An Experimental Study Of Epimedium Intervention Steroid-induced Avascular Necrosis Of The Femoral Head

BackgroundOsteonecrosis of the femoral head(ONFH) were the pathological process of femoral head blood supply destruction or bone cells apoptosis due to different pathogenesis. In Clinical it can be divided with traumatic and nontraumatic types, the former has a clear mechanism, while the latter is a complication of systemic disease, but the pathogenesis is still unknown. Steroid-induced avascular necrosis of the femoral head(SANFH), is nontraumatic osteonecrosis of the femoral head, occurred in long-term or high-dose usage more than the physical requirement of adrenal cortical hormone, which is the most common type of ONFH.The main clinical manifestations is hip pain, activity limitation, limp, the late high disability rate, seriously impact on health and quality of life in patients. In clinical it is common seen in autoimmune diseases, pediatric renal disease, rheumatoid arthritis, organ transplantation with long-term use of hormones. According to the domestic and foreign statistics, steroid-induced avascular necrosis of the femoral head accounted for nearly half of ONFH, but the patients with ONFH in need of treatment are500～7500000, the annual new patients are15～200000, and non-traumatic ONFH mostly occurs in young adults in our country. At present, hormone therapy is adopted mainly for the treatment of autoimmune diseases, especially in2003when hormones played an important role in the fight against atypical pneumonia (SARS). At the same time, it also caused more avascular necrosis of the femoral head, and the hormone application is substandard, which caused more and more hormone induced necrosis of femoral head. Because of its unclear pathological mechanism,a variety of treatment methods are uncertain. The TCM is based on its concept of holism, guided by treatment based on syndrome differentiation, has its unique advantages in the understanding and treatment of disease. The Epimedium is based on TCM theories that invigorate kidney yang, strengthen bones and muscles, antirheumatism. Combined with modern medical research, the drug can improve bone metabolism and promote osteoblast generation. On the basic understanding of SANFH, We proceed to develop a safe and effective drug, better serve clinical.The patients who must take hormone will take Epimedium at the same time, which will intervene the natural process of disease, change the evolution and destructive process of disease, and can greatly reduce the incidence and disability rate, ultimately improve the function or postpone artificial joint replacement, improve the life quality of the patients, alleviate pain, and reduce the economic burden of the patients.Existing problemsIn modern medicine, Studies on pathological mechanism of SANFH is still in the experimental stage. It is rarely reported at home and abroad and many conclusion are established at the hypothesis level. There is no specific methods in treatment, and there is no more effective drugs rather than joint replacement, and it is often difficult for patients to accept the risk of surgical operation. Though it takes certain effect and has significant effect, small toxic and side effect, and can be accepted by majority of patients, TCM treatment also has many problems. It mainly reflected in two aspects: one is that the etiology and pathogenesis of SANFH and syndrome differentiation have not been formed a unified theory and knowledge, another is that it is rare in animal studies and the mechanism research of traditional Chinese medicine treatment, though the clinical reports are very common. Therefore, we inheritate and make use of traditional Chinese medicine theory on the treatment of SANFH, and should pay attention to make further researches on TCM treatment of SANFH with multiple pathways and multidisciplines by means of modern science.ObjectiveThe following problems shoule be solved through a series of researches:1develope models of steroid-induced avascular necrosis of femoral head of rabbit, revealed the relationship between the PPARy, ALP, osteocalcin mRNA and osteocalcin in bone trabecular area ratio, the rate of empty lacunae, the average diameter of fat cells and steroid-induced avascular necrosis of femoral head.2The extraction of Herba Epimedii affect on animal model of avascular necrosis of the femoral head, revealed the relationship between PPARy of steroid-induced avascular necrosis of the femoral head and adipocyte differentiation in the intervention of Epimedium.3The extraction of Herba Epimedii affect on animal model of avascular necrosis of the femoral head, revealed the relationship between ALP, osteocalcin mRNA of steroid-induced avascular necrosis of the femoral head and the transformation activity of osteoblasts in the intervention of Epimedium.4The extraction of Herba Epimedii affect on animal model of avascular necrosis of the femoral head, observed the change of trabecular area ratio, the rate of empty lacunae, fat cell average diameter of steroid-induced avascular necrosis of the femoral head, in order to determine the mechanism of prevention and treatment by EpimediumThe first part:the preparation of rabbit model of steroid-induced femoral head necrosis and epimedium extractMethods130healthy adult New Zealand white rabbits, male and female, weighted in2.5-3.Okg, feed by grain and single cage, provided by the animal experimental center of Henan province. Extraction of Herbal Epimedium, the composition of epimedium herb extract suspension. Containing crude drug2.3g/ml, produced by preparation room of The First Affiliated Hospital of Henan University of Traditional Chinese Medicine. 