杜仲汤调控AKT/FOXO1信号通路对成骨细胞骨形成的影响OA

Objective:To explore the mechanism by which Duzhong(杜仲)Decoction promote bone formation of osteoblasts.Methods:The blank serum and the Duzhong Decoction-containing serum were prepared.The osteoblast inju-ry model was induced by H2 O2.Osteoblasts were allocated into control(blank serum),model,and 5%,10%,and 15%Duzhong Decoction-containing serum groups.The proliferation of osteoblasts was detected by the CCK8 method.Bio-chemical assay kits were used to measure the changes in cell oxidation indicators.The alkaline phosphatase(ALP)as-say kit was used to measure ALP activity.Mineralization of osteoblasts was observed by Alizarin red staining.qPCR was employed to determine the mRNA levels of Runx2,osteoprotegerin(Opg),and bone morphogenetic protein-2(Bmp2)in osteoblasts.Western blotting was performed to determine the protein levels of BMP-2,collagen type I(COL1),phospho-rylated protein kinase B(p-AKT),AKT,forkhead box protein O1(FOXO1),and phosphorylated(p)-FOXO1 in osteo-blasts.Meanwhile,the phosphatidylinositol 3-kinase(PI3K)/AKT pathway inhibitor LY294002 was used to further clar-ify the regulatory mechanism.Results:Compared with the control group,the model group showed decreased proliferation of osteoblasts(P<0.01),increased MDA content,weakened activities of superoxide dismutase(SOD)and ALP(P<0.01),reduced formation of mineralized nodules(P<0.01),and down-regulated mRNA levels of Runx2,Opg,and Bmp2(P<0.05 or P<0.01)and protein levels of BMP-2,COL1,p-AKT,and FOXO1(P<0.05 or P<0.01).Compared with the model group,Duzhong Decoction-containing serum promoted the proliferation of osteoblasts(P<0.05),decreased the MDA content,enhanced the SOD activity(P<0.01),increased the activity of ALP,boosted cell mineralization(P<0.05),and up-regulated the mRNA levels of Runx2,Opg,and Bmp2(P<0.05)and the protein levels of BMP-2,COL1,p-AKT,and FOXO1(P<0.01).The inhibitor LY294002 effectively inhibited the expression of p-FOXO1 and p-AKT and down-regulated the phosphorylation level of AKT/FOXO1 pathway.Conclusion:Duzhong Decoction regulates the AKT/FOXO1 signaling pathway to ameliorate oxidative damage and promote bone formation of osteoblasts,thereby pre-venting osteoporosis.