| Objective: Periodontitis is a worldwide chronic disease in periodontal supportive tissues which characterized as bacteria infection,inflammation infiltration and tissue destruction.Increasing evidences show that compared to bacteria infection,inflammatory and immune response play a dominant role in occurrence and develop of periodontitis.Therefore,its urgent to explore chemical agents which could efficiently control periodontal inflammatory response in order to achieve better tissue regeneration.Mesenchymal stem cells show pivotal regulatory effects in inflammatory diseases and autoimmune diseases.Previous study reported that rather than anti-bacterial effects,azithromycin promotes osteogenic differentiation of PDLSCs in inflammatory micro-environment.However,the underlying mechanisms remains unclear.Whether azithormycin could affect apoptosis and autophagy is still unknown.This study aimed to investigate the effect and underlying mechanisms of azithromycin on osteogenic differentiation of periodontal ligament stem cells(PDLSCs)in stimulated with TNF-?,and whether cell apoptosis and autophagy are involve in this process.Methods: 1.MTS assay was used to dectect the proliferation of PDLSCs.Alkaline phosphatase staining,alkaline phosphatase activity,alizarin red staining assays were perfomed to test the osteogenic differentiation of PDLSCs.RT-qPCR analysis were preformed to detect the mRNA levels of KDM2 A,KDM2B and EZH2.Western Blotting were utilized to test p-p65,p-I?B-?,caspase-8 and caspase-3.2.To investigate azithromycin affect cell autophagy and apoptosis in inflammatory micro-environment,PDLSCs were treated with azithromycin,3-MA and CQ respectively.Western Blotting assays were used to detect the LC3,p62,Bcl-2 and Bax protein level.Immunofluorescence were performed to detect the LC3 marker.Results: 1.Alkaline phosphatase staining,alizarin red staining,alkaline phosphatase activity showed decreased in PDLSCs when treated with 100ng/ml TNF-?.RT-qPCR analysis showed that the KDM2 A,KDM2B and EZH2.mRNA profile were increased in stimulation with TNF-?.Azithromycin increased the alkaline phosphatase staining,alizarin red staining and alkaline phosphatase activity in the presence of TNF-?.Western Blotting results showed that 100ng/ml TNF-?increased the protein levels of p65,p-p65,I?B-?,p-I?B-?,caspase-8 and caspase-3.Annexin V assays showed that the early and late apoptosis rate was increased in PDLSCs stimulated with 100ng/ml TNF-?,and decreased in pretreatment with azithromycin.2.20ng/ml TNF-?showed an increased in the ratio of LC3II/LC3I.50,100ng/ml TNF-?decreased LC3II/LC3I and increased p62 expressions.Azithromycin showed an increased in the ratio of LC3II/LC3I and decreased in p62 in PDLSCs stimulated with 20,50,100ng/ml TNF-?;Bcl-2 protein expression increased in PDLSCs stimulated with 20ng/ml TNF-?,while significantly decreased in PDLSCs stimulated with 50,100ng/ml TNF-?.Bax protein level increased in PDLSCs treated with100ng/ml TNF-?.Pretreatment with azithromycin,Bcl-2 protein level increased and Bax protein level decreased in the presence of TNF-?.Immunofluorescence showed that 20ng/ml TNF-?increased the expression of LC3 and puncta slightly,and50,100ng/ml TNF-?decreased LC3 and puncta.Azithromycin and CQ increased the expression of LC3 and puncta,while 3-MA decreased the LC3 and puncta.Conclusions:1.Azithromycin promoted PDLSCs osteogenic differentiation in an inflammation micro-environment by inhibiting the NF-?B signaling pathways.This role of azithromycin may associate with the suppression of TNF-?-induced apoptosis.1.20ng/ml TNF-?induced autophagy and inhibited the occurrence of apoptosis.Azithromycin played a synergistic role;50,100ng/ml TNF-?inhibited autophagy of PDLSCs and induced cell apoptosis.However,azithromycin can reverse the effect of TNF-?.3.Azithromycin promoted autophagy of PDLSCs,which may be one of the mechanisms for promoting osteogenic differentiation of PDLSCs in inflammatory microenvironment. |
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