| PurposeBone marrow mesenchymal stem cells have a metabolic balance between osteogenic and adipogenic differentiation.Previous studies have found that hyperlipidemia causes reduced bone formation and poor osseointegration around implants in rats.Sdccag3 is differentially expressed in hyperlipidemia rats,and it can be beneficial to the osteogenesis disorder caused by dyslipidemia,but the pathway mechanism of Sdccag3 to promote osteogenic differentiation and its influence on the steady state of osteogenic and adipogenic differentiation have not been studied.In this experimental study,① to verify the effect of hyperlipidemia on the homeostasis of osteogenesis and adipogenesis of rat implants and bone marrow mesenchymal stem cells;② to explore the effects of Sdccag3 on the homeostasis of osteogenesis and adipogenesis differentiation under the control of Sdccag3 changes and the functional role of Sdccag3;③ Clarify the specific pathway mechanism of Sdccag3 regulating bone metabolism.Methods(1)Explore that high-fat and Sdccag3 affect the differentiation of BMSCs:①Osteogenesis induced rat bone marrow mesenchymal stem cells(BMSCs)in a high-fat environment,to detect the expression of osteogenic and adipogenic differentiation indicators and LRP5,LRP6,β-catenin,Wnt5a,Wnt5b in the Wnt pathway by RT-PCR and Western blot during the differentiation process;②ALP,Alizarin Red and Oil Red O staining to observe the effect of high-fat environment on the osteogenic and adipogenic differentiation of BMSCs;③After knock-down/over-expression Sdccag3 in BMSCs,RT-PCR and Western blot were used to detect the changes in the expression of osteogenic and adipogenic differentiation indicators and Wnt pathway related factors during the differentiation process;④To observe the effect on the osteogenic and adipogenic differentiation of BMSCs in morphology.Immunofluorescence was used to detect the expression relationship of Sdccag3 and LRP5.(2)Explore that hyperlipidemia and Sdccag3 affect bone metabolism in rats:①Successfully constructed a hyperlipidemia rat model,implanted bilateral femurs,samples were taken 4 weeks later,RT-PCR was used to detect the expression levels of osteogenic and adipogenic differentiation indicators in gene levels and the gene expression levels of LRP5,LRP6,β-catenin,Wnt5a,and Wnt5b in the Wnt pathway;②Micro-CT,quantitative analysis of bone morphology,and hard tissue section staining to detect bone formation and fat formation around the implant;③In hyperlipidemia rats after knock-down/over-expression of Sdccag3,detect the effect on the expression of osteogenic and adipogenic differentiation indicators and Wnt pathway related factors;④ detect the effect on bone formation and fat formation around the implant from morphology.(3)To explore that LRP5 affects the expression of Sdccag3 and bone metabolism:Knockdown expression of LRP5 in hyperlipidemia rats,and four weeks after bilateral femoral implants were implanted,RT-PCR detects the effect on the expression of Sdccag3 gene,and detects gene expression changes of osteogenesis and adipogenic differentiation indicators and Wnt pathway related factors.Micro-CT,bone morphology quantitative analysis,and hard tissue section staining to detect bone formation around the implant.Results(1)High-fat environment inhibits osteogenic differentiation of BMSCs and promotes adipogenic differentiation:After high-fat osteogenic induction of BMSCs,the expression of Sdccag3,LRP5,LRP6,β-catenin,Wnt5a,Wnt5b decreased(P<0.05).The expression of osteogenic differentiation indicators ALP and Runx2 decreased(P<0.05),and the expression of adipogenic differentiation indicators FABP4 and PPAR-γ increased(P<0.05).And compared with the ordinary osteogenic induction group,immunofluorescence showed that the expression of Sdccag3 and LRP5 protein in the cell decreased.The number of mineralized nodules and ALP activity decreased,and the number of lipid droplets increased.The osteogenic differentiation of BMSCs was weakened,and the adipogenic differentiation was enhanced.