Effect Of Kaempferol In Eucommia Ulmoides On Osteogenesis In Ovariectomized Rats And Its Relationship With MTOR Signaling Pathway

The first partObjective:To observe the effect of kaempferol(Kae)on osteogenesis induced by bone marrow mesenchymal stem cells in ovariectomized rats.Method:First,ovariectomized rat model and adult female Sprague-Dawley(SD)rats were obtained.After 8 weeks of routine feeding,bone marrow mesenchymal stem cells(BMSCs)from the femurs and tibias of normal SD and OVX rats were flushed out with PBS in a biosafety cabinet.Isolated cells were routinely cultured and passaged.After the P3 generation of BMSCs was harvested,they were randomly divided into three groups as follows:(1)Control group(Control):rat bone marrow mesenchymal stem cells from the normal SD rats.(2)Osteoporosis model group(OVX):rat bone marrow mesenchymal stem cells from ovariectomized model rats.(3)Kae intervention group(OVX+Kae):rat bone marrow mesenchymal stem cells were incubated with Kae.The BMSCs in all the group were inoculated with osteogenic induction medium and differentiated into osteoblasts.MTT assay was used to detect the cytotoxicity of Kae.Alizarin red staining was used to observe the induction of osteogenesis and the activity of alkaline phosphatase(ALP)was tested in the cells after induction of osteogenesis.The relative transcription levels of mRNA of Runx2 and Osterix were detected by qPCR and the protein expression levels of Runx2 and Osterix were detected by Western blot.Result:1.Different concentrations of Kae have different degrees of cytotoxicity to rat bone marrow mesenchymal stem cells,and have a significant dose-dependent.When the concentration of Kae is 100 μM,the cytotoxicity reaches its maximum.2.Alizarin red staining showed that Kae had the best effect on inducing osteogenesis when the concentration of Kae was 10 μM.3.Kae can significantly increase ALP activity during osteogenesis induced by bone marrow mesenchymal stem cells in ovaiectomized rats.4.Kae can significantly up-regulate the relative transcriptional levels of Runx2 and Osterix in cells of each group.5.Kae can significantly up-regulate the protein expression of Runx2 and Osterix in cells of each group.Conclusion:1.Kae promote bone marrow mesenchymal stem cells to differentiate into osteoblasts and improve bone metabolism in ovariectomized rats.2.The mechanism of Kae improving bone metabolism may be related to its ability to regulate the gene or protein expression levels of Runx-2 and Osterix.The second partObjective:To investigate the mechanism of Kae promoting osteogenesis induced by bone marrow mesenchymal stem cells in ovariectomized rats and its relationship with mTOR signaling pathway.Method:First,ovariectomized rat model and adult female Sprague-Dawley(SD)rats were obtained.After 8 weeks of routine feeding,bone marrow mesenchymal stem cells(BMSCs)from the femurs and tibias of normal SD and OVX rats were flushed out with PBS in a biosafety cabinet.Isolated cells were routinely cultured and passaged.After the P3 generation of BMSCs was harvested,they were randomly divided into five groups as follows:(1)Control group:BMSCs derived from normal rats were inoculated with osteogenic induction medium and differentiated into osteoblasts;(2)OVX group:BMSCs derived from OVX rats were inoculated and induced as above;(3)OVX + Kae group:BMSCs derived from OVX rats were incubated with Kae and induced as above;(4)OVX + Kae + Rapa group:BMSCs derived from OVX rats were incubated with Kae and Rapa and induced as above;(5)OVX + Rapa group:BMSCs derived from OVX rats were incubated with Rapa and induced as above.Alizarin red staining was used to observe the induction of osteogenesis and to detect the activity of alkaline phosphatase(ALP)in the cells after induction of osteogenesis.The relative transcriptional levels of mRNA of Runx2,Osterix,4E/BP1 and S6K1 were detected by qPCR,and the protein expression levels of Runx2,Osterix,4E/BP1,p-4E/BP1,S6K1 and p-S6K1 were detected by Western blot.Result:1.After intervention with Kae,the number of calcium nodules increased significantly(p<0.05 vs.OVX),while the number of calcium nodules decreased significantly in OVX + Kae + Rapa group and OVX + Rapa group(p<0.05 vs.OVX+Kae).2.After intervention with Kae,ALP activity increased significantly(p<0.05 vs.OVX),while ALP activity decreased significantly in OVX + Kae + Rapa group and OVX + Rapa group(p<0.05 vs.OVX + Kae).3.Kae could significantly increase the relative transcriptional levels of mRNA of Runx2 and Osterix in cells(p<0.05 vs.OVX),while the relative transcriptional levels of Runx2 and Osterix in OVX + Kae + Rapa and OVX + Rapa groups were significantly decreased(p<0.05 vs.OVX + Kae).4.Kae could significantly decrease the relative transcriptional levels of mRNA of 4E/BP1 and increase S6K1’s in cells(p<0.05 vs.OVX),while the relative transcriptional levels of 4E/BP1 and S6K1 in OVX+Kae+Rapa and OVX+Rapa groups were significantly reversed(p<0.05 vs.OVX+Kae).5.Kae intervention could significantly increase the protein expression of Runx2 and Osterix in cells(p<0.05 vs.OVX),while the expression of Runx2 and Osterix in OVX + Kae + Rapa and OVX + Rapa groups decreased significantly(p<0.05 vs.OVX+Kae).6.Kae could significantly downreguleted the protein expression levels of p-4E/BP1 and upregulated p-S6K1’s in cells(p<0.05 vs.OVX),while the expression levels of 4E/BP1 and S6K1 in OVX+Kae+Rapa and OVX+Rapa groups reversed significantly(p<0.05 vs.OVX+Kae).Conclusion:The mechanism of Kae promoting bone marrow mesenchymal stem cells to differentiate into osteoblasts in ovariectomized rats may be related to its regulation of mTOR-RUNX2/Osterix signaling pathway.The third partObjective:To observe the effect of Kae on bone metabolism in ovariectomized rats and to explore the relationship between its mechanism and mTOR signaling pathway.Method:Thirty SD rats were randomly divided into five groups as follows:(1)sham group:rats were sham-operated and fat tissue around the ovaries was removed;(2)OVX group:rats were ovariectomized;(3)Kae treatment group(OVX + Kae):continuous administration of Kae(100mg/kg/d)via gavage for 8 weeks in ovariectomized rats;(4)Kae and Rapa administration group(OVX + Kae +Rapa),rats were operated as(3)and injected intraperitoneally with Rapa 0.2mg/kg/d for 8 weeks;(5)Rapa group(OVX + Rapa):OVX rats were injected intraperitoneally with Rapa 0.2mg/kg/d for 8 weeks.Rats in each group were sacrificed at the 8th week.Femurs were taken and scanned with Micro-CT.Result:1.Kae can significantly reduce the number of trabeculae in femoral metaphysis and the thickness of trabeculae in OVX-induced rats.However,the positive effect of Kae was reversed in rapamycin-treated rats.2.Kae can significantly improve BV/TV,SMI,Tb.N,Tb.Th,BMD and other bone morphological parameters in OVX rats,while the positive effects of Kae in OVX+Kae+Rapa group and OVX+Kae group were significantly reversed.Conclusion:1.Kae improves bone mineral density and morphological parameters of ovariectomized rats,promotes bone formation.2.The anti-osteoporosis mechanism of Kae may be related to mTOR signaling pathway.