Administration Of Erythropoietin Exerts Protective Effects Against Glucocorticoid-induced Osteonecrosis Of The Femoral Head In Rats

Objectives:1. To study erythropoietin’s effect on glucocorticoid-induced apoptosis of osteoblast of rats in vitro and its probable molecular mechanism.2. The present study was implemented to evaluate these anti-apoptotic and tