Curcumin Promotes Osteogenic Differentiation Of Periodontal Ligament Stem Cells Through The PI3K/AKT/Nrf2 Signaling Pathway

Objectives:Curcumin,a polyphenolic phytochemical,has been widely studied due to its wide-ranging pharmacological activities and trustworthy safety.The aim of this study was to investigate the effect of curcumin on the osteogenic differentiation of periodontal ligament stem cells(PDLSCs)and its underlying potential mechanism.Methods:The tissue explant adherence method was used to isolate PDLSCs.Clonogenic experiment was used to evaluate the self-proliferation of PDLSCs,Flow cytometry was used to evaluate the specific markers of MSCs,Alizarin Red staining and Oil Red O staining were applied to confirm the multiple differentiation potential of the cells.And then,PDLSCs were treated by curcumin,CCK8 assays were used to evaluate the effect of curcumin at different concentrations on cytotoxicity,and ALP activity assays,ALP staining and Alizarin Red staining were used to measure the osteogenic differentiation ability,and the optimal concentration was selected.In order to clarify its mechanism,firstly,western blot was used to detect the activation effect of curcumin on PI3K/AKT and Nrf2 signals.Next,PDLSCs were treated with LY294002(a PI3K inhibitor)and siNrf2 respectively in the presence of curcumin,the PI3K/AKT and the Nrf2 signaling pathway were interfere,western blot was applied to evaluate the AKT phosphorylation levels and the Nrf2 levels.Finally,western blot,RT-PCR,ALP activity assays,ALP staining and Alizarin Red staining were used to detect the changes of osteogenesis-related indicators,and proved weather the potential effect of curcumin on osteogenic differentiation is related to the PI3K/AKT/Nrf2 signals.Results:PDLSCs were successfully cultured by method of tissue block.The result of Clonogenic experiment reveal that PDLSCs possess the great capacities of proliferation,Flow cytometry proved that PDLSCs were positive for the specific markers of MSCs(CD73,CD44,CD105 and CD73).Besides,Oil Red O staining,Alizarin Red staining showed the multiple differentiation ability of PDLSCs.The results of drug screening reveal that curcumin at low concentration(0.001,0.01,0.1,1?M)had no cytotoxicity and could promote osteogenic differentiation of the PDLSCs,0.1 ?M curcumin had the strongest effects and this concentration was selected for the follow-up experimental study.The results of Western blot showed that curcumin could activate the PI3K/AKT and Nrf2 signals and the protein expression levels of p-AKT and Nrf2 were increase.LY294002 was added to inhibit the PI3K/AKT signaling pathway,and the expression of p-Akt,Nrf2 all decreased.In addition,curcumin exposure dramatically increased the expression of osteogenesis related indexes,LY294002 was added to inhibit the expression of osteogenesis related indexes.In addition,siNrf2 was used to block the Nrf2 pathway;the protein expression of Nrf2 decreased while p-AKT had not show a downward trend,and the effect of osteogenesis was be inhibited by siNrf2.Conclusion:Curcumin at an appropriate concentration had no cytotoxicity and could promote osteogenic differentiation of the PDLSCs,and the effect is related to the PI3K/AKT/Nrf2 signaling pathway.Providing more theoretical support for the application of curcumin in the osteogenic differentiation of stem cells.