| Objective:To detect the expression of Notch signal pathway related receptors and ligands in the femoral head of young rabbits with early steroid-induced avascular necrosis of the femoral head,to compare the effects of different doses of prednisolone acetate on the expression of Notch signal pathway related receptors and ligands in human osteoblasts,and to explore the relationship between Notch signal pathway and glucocorticoid-induced avascular necrosis of the femoral head in young rabbits.Methods:1.60 healthy 2-month-old New Zealand white rabbits,half male and half female,between weight 1.5kg2.0,were randomly divided into hormone injection group（n=48）and control group（n=12）.The hormone injection group was injected with 7.5mg/kg prednisolone acetate into the gluteal muscle,and the control group was injected with the same amount of normal saline into the gluteal muscle for 8 weeks.After 8 weeks,all the experimental animals were killed and the bilateral femoral heads were immediately removed and preserved at-80℃.Combined with the CT scan of the experimental animals during the modeling period,and the pathological results,the experimental animals were divided into hormone injection disease group,hormone injection non-disease group and control group.ELISA and RT-PCR were used to detect the expression of Notch1,Notch2,Jagged1,DLL1,DLL4 and Notch signal pathway target gene Hes1 in the femoral head of each group.2.Human osteoblasts were cultured in vitro and passaged.Hormone intervention group:10-8mol/L,10-7mol/L,10-6mol/L acetate prednisolone acetate;Notch signal pathway inhibitor group:DAPT,50μm;Notch signal pathway activator group Jagged1（188mur204）,50μm;blank control group,ensure that the concentration of DMSO in each group was 0.1%,cultured for 72 hours respectively.ELISA and RT-PCR were used to detect the expression of Notch1,Notch2,Jagged1,DLL1,DLL4 and Notch signal pathway target gene HES1 in each group,and flow cytometry was used to detect the apoptosis of human osteoblasts in each group.Results:1.Of the 48 experimental animals in the prednisolone acetate injection group,6were killed and 10 died in the course of the experiment,and 32 survived 8 weeks later,of which 6 were found to be positive for osteonecrosis of the femoral head,and all of them were unilateral,and the incidence of osteonecrosis of the femoral head was 18.75%.There were 12 animals in the control group,3 animals were killed in the course of the experiment,and there was no death.Glucocorticoid injection onset group（n=6）,corticosteroid injection non-morbidity group（n=26）,control group（n=9）.The expressions of Notch1,Jagged1,DLL1and DLL4 were158.01±54.81/219.53±25.16/257.88±17.96,2.26±0.33/3.40±0.53/4.05±0.39,2.05±0.07/2.93±0.46/3.30±0.35,2.56±0.82/5.08±1.14/6.58±1.02.The incidence group was lower than the non-disease group and the control group,and the difference was significant.（F=18.61,32.19,17.71 and 25.02,P<0.01）;The expression levels of Notch2 were8.42±0.59/6.01±0.93/4.90±0.68,the incidence group was higher than the non-disease group and the control group,and the difference was significant.（F=25.02,P<0.01）.The relative expression levels of the target gene HES1 were 0.43±0.11/0.80±0.27/1.14±0.35.The incidence group was lower than the non-disease group and the control group,and the difference was significant.（F=12.49,P<0.01）.2.Human osteoblasts were cultured in vitro.The apoptosis rates（%）of control group,prednisolone acetate intervention group:10-8mol/L,10-7mol/L,10-6mol/L,Notch signal pathway inhibitor group and Notch signal pathway activator group were6.08±1.07/11.90±1.40/20.78±2.04/31.00±0.40/27.09±0.86/6.23±1.78,the apoptosis rate of prednisolone acetate intervention group:10-8mol/L,10-7mol/L,10-6mol/L,Notch signal pathway inhibitor group was higher than that of the control group,and the difference was significant.（F=182.06,P<0.01）.The expression levels of Notch1 and Notch2 in each group were9.01±0.16/7.89±0.07/6.71±0.30/5.17±0.34/9.07±0.21/9.15±0.21,7.16±0.04/6.13±0.04/5.84±0.14/5.08±0.04/7.17±0.06/7.22±0.11,compared with the control group,the expression of prednisolone acetate intervention group:10-8mol/L,10-7mol/L and 10-6mol/L decreased in a dose-dependent manner,and the difference was significant.（F=143.70 and 335.71,P<0.01）.The expressions of Jagged1,DLL1 and DLL4 in each group were23.92±0.51/23.00±0.16/17.28±0.66/12.48±0.26/8.51±0.49/26.56±0.41,0.68±0.02/0.61±0.01/0.44±0.01/0.24±0.04/0.24±0.04/0.72±0.01,1.14±0.02/0.96±0.01/0.76±0.03/0.57±0.03/0.81±0.04/1.27±0.03,compared with the control group,the expression of prednisolone acetate intervention group:10-8mol/L,10-7mol/L and 10-6mol/L decreased in a dose-dependent manner and the expression of Notch signal pathway inhibitor group decreased compared with the control group,while the expression of Notch signal pathway activator group increased compared with the control group,the difference was significant.（F=401.45,215.87and 243.21,P<0.01）.The relative expression of HES1 in each group was1.00±0.14/0.81±0.09/0.54±0.05/0.45±0.02/0.53±0.08/1.30±0.13,compared with the control group,the prednisolone acetate intervention group:10-8mol/L,10-7mol/L and 10-6mol/L and Notch signal pathway inhibitor group were decreased while the expression of Notch signal pathway activator group was increased,the difference was significant.（F=35.33,P<0.01）Conclusion:1.Glucocorticoid can inhibit the expression of Notch signal pathway in human osteoblasts,which may promote the apoptosis of human osteoblasts by inhibiting the expression of Notch signal pathway.2.Glucocorticoid may inhibit the expression of Notch signal pathway receptors Notch1,ligand Jagged1,DLL1,DLL4 and target gene HES1 in the femoral head of young rabbits,increase the expression of receptor Notch2,to reduce the osteogenic ability of the femoral head of young rabbits,enhance the ability of bone resorption and adipogenesis in the femoral head,and induce apoptosis of femoral head osteocytes,resulting in avascular necrosis of the femoral head of young rabbits. |
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