| Objective:To detect the expression of TGF-βsignal pathway related factors in early steroid-induced osteonecrosis of the femoral head of young rabbits,to compare the effects of different doses of prednisolone acetate on the expression of TGF-βsignal pathway related factors in human osteoblasts,and to explore the relationship between TGF-βsignal pathway and steroid-induced osteonecrosis of the femoral head.Methods:1.60 8-week-old New Zealand white rabbits,between weight 1.5～2.0kg,were randomly divided into experimental group（Eg,n=48）and control group（Cg,n=12）.The Eg was injected with 7.5mg/kg prednisolone acetate into the gluteal muscle,and the Cg was injected with the same amount of normal saline into the gluteal muscle for 8weeks,and 50000 U penicillin was injected once a week to prevent infection.After 8 weeks of experiment,all the experimental animals were performed by computed tomography（CT）then sacrificed and bilateral femoral heads harvested,frozen in the refrigerator at-80℃for follow-up examination.ELISA and RT-PCR were used to detect the expression of TGF-β1,TGF-β2,Smad2,Smad3 and the transcription factor Runx2 in the femoral head.2.Human osteoblasts were cultured in vitro and passaged.Prednisolone acetate intervention group（PIg）:10-8mol/L,10-7mol/L,10-6mol/L prednisolone acetate;TGF-βsignal pathway inhibitor group（TIg）:5μmol/L SB 431542;TGF-βsignal pathway activator group（TAg）:10μM SRI-011381 hydrochloride;control group（Cg）,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 TGF-β1,TGF-β2,Smad2,Smad3 and the transcription factor Runx2 in each group,and flow cytometry was used to detect the apoptosis of human osteoblasts in each group.Results:1.In Eg（n=48）,6 rabbits were sacrificed,32 survived,6 experimental animals had positive avascular necrosis（disease group,Dg）,and 26 were negative（non-disease group,NDg）at an incidence of 18.75%（6/32）.Elisa results showed that in Cg,NDg and Dg the expression of TGF-β1（pg/m L）were 77.12±14.62、90.17±11.90、126.14±25.66,and the difference was significant.（t=3.35,P<0.05;t=4.24,P<0.01）.The expression of TGF-β2（pg/m L）were 74.54±7.63、89.24±9.51、109.74±16.45,and the difference was significant.（t=4.12、5.65,P<0.01）.The expression of Smad2（ng/m L）were 17.74±2.72、23.82±3.58、31.28±3.88,and the difference was significant.（t=4.54、7.99,P<0.01）.The expression of Smad3（ng/m L）were 1.76±0.52、2.39±0.45、3.53±0.47,and the difference was significant.（t=5.60、6.71,P<0.01）.PT-PCR showed that in Cg,NDg and Dg the expression levels of Runx2 were 1.02±0.17、1.27±0.14、1.72±0.11 and the difference was significant.（t=7.60、8.91,P<0.01）.2.Human osteoblasts were cultured in vitro.The apoptosis rate（%）of human osteoblasts in Cg,PIg:10-8mol/L,10-7mol/L,10-6mol/L,TIg and TAg were 6.36±1.61、13.53±0.38、20.72±0.45、30.17±1.35、24.58±1.20、5.72±0.01,the apoptosis rate of PIg and TIg was higher than that of Cg,and the difference was significant（F=288.55,P<0.01）.Elisa showed that:The expression of TGF-β1（ng/m L）in each group were 169.29±1.78、148.90±2.02、127.66±3.16、114.85±2.54、175.52±5.90、173.33±3.86,compared with the Cg,the expression in the PIg decreased in a dose-dependent manner,and the difference was significant.（F=161.17,P<0.01）;The expression of TGF-β2（ng/m L）in each group were 313.08±5.27、286.63±5.17、231.45±13.69、159.01±3.45、311.35±11.48、310.78±6.22,compared with the Cg,the expression in the PIg decreased in a dose-dependent manner,and the difference was significant.（F=163.74,P<0.01）;The expression of Smad 2（ng/m L）in each group were 395.66±3.61、369.06±2.30、342.75±1.81、293.30±3.05、312.39±4.45、399.13±6.09,compared with the Cg,the expression in the PIg decreased in a dose-dependent manner,the expression in the TIg was lower than that in the Cg,and the difference was significant.（F=390.35,P<0.01）;The expression of Smad 3（ng/m L）in each group were 89.71±0.79、85.10±0.87、78.19±0.82、67.67±0.60、82.32±1.12、90.14±0.44,compared with the Cg,the expression in the PIg decreased in a dose-dependent manner,the expression in the Tig was lower than that in the Cg,and the difference was significant.（F=330.07,P<0.01）.RT-PCR showed that:The relative expression of Runx2 in Cg,PIg,TIg and TAg were 1.00±0.15、0.73±0.06、0.57±0.07、0.28±0.10、0.51±0.04、1.45±0.29,the expression of PIg and TIg was lower than that in Cg,while the expression of TAg was higher than that in Cg,and the difference was significant.（F=25.47,P<0.05）.Conclusion:Glucocorticoid can promote the apoptosis of human osteoblasts in vitro by down-regulating the expression of TGF--βsignal pathway,while in the model of steroid-induced osteonecrosis of femoral head in young rabbits,the expression of TGF--βsignal pathway is up-regulated,which may have the effect of delaying osteonecrosis of femoral head and/or repairing necrotic bone tissue. |
