Kaempferol Promotes Osteogenic Differentiation Of Mouse Bone Marrow Mesenchymal Stem Cells Under Traction Force Through MTORC1 Signaling

Background:Kaempferol(Kae)is a typical flavonol with obvious osteogenic effect.Long-term consumption of food rich in flavonol can improve bone health.Disraction osteogenesis is often used to treat bone structural deformities caused by various reasons and bone loss caused by tumor,trauma and infection.Some studies have shown that Kae can promote the formation of new bone by promoting the expression of osteogenic genes in vitro and in vivo,but there are few reports on the application of Kae in distraction osteogenesis.Therefore,this study uses a multi-cell stretching device to exert periodic uniaxial tension on the bone marrow mesenchymal stem cells(BMMSCs)of mice treated by Kae,while using tool drugs to inhibit the expression of endogenous mammalian target of rapamycin complex 1(mTORCl)signal pathway to explore the role of kaempferol in the process of promoting cell osteogenic differentiation under tension,and provide evidence for the clinical application of distraction osteogenesis.Materials and Methods:1.Effect of kaempferol on osteogenic differentiation of mouse bone marrow mesenchymal stem cells under tensionMouse BMMSCs were cultured,identified and stressed in vitro(10%uniaxial dynamic tension),and the appropriate concentration of kaempferol was selected through the alizarin staining,cell count and cytotoxicity test for the next step.The endogenous mTORC1 signal was changed by the tool drug pp242,and the cell traction unit was divided into four groups(group A:control group;group B:inhibition of mTORC1 signal group;group C:Kae treatment group;group D:inhibition of mTORCl signal group after Kae treatment).The alkaline phosphatase activity(ALP),osteocalcin(OCN)expression and the relative content of intracellular calcium ions in the four groups were measured at 4 hours after traction.2.mTORCl signal pathway mediates the mechanism of kaempferol promoting osteogenic differentiation of BMMSCs under tensionWestern blot was used to detect the phosphorylation expression of mammalian target of rapamycin(mTOR),ribosomal protein S6 kinases(S6K),4E/BP1 and the expression of osteogenic transcription factors Runx2 and Osterix in the above four groups of cells.qRT-PCR was used to detect the mRNA expression level of the above factors.Result:1.Effect of kaempferol on osteogenic differentiation of mouse bone marrow mesenchymal stem cells under tensionThrough alizarin staining,cell count and cytotoxicity test,10 μMol/L kaempferol has little inhibitory effect on cells and has the strongest osteogenic ability.Four hours after the end of the stress,the results of the relative contents of ALP,OCN and intracellular calcium ions in the four groups were different,among which the contents of ALP and OCN in group C were the highest,and the relative contents of intracellular calcium ions were the lowest.The difference was statistically significant(P<0.05).2.mTORC1 signal pathway mediates the mechanism of kaempferol promoting osteogenic differentiation of BMMSCs under tensionFour hours after the end of the stress,the protein phosphorylation expression of mTOR,S6K,4E/BP1,the protein expression of Runx2,Osterix and the mRNA expression results of the above factors in the four groups were different,among which the mRNA level and protein phosphorylation expression of mTOR,S6Kin Group C were higher than those in other groups,the mRNA level and protein expression of Runx2 and Osterix were higher than those in other groups,and the 4E/BP1 mRNA level and protein phosphorylation expression were lower than those in other groups,the mRNA levels’ difference were statistically significant(P<0.05).Conclusion1.Kae can promote bone formation of mouse BMMSCs cells under tension.2.Kae promotes osteogenic differentiation of mouse BMMSCs under tension through mTORC1 signal pathway.