Preventing Steroid Induced Osteonecrosis Of Femoral Head With Liuweidihuang Pills And Molecular Evaluation

Objective Steroid-induced Osteonecrosis of Femoral Head(ONFH),is the disease that the blood supply of femoral head was damaged or bone cytomorphosis caused by hormone administration lead to death of active component . It was a kind of common and stubborn disease in orthopedics.Long-term or high doses of glucocorticoid, particularly in the treatment of autoimmune diseases induced by ONFH, its incidence was increasing year by year trend,it came to the first in non-traumatic Osteonecrosis of femoral head. It was slow-moving in clinical treatment of ONFH, we always carry out the treatment until necrosis of bone, seriously affecting the work and quality of life in patients, because of its pathogenesis is not clear, is still no effective preventive measures, the final line of most of the need for total hip replacement. Therefore, intervention, blocking the progress of ONFH caused by use of hormones, its pathogenesis and control of technology research, has important significance. This experiment, the establishment of animal model of steroid-induced necrosis of femoral head , the application Liuweidihuang pills to intervene,to evaluate the effects and molecular mechanism of Liuweidihuang pills in preventing steroid induced osteonecrosis of femoral head (ONFH).Methods Thirty-six adult Kunming mice , weighing 40 ~ 50 g, an average of 46g, were randomly divided into three groups(n=12): control group, model group and prevention group. Three groups of animals, body weight and the number of male and female, t-test no statistically significant difference (P>0.05). Blank control group (group A), non-preparation of models; horse serum + hormone group (group B), horse serum + hormones + Liuweidihuang pills group (group C). B, C mice by 10 mL / kg intraperitoneal injection of horse serum; 2 weeks after the interval by 5 mL / kg intraperitoneal injection of horse serum again, at the same time intramuscular injection of prednisolone acetate 45 mL / (kg ? d ), a total of 5 d, preparation of steroid-induced ONFH mouse model. group C were fed with Liuweidihuang pills 2 g / (kg ? d). Observed the general situation in mice after treatment , the mice were sacrificed in the 1st hormone 2,4,8 weeks after injection and generally observe the femoral head,then take bilateral femoral head and the liver histological and immunohistochemical staining, TUNEL assay bone cell apoptosis. Statistical analysis using SPSS13.0 package for analysis. Data are mean±standard deviation that was used to compare between the two groups analysis of variance was used to compare pairwise t test, P value less than 0.05 was considered statistically significant.Results All the mice had no significant change after the first time intraperitoneal injection of horse serum ; About 6 hours after second intraperitoneal injection of horse serum , groups B, C of mice appeared to reduce food intake, apathetic, unresponsive, activities such phenomena as low. Group C of mice gradually improved about 1 week , Group B of mice gradually improved about 2 week . group A of mice no significant changes.All mice survived throughout the experiment period except two death on 7,11 days after second injection of horse serum intraperitoneally in model group and one death on 24 hours after second injection of horse serum intraperitoneally in prevention group.Group A at different time points, thick trabeculae, complete, ordered rules, bone cells are visible, occasionally empty lacunae. The fat cells is relatively small and morphologically normal between marrow . The bone trabeculae of group B is thinner than normal after 2 weeks of 1st steroid injection , partly broken bone cells increase in empty lacunae, some bone cells, margination, condensation .Fat cell diameter increases and increase in the number between marrow ; 4,8 weeks trabecular bone thinning, sparse breaks in increasing bone fragments can be seen the emergence of the rate of empty lacunae increased. Group C was no significant change in trabecular bone than group A at different time points,empty lacunae is rare.the percentage of empty osteocyte lacunae was significantly higher than that in control and prevention group(P <0.01).Immunohistochemical staining:In group A , OPG brown granules more common, OPGL brown granules less at different time points.In group B , OPG expression was significantly decreased and OPGL expression was significantly increased(P <0.01).Apoptosis analysis showed that: Group B are visible in the nucleus appeared more positive cells in brown granules at different time points , which apoptotic index increased with time; group A, C , occasionally positive cells at different time points, the cells no significant changes in AI.Apoptosis analysis showed that apoptosis index in model group was significantly higher than that in control and prevention group(P <0.01).Conclusion1. Liuweidihuang pills has a significant preventive effect to lipid metabolism disorder of the liver caused by hormone application.2. Liuweidihuang pills has the improvement of bone metabolism and protect bone cells, by inhibiting hormone on osteoblast activity to prevent bone resorption, promoting bone formation and bone repair.3. Liuweidihuang pills can antagonism the impact of hormones to OPG and OPGL protein expression, thus contribute to the proliferation of osteoblast cells, inhibit the activity of osteoclasts and effectively improve bone metabolism and status of bone biomechanics. 4. Steroids can lead to significantly increased apoptosis of bone cells,but Liuweidihuang pills can significantly reduce the apoptosis of bone cells and protect bone cells.Thus,Liuweidihuang pills can prevent steroid induced osteonecrosis of femoral head by improving lipid metabolism, releiving bone lesion, and protecting against cell death.