(2)Knock-down/over-expression of Sdccag3 regulates the osteogenic and adipogenic differentiation of BMSCs:① After low expression of Sdccag3 in BMSCs,the expression of LRP5 protein in the cells decreased(P<0.05).The expression of Sdccag3,LRP5,LRP6;β-catenin,Wnt5a,Wnt5b and osteogenic differentiation indicators ALP,Runx2 decreased(P<0.05).The expression of adipogenic differentiation indicators FABP4,PPAR-y increased(P<0.05).The number of mineralized nodules and ALP activity in the Sdccag3-knockdown group decreased and the number of lipid droplets formed increased compared with the LV16 group.The osteogenic differentiation of BMSCs was weakened,and the adipogenic differentiation was enhanced.② After Sdccag3 was overexpressed in BMSCs,the expression of LRP5 protein in cells increased(P<0.05).The expression of LRP5,LRP6,β-catenin,Wnt5a,Wnt5b and osteogenic differentiation indicators ALP,Runx2 expression increased(P<0.05).The expression of adipogenic differentiation indicators FABP4,PPAR-γ decreased(P<0.05).The number of mineralized nodules and ALP activity in the Sdccag3-overexpression group increased and the number of lipid droplets formed decreased compared with the LV17 group.The osteogenic differentiation of BMSCs was enhanced,and the adipogenic differentiation was weakened.(3)Osteogenesis of bone tissue around implants in rats with hyperlipidemia decreases,and fat formation increases:The hyperlipidemia rat model was successfully established.In the rats of high-fat group,the expression of Sdccag3,LRP5,LRP6,β-catenin,Wnt5a,and Wnt5b in the bone tissue decreased(P<0.05).The expression of osteogenic differentiation index ALP and Runx2 decreased(P<0.05).The expression of adipogenic differentiation index FABP4,PPAR-γincreased(P<0.05).And rats in the high-fat group had less new bone formation around the implant,decreased bone mass,and increased fat formation compared with the normal group.(4)Knock-down/over-expression of Sdccag3 affects osteogenesis and fat metabolism of rat bone tissue:①Compared with the LV16 group,the Sdccag3-knockdown group had decreased the expression of Sdccag3 in the bone tissue(P<0.05).The expression of LRP5,LRP6,β-catenin,Wnt5a,Wnt5b decreased(P<0.05).The expression of osteogenic differentiation indicators ALP and Runx2 decreased(P<0.05).And the expression of adipogenic differentiation indicators FABP4 and PPAR-y increased(P<0.05).And the formation of new bone around the implant decreased,the bone mass decreased,and fat formation increased.②Compared with the LV17 group,the expression of Sdccag3 in the bone tissue increased(P<0.05),the expression of LRP5,LRP6,β-catenin,Wnt5a and Wnt5b increased(P<0.05),the expression of osteogenic differentiation indicators ALP and Runx2 increased(P<0.05),and the expression of adipogenic differentiation indicators FABP4 and PPAR-y decreased(P<0.05)in the Sdccag3-overexpression group.And the Sdccag3-overexpression group had increased formation of new bone around the implant,increased bone mass and decreased fat formation.(5)Low expression of LRP5 regulates Sdccag3 and affects bone metabolism in rats:In the LRP5-knockdown group,the expression of LRP5 and Sdccag3 in the bone tissue was reduced(P<0.05).And the expression levels of LRP6,β-catenin,Wnt5a,and Wnt5b decreased(P<0.05).The expression of osteogenic differentiation indicators ALP and Runx2 decreased(P<0.05),and the expressions of adipogenic differentiation indicators FABP4 and PPAR-γ increased(P<0.05).The formation of new bone around the implant and the bone mass were reduced compared with the LV16 group.Conclusion1.Hyperlipidemia inhibits the formation of new bone and promotes fat formation around the implant.2.Over-expression of Sdccag3 promotes bone formation and inhibits fat formation around implants in BMSCs and hyperlipidemia rats in a high-fat environment.3.Sdccag3 plays a role in regulating the balance of osteogenesis and adipogenesis through Wnt pathway. |
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