorescence of LC3 and p-NF-?B were detected in tooth samples with caries.Results:The odontoblastic differentiation capacity was enhanced with LPS stimulation.The expression of autophagy markers LC3,Atg5,Beclinl and TFE3 increased time dependently,as well along with the amount of autophagosomes and autophagy fluxes.This result suggests that autophagy was enhanced in odontoblastscultured with mineralized-induced medium containing LPS.To confirm the role of autophagy in differentiated odontoblasts with LPS stimulation,chloroquine(CQ)or rapamycin were used to either block or enhance autophagy.The number of mineralized nodules decreased when autophagy was inhibited,but they increased obviously with rapamycin treatment.The expression levels of DSP,DMP1 and OSX were downregulated with CQ treatment but were upregulated when autophagy was enhanced.NF-?B expression was negatively related to autophagy and could inhibit odontoblast differentiation.Furthermore,p-NF-KB and LC3 colocalization was detected in cells stimulated with LPS.The nucleus translocation of p-NF-?B in odontoblasts was enhanced when autophagy was inhibited by Atg5 siRNA.In addition,the colocalization of p-NF-?B and LC3 in odontoblasts and sub-odontoblastic layers was observed in caries teeth with reactionary dentin.Conclusion:Our findings provide a novel insight into the role of autophagy in regulating odontoblastic differentiation by suppressing NF-?B activation in inflammatory environments.