The Effect Of TLR4 Activation With LPS On Bone Morphogenetic Protein 9-induced Osteogenic Differentiation Of Mesenchymal Stem Cells

Objective: To study the effect of TLR4 activation with LPS on bone morphogenetic protein 9(BMP9)-induced osteogenic differentiation of mesenchymal stem cells(MSCs)and detect the possible mechanism.Methods: Firstly,the endogenous expression of TLR4 in MSCs was detected by RT-PCR and Western blot.The expression change of TLR4 in MSCs infected with Ad-GFP and Ad-BMP9 was detected by Western blot.The expression of IL-1β and IL-6 were detected by RT-PCR.Subsequently,MSCs infected with Ad-GFP or Ad-BMP9 was stimulated with LPS.The early osteogenic differentiation capability of MSCs was detected by ALP staining and quantitative assay;the calcium deposition was detected by alizarin red S staining;the expression of later osteogenic differentiation marker gene OPN was determined by RT-PCR and Western blot;the activation change of Smad1/5/8 signaling pathway was detected by Western blot and luciferase reporter assay;the expression of Runx2 was detected by RT-PCR and Western blot.Next,the MSCs was pretreated with NAC,and then stimulated with LPS.The intracellular ROS level was determined by DCFH-DA using a fluorescence microscope and flow cytometer,and the expression of Nrf2,SOD1 and SOD2 were detected by Western blot.Then,the MSCs was pretreated with BAY11-7082,and then stimulate with LPS.The intracellular ROS level was determined by DCFH-DA using a flow cytometer.Lastly,MSCs were infected with Ad-BMP9 in the presence of BAY11-082 or NAC,and then stimulated with LPS.The osteogenic differentiation capability change of MSCs was detected by ALP staining and quantitative assay,calcium deposition and the expression of OPN;the activation change of Smad1/5/8 signaling pathway was detected by Western blot,luciferase reporter assay and ICC;the expression of Runx2 was detected by RT-PCR and Western blot.Results: The endogenous expression of TLR4 was detectable in MSCs.BMP9 could inhibit the expression of TLR4 but not IL-1β and IL-6 in MSCs.Activation TLR4 with LPS could inhibit BMP9-induced ALP activity,calcium deposition,OPN expression,Smad1/5/8 activation and Runx2 expression.TLR4 activation with LPS increased intracellular ROS level in MSCs,and NF-κB played a role in this process.The impaired effect of TLR4 activation on BMP9-induced osteogenic differentiation could be partly blocked by BAY11-7082 or NAC.Conclusion: TLR4/NF-κB/ROS signaling triggered by LPS could suppress BMP9-induced osteogenic differentiation of MSCs.