| Objective:To investigate the effect and mechanism of soy isoflavone on the treatment of steroid induced necrosis of the femoral head.Methods:1. Dexamethasone sodium phosphate solution (10 mg/kg) was injected into bilateral gluteus in the rabbites, every 3 days for 14 times. General observation was done after modelling. Osteonecrosis was verified by pathological observation and MRI findings at 6 weeks.2. Rat BMSCs cells were isolated by whole bone marrow adherent culture method, and cultured, identified and discriminatory.3. Proliferation ability of the bone marrow mesenchymal stem cells was determined by MMT method.4. The relative expression of Runx2, Col 1, Osteocalcin mRNA was detected by RT-PCR.5. The activity of ALP and TG in tissues or cells was detected by Reagent kit.6. Western blot to detect the expression of BMSCs in the PPARy protein.Results:1. After 6 weeks, rabbits did not show obvious changes in control group; increased hair removal, decreased food intake, and slight limp were observed in model group. The MRI results showed normal shape of the bilateral femoral head and no abnormal signals in control group; irregular shape of the bilateral femoral head and a slice of irregular abnormal signals were observed, and necrosis and cystolization of the subchondral bone and sparse changes of trabecular bone were shown in model group. General observation from coronal section of femoral head showed smooth red cartilage surface in control group; on the contrary, the cartilage surface of the femoral head became dull, thin even visible hemorrhage under articular cartilage and necrosis of the femoral head were observed. The histopathological examination indicated that trabecular bone of the femoral head in control group was massive, thick, and close, and osteocytes in the bone lacunae had normal shapes. The osseous trabecular became thinner and broken; karyopyknosis of osteocytes and bone empty lacunae could be obviously seen in model group.2. BMSCs cultured for 2 days, we can see a large number of round cells, only a small quantity of spindle cells. After 5 days, the adherent cells fusiform shape, form colonies value-added, the cells are arranged in a daisy-like or spiral-shaped. 8-10 days later, the cells remain fusiform. BMSCs nuclear staining deep blue. The third passage BMSCs phenotype identification, flow cytometry test results showed that BMSCs surface antigen markers CD44, CD90 positive, and CD34, CD45 negative. BMSCs into bone induction seen significant calcified nodules. After induction into the cartilage, cartilage matrix formation.3. The expression of Runx2, Col I and OC mRNAs was increased in the bone tissue of early steroid-induced avascular necrosis of femoral head in rabbits treated with the soy isoflavone for 8 weeks than that of in the models, while the expression of PPARγ mRNA was decreased.4. Soy isoflavones significantly increased cell proliferation of BMSCs in time-and dose-dependent manners. Cell proliferation of BMSCs was significantly increased treatment with 20 μM genistein for 2-3 days.5. The expression of Runx2, Col I and OC mRNAs were significantly enhanced by treatment with Soy isoflavones. The activities of ALP and TG was significantly improved by treatment with 20 μM Soy isoflavones, respectively. Furthermore, pretreatment with 20 μM Soy isoflavones significantly activated the PPARy protein expression.6. Up-regulation of PPARy could significantly inhibited the effect of Soy isoflavones on cell proliferation of BMSCs.7. Up-regulation of PPARy could significantly suppressed the Runx2, Col I and OC mRNAs expressions, decreased ALP activity and promoted TG activity of BMSCs.Conclusion:Soy isoflavones regulate the differentiation of BMSCs into osteoblasts by regulating the PPARγ pathway to treatment the Steraid-induced Avasadar Necrosis of Femoral Head. |
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