| Objective: To observe the related protein expression in Wnt signaling pathway in the process of steroid-induced avascular necrosis of femoral head rabbit models under hypoxia condition,and the effect of cynomorium on Wnt signaling pathway in SANFH rabbit models in hypoxia state,explore its treatment mechanism for steroid induced femoral headnecrosis in order to provide scientific basis for clinical application of this therapy.Methods:Experiment 1: 24 healthy adult New Zealand white rabbits were randomly divided into normal control group,2 week model group and 4 week model group,8 rabbits in each group.Except the normal control group,lipopolysaccharide is injected into the rabbits of the model groups using ear vein injection of 20?g/kg in the 1d,24 h after repetition.At the second injection of LPS,methylprednisolone is injected using intragluteal injection with the dose of 20 mg/kg,then methylprednisolone is injected twice again,per 24 h.Before the first injections of LPS,penicillin is injected everyday by intramuscular injection of 4 x 105U/rabbit for three times for infection prevention.After that,the penicillin Injection instead of twice a week.At the end of the experiment,two rabbits of each group are sacrificed randomly for contrast observation on histopathology by imaging examination and light microscope to prove successful modeling.At the with the corresponding time,the blood of femoralarteries were extracted for monitoring Partial Pressure of Oxygen in each group respectively.Then making materials from the femoral head and the femoral head were isolated and cultured osteoblasts by enzyme digestion and tissue block method.Western blot was used to detect the expression of ?-catenin,Gsk 3? and other signaling molecules in the classical Wnt pathway.Experiment 2: The model making method is same with experiment 1,and all of the groups were given regular feeding.Four weeks after modeling,model groups began to give drug intervention for 8 Weeks.The low dose group of Cynomorium was given extract in the dose of 60 m L/kg·d,equivalent to the 138 g crude drug,twice by gavage.The high dose group to give 120 m L/kg·d,equivalent to the crude drug 276 g,twice by gavage.The placebo treated with normal saline by 100 m L/kg·d,twice by gavage.After 8 weeks of intervention,blood and sampling were drawn.Western blot was used to detect the expression of ?-catenin,Gsk 3? and other signaling molecules in the classical Wnt pathway.All the data collected were analyzed by SPSS18.0 statistical analysis software in the whole experiment process.The measurement data were expressed as mean plus or minus standard deviation((?)±s).Results:Experiment 1:(1)In the first week of modeling,4 week model group had 1 rabbit died of endotoxin poisoning.In the second week,the 2 weeks model group had 1 rabbit died of anaesthesia.Imaging observation and light microscope observation shown that the modeling were successful.(2)Histopathological observation: In the normal group,the trabecular bone structure,bone cell,marrow cavity structure were normal.The 2 weeks model group: bone trabeculae distribution still neat,but intra bone marrow hematopoietic cells decreased,the number of fat cells in between the trabecular bone and medullary cavity was gradually increased,form bigger,empty bone lacunae were compared with normal control group increased.The 4 weeks model group: the bone trabecula disturbance,sparse,fat cells increased significantly,the hematopoietic cells decreased significantly,the number of empty bone lacunae was significantly higher than the 2 weeks model group.(3)Arterial oxygen pressure(Pa02)detection:In the first day of the experiment,the Pa02 of all rabbits no obvious difference.At the end of the second week,there was no significant difference between the 2 week model group and 4 week model group,but were significantly lower than the normal group(P<0.05).In the fourth week,the 4 week model group was significantly lower than that of the 2 week model group(P<0.05),significantly lowest than the control group(P<0.05).(4)The Western blot shows that in the rabbit models of steroid induced necrosis of the femoral head,?-catenin,p-Gsk 3?,Cyclin D1 protein expression level with femoral arterial oxygen partial pressure decreases,Gsk 3? increases.Compared with the blank control group,difference has statistical significance(P<0.05).Experiment 2:(1)In the first week of modeling,The low dose group of Cynomorium has1 rabbit died of endotoxin poisoning.At the first week of administration,The high dose group of Cynomorium had 1 rabbit died of gavage.At the 6th week of administration,The placebo group had 1 rabbit death.Imaging observation and light microscope observation shown that the modeling were successful.(2)Histopathological observation: normal control group showed normal.The placebo group: bone trabecular structure disorder,sparse,part of the fracture,fat cells increased significantly,the hematopoietic cells decreased significantly,and bone karyopyknosis,empty bone lacuna increased significantly.Cynomorium songaricum Rupr in small dose group: bone trabecula were still relatively sparse,hematopoietic cells increased,fat cells is relatively reduced,empty bone lacunas quantity is also reduced.High dose group of Cynomorium necrosis of the femoral head were significantly improved,basically clear trabecular structure,majority of bone cell morphology and structure of the normal,empty bone lacuna significantly decreased,still visible a few necrotic bone cells,intramedullary hematopoietic cells are abundant,fat cells decreased significantly.(3)As in Experiment 1,the Pa02 of all rabbits no obvious difference.In the fourth week,there is no obvious difference between the low dose group of Cynomorium,high dose of group Cynomorium and placebo group,but significantly lower than the normal group(P<0.05).In the 12 th week,the low dose group of Cynomorium was higher than the placebo group(P<0.05),and significantly lower than the high dose of group Cynomorium(P<0.05).(4)Western blot showed in rabbit model of steroid induced necrosis of femoral head in giving Cynomorium intervention,the increase in the expression of ?-catenin,p-Gsk 3? and cyclin D1,Gsk 3? protein expression inhibited and in a dose-dependent manner,compared with the blank group,the difference was statistically significant(P<0.05).Conclusions:In the pathological process of SANFH,the osteoblasts in femoral bone in the ischemia and hypoxia state for a long time.The expression of Wnt signaling pathway evitably be influenced in this condition.And cynomorium has the functionality ofantioxidation,Oxygen-resistance and anti-aging,which can regulate the expression of Wnt signaling pathway in SANFH rabbit models.And also showed dose-dependent manner. |
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