The Role And Mechanism Of DKK-1/wnt/?-catenin Signal Axis In Alcoholic Necrosis Of Femoral Head

Objective: To investigate whether alcohol exposure in vivo and in vitro altered the expression of osteogenesis and lipogenesis in bone tissue by affecting the inhibitor Dkk-1 of the Wnt signaling pathway.Methods: Primary bone marrow mesenchymal stem cells of SD rats were isolated for identification and culture.After induction of osteogenic and lipogenic differentiation,ethanol exposure intervention was conducted according to concentration gradient(0mmol/L,10mmol/L,50mmol/L,100mmol/L).WB technology was applied to detect the expression of the contents of n-catenin in Dkk-1 and Wnt pathway and the downstream osteogenic and lipogenic proteins.Bone marrow mesenchymal stem cells were transfected with si Dkk-1lentivirus,and further osteogenesis and lipid culture were performed under ethanol exposure.WB technology was applied to observe the expression changes of target proteins and downstream proteins.Thirty SD rats aged 4-6 weeks were divided into two groups,one group was given food grade alcohol(ethanol concentration of 56°)and the other group was given normal saline as the control group.At the end of 24 weeks,10 rats in each group wererandomly selected to take out femoral head tissues for HE staining,immunohistochemistry and immunofluorescence experiments.Histopathological changes and changes of target proteins and downstream related proteins in tissues were observed.Finally,in vivo and in vitro immunofluorescence was performed to verify the consistency.Results :(1)The Rat bone marrow mesenchymal stem cells were successfully extracted and exposed to ethanol during osteogenic and lipogenic differentiation.Oil red O staining and alizarin red staining demonstrated that the expressions of osteogenic and lipogenic cells were gradient dependent and the two were reversed.WB detection found that Dkk-1 had ethanol concentration gradient dependence(P<0.05),and inhibited the expression of catenin protein and osteoblast-related protein Runx2(P<0.05)and promoted the expression of lipid-related protein PPAR-?(P<0.05).(2)After si Dkk-1 lentivirus transfection,Dkk-1 at the same ethanol concentration was successfully inhibited,and the cell staining results and the expression results of osteogenic and lipid-related proteins were reversed.(3)After 24 weeks in animal experiments,HE staining pathological results showed sparse trabeculae,necrosis of the surrounding bone marrow,and high expression of Dkk-1 in the alcohol group(P<0.05).The results of immunohistochemistry and immunofluorescence were consistent with those of in vitro experiments,and both in vivo and in vivo experiments of alcohol group were inhibited(P<0.05).Conclusion :(1)Under alcohol exposure,the Wnt signaling pathway inhibitor Dkk-1has a positive concentration dependent change on ethanol.(2)The change of Dkk-1 induced by alcohol affects the osteogenic and lipogenic abilities of bone marrow mesenchymal stem cells and bone tissues in femoral head,which can inhibit osteogenic differentiation and stimulate lipogenic differentiation.(3)Dkk-1 may be involved in the occurrence and development of alcohol-induced femoral head necrosis in animal models.