2The model:After one week of feeding, the animal were randomly divided into two groups, control group10, model group20, the body weight and sex differences of two groups showed no statistical significance by t test. Ensure that no statistically significant test. All animal model were first applied to horse serum10mL/kg by rabbit ear vein injection,3weeks after the interval, horse serum was reduced6mL/kg by rabbit ear vein injection again; after2weeks interval, intraperitoneal injecte methylprednisolone,45mg/kg for3D, one time daily. At the period of hormone injection, every animal was injected penicillin100000units intraperitoneal every day, consecutive7D, prevention of infection.2Results2.1confirmed that the model was successfulAfter model ing,selected one model animal and one normal animal randomly, killed them by air embolism method, anatomy femoral head,made pathologic slices, observation confirmed femoral head necrosis model was successful.2.2animal grouping and drugAnimal was feed conventional in blank group. To the last injection of hormone, from the remaining19animal, randomly selected16rabbits,which were divided into two groups:model group and experimental group. Selected8animals to blank group,(the remaining4animal were executed by air embolism method).at this point, the experimental group, blank group, model group and experiment group, each group has8only animals. Dosing method and sample collection:since the beginning of the first day, blank group and model group animal were fed to10ml normal salin every daye, the test group anima lwas fed to10ml extraction of Herba Epimedii gavage every day. continued in a total of8weeks. The second part:An experimental study of Epimedium intervention Steroid-induced avascular necrosis of the femoral headThe first Experiment:effects of Epimedium on steroid-induced avascular necrosis of the femoral head OSTEmRNA, PPARy mRNA1methodAt the end of the experiment, three groups were selected respectively8animals after eighth weeks, after anesthesia rapidly anatomy femoral head and femur, extracted5ml red bone marrow of proximal femur epiphysis at near, Implantated them in aseptic centrifugal tube with DMEM culture liquid,taked them to the laboratory. saved for inspection. Extracted marrow total RNA by Trizol method, FQ-PCR press kit, detected the expression of PPAR y mRNA and Osteocalcin mRNA. Set the PCR primers, also set the template and blank control. The detection is carried out in ABI7300fluorescence quantitative PCR instrument. Amplified reaction conditions:50℃2min.95℃5min,95℃15s,60℃45s,40cycles; after the PCR reaction, the PCR products were analyzed for specificity by agarose gel electrophoresis, two genes mRNA relative expression of femoral head tissues were analysed by calculating formula mRNA=2-ΔCt2ResultsThe expression of PPAR y mRNA in The test group and the control group cells was lower (expression value was2.31±0.29and1.84±0.15), there are no significant differences between the two groups (P>0.05). The expression of PPAR y mRNA in model group was Higher (4.75±0.44cells), the difference had statistical significance between the model group and experimental group, blank group (P<0.05).The expression of osteocalcin mRNA is lower in model group (6.86±1.05),It was highly expressed in experimental group and blank group (12.70±1.27expression values and14.07±0.80). Compared with the model group, the differences were statistically significant (P<0.05), and the difference between the experimental group and the control group had no statistical significance (P>0.05). The experimental results showed that, the expression of PPARy mRNA in the model group was higher, Compared with the blank group and experimental groups,it had statistical significance (P<0.05); the expression level of Osteocalcin mRNA in the model group were significantly lower than those of control group and experimental group (P<0.05).The second Experiment:effects of Epimedium on trabecular area ratio, the rate of empty lacunae, average diameter of fat cells, ALP activity of steroid-induced avascular necrosis of the femoral head1methodRemoved the femur, washed them with warm saline several times, in the clean bench broke femur at both ends with tongs, flushed the marrow cavity repeatedly with10ml DMEM culture liquid, beated repeatedly, the clumps of cells were blowed uniform. Filtration, after centrifugation, addited DMEM medium containing10%FBS in75cm2cell culture bottle in2×106/ml cell density, cultured in37℃,5%CO2, the moisture saturated CO2incubator.80%cells confluence after passage. Take the second generation MSCs, per hole1×105cells was inoculated into6well plates, as the experiment