The Experimental Study Of The Effects And Mechanisms Of Vitamin E On Steroid-induced Osteonecrosis Of The Femoral Head

PurposeThe aim of this study is to investigate the effects and possible mechanisms of vitamin E on osteocyte apoptosis and DNA oxidative damage in bone marrow hemopoietic cells at early stage of steroid-induced osteonecrosis of femoral head on molecular and histomorphological level.To explore effect of vitamin E at different doses on the osteocyte apoptosis of rabbits with steroid-induced avascular osteonecrosis of the femoral head(SANFH)and to provide relevant method for prevention and early treatment on SANFH.MethodEffects of vitamin E on osteocyte apoptosis and DNA oxidative damage in bone marrow hemopoietic cells at early stage of steroid-induced femoral head osteonecrosis1.Thirty-six chinese white rabbits were randomly divided into three groups(model group,vitamin E group,and control group),each with 12 rabbits.Model group: this group was first injected with an intravenous dose of lipopolysaccharide(LPS),10?g/Kg,and then with an intramuscular dose of methylprednisolone(MPS),20mg/kg per time,three times at an interval of 24 h in order to establish rabbit models of osteonecrosis.Vitamin E group: this group was treated same as the model group,and were daily fed with vitamin E(0.6g/Kg/d)since the beginning of modeling.Control group: this group was treated with equal normal saline in the same way and at the same time as the aforementioned two groups.In order to prevent infection,all the rabbits were injected with penicilinium(800,000 U/rabbit)twice a week.During the Experiment,general condition of each animal was observed(such as mental state,appetite and weight,etc.)Two time points,weeks 4,and 6,were selected after modeling was completed.After animals were sacrificed(each n =6),bilateral femoral head were conventional fixed,decalcified,embedding slices under test.2.Each specimen tissue morphology was observed under the microscope.Osteonecrosis was verified by histopathology with haematoxylin-eosin(HE)staining.Empty bone lacuna was counted.3.The apoptosis rate of osteonecrosis was observed by terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assay.4.The apoptosis Expressions of caspase-3,bcl-2 and bax were analyzed by immunohistochemistry(S-P).Bcl-2,bax and caspase-3 mRNA and protein levels were analyzed by real time quantitative PCR.5.The DNA oxidative damage of bone marrow hematopoietic cells were analyzed by immunohistochemistry.Effect of vitamin E at different doses on the osteocyte apoptosis with steroid-induced avascular osteonecrosis of the femoral head at the early stage1.Seventy-five chinese white rabbits were randomly divided into five groups.The model group(n=15)was injected with lipopolysaccharide(LPS,10 ?g/Kg)and then methylprednisone(MPS)(20mg/kg)was given 3 times with 24 hour interval.The vitamin E group(n=45)divided into three groups: high-dose group(1.2g/Kg/d),middle-dose group(0.6g/Kg/d),low-dose group(0.3g/Kg/d).These group was treated same as the model group,and were daily fed with vitamin E at different doses since the beginning of modeling.Control group(n=15): this group was treated with equal normal saline in the same way and at the same time as the aforementioned two groups.During the Experiment,general condition of each animal was observed(such as mental state,appetite and weight,etc.)Three time points,weeks 4,6,and 8,were selected after modeling was completed.2.Each specimen tissue morphology was observed under the microscope.Osteonecrosis was verified by histopathology with haematoxylin-eosin(HE)staining.HE staining empty bone lacuna was counted.3.TdT-mediated dUTP nick end labeling(TUNEL)assay was used to detect apoptosis and apoptosis index was counted.Results:1.histopathological observation(HE)Model group:bone trabeculae were sParser and thinner,structurally disordered,of fragments;the number of bone cells reduced;and empty bone lacunae increased.Vitamin E group: middle-dose group and low-dose group of vitamin E: bone trabecular structure was irregular.Part of the arrangement was sparse and clutter.Increased numbers of empty bone lacunae were observed.High-dose group of vitamin E: bone trabecular structure was approximately integral.There was little empty bone lacunae.Control group: bone trabeculae were very intact,well orderly,dense and plump,and osteocytes were visible,of big and central cell nucleus.After the modeling for weeks 4 and 6 respectively,the vacant bone lacunae rates in model group were higher than that in vitamin E group(P<0.05).The empty bone lacunae rates in vitamin E group and model group were higher than that in control group(P<0.05).The empty bone lacunae rates in high-dose group of vitamin E were lower than that in middle-dose and low-dose group of vitamin E(P<0.05).2.Detection of apoptosis(TUNEL)The osteocyte apoptosis indexes in model group were higher than that in vitamin E group(P<0.05).The apoptosis indexes in vitamin E group and model group were higher than that in control group(P<0.05).The apoptosis indexes in high-dose group of vitamin E were lower than that in middle-dose and low-dose group of vitamin E(P<0.05).3.Immunohistochemical resultsCompared with the control group,the bcl-2 positive cell rate at week 4 in femoral head tissues in model group decreased,the bax and caspase-3 positive cell rate increased(P<0.05).The proportion of bcl-2 positive cells of vitamin E group was higher than that in model group,but lower than that in control group(P<0.05);Besides,the bax and caspase-3 positive cell rate in vitamin E group was lower than that in model group,but higher than that in control group(P<0.05).4 Assessment of DNA oxidative damage in marrow bone hematopoietic cellsthe DNA oxidative damage rate at week 4 in bone marrow hemopoietic cells in vitamin E group was lower than that in model group,and higher than that in control group(P<0.05).5.Result of PCRAt week 4,compared with vitamin E group,bcl-2 m RNA level in model group decreased,but bax and caspase-3 m RNA levels of femoral head tissue of rabbits in model group increased(P <0.05).Compared with the control group,bax and caspase-3 m RNA levels of specimen of rabbits increased significantly and bcl-2 m RNA level decreased significantly in the model and vitamin E group(P<0.05).6.Among different dose groups of vitamin E,the high-dose group achieved significant differences(P < 0.05).Conclusion:1.In the early period in steroid induced avascular necrosis of femoral head,large dose of hormone may inhibit the expression of bcl-2,promote the expression of bax and caspase-3 and result in femoral head necrosis.2.Vitamin E is effective in intervening in apoptosis through decreasing caspase-3 and bax expression and up-regulating bcl-2 expression at early stage of steroid-induced femoral head necrosis in rabbit models.3.Vitamin E is effective in alleviating DNA oxidative damage in bone marrow hemopoietic cells at early stage of steroid-induced osteonecrosis of the femoral head.4.The high-dose of vitamin E can effectively improve the bone cell apoptosis in the steroid induced avascular necrosis of the femoral head.