Od. Observed the morphology, growth and differentiation of MSCs daily under inverted microscope. At the tenth day of the experiment, a part of the cell was cracked by repeated freezing and thawing method, then centrifuged to assay the activity of ALP cells with ALP kit; At the twenty-first day of the experiment, the remaining cells was stained by oil red "O". the fat cells were observed under light microscope and counted the number.2Results2.1The activity of intracellular alkaline phosphatase (ALP) was determined: compared model group and blank control group, experimental group, P<0.05, ALP decreased significantly, there was statistical significance, explained that, the metabolism of bone cell wsa reduced, bone resorption increased; compared experimental group with the control group, P<0.05, there was no significant changes in ALP activity, no statistical significance2.2trabecular area ratio, the rate of empty lacunae, average diameter of fat cell were statisticed.the trabecular area ratio in test group and the blank group cell was higher (42.25±3.86expression values and55.75±3.5), there was no significant differences between the two groups (P>0.05). In model group, trabecular bone area was at a low level (29.75±0.75), and the difference between the model group and experimental group, blank group had statistical significance (P<0.05). In The test group and the control group cells, missing bone lacuna rate was lower (expression value was13.75±1.50and10.25±2.26), there was no significant differences between the two groups (P>0.05),In model group, vacancy lacunaes rate was higher (20.50±2.08), and the difference between the model group and experimental group, blank group was statistically significant (P<0.05). In the test group and the blank group, fatty cell average diameter was at a lower level (38±2.94expression values and29.50±3.10), there was no significant differences between the two groups (P>0.05).The average diameter of fat cells in model group was at higher level (52.25±4.78), and the difference between the model group and experimental group, blank group had statistical significance (P<0.05).The third part:The clinical study of Epimedium treating steroid-induced avascular necrosis of femoral head1methodFrom2010November to2012November, a total of30patients with steroid-induced avascular necrosis of the femoral head were included in the study. The test group and the control group were15cases in each.The treatment methods in test group:modified core decompression and strengthen the mechanical support operation plus Herba Epimedii, The treatment methods in control group:simple modified core decompression and strengthen the mechanical support operation. Patients were from the Department of orthopedics of the First Affiliated Hospital of Henan University of Traditional Chinese Medicine 2Results2.1At3,6months,compared hip joint function:the rank sum test, the two groups before and after treatment of hip joint function itself, there were significant differences, P<0.01, two kinds of treatment for avascular necrosis of the femoral head had good effect; the comparison between two groups, there was significant difference. P<0.05, the hip joint Harris score of the experimental group was significantly higher than that of the control group.2.2At3,6months, compared curative effect:by CMH test, compare the excellent rate of curative effect of two groups,p<0.05, the excellent and good rate was86.67%in the test group,in the control group, the excellent and good rate was60%, the excellent and good rate in the experimental group was better than control group.2.3At36months, compared treatment imaging:by CMH test, compared two group imaging, p<0.01,in trial group,3cases improved, accounted for20%,11cases were stable, accounted for73.33%,1case was unstable,accounted for6.67%,0cases was worse, accounted for0%;in the control group,1case was improved,accounted for6.67%,11cases were stable, accounted for73.33%,2cases were unstable, accounted for20%,1case was worse, accounted for6.67%, the imaging in test group were better than the control group.Conclusions1by horse serum plus methylprednisolone method can model rabbit avascular necrosis of the femoral head of hormone.2Epimedium can inhibit expression of PPAR y mRNA in steroid-induced femoral head necrosis, prevent bone marrow stromal stem cells differentiate into adipocytes. Epimedium can restrain the low expression level of Osteocalcin mRNA caused by hormone.3hormone can reduce the activity of ALP in the bone tissue, Epimedium can restrain decrease of ALP activity induced by hormone, hormone can increase empty bone lacuna rate of femoral head, increased fat cell diameter, decreased trabecular bone area ratio, Epimedium can improve the above changes. 4modified core decompression with bone fibula support hollow screw stabilization surgery combined with traditional Chinese medicine Epimedium Decoction orally and simple modified core decompression with bone fibula support hollow screw fixation all can improve the hip joint function, prevent the femoral head collapse, promote the repair of femoral head necrosis to a certain extent. By Comparation, the former was better than the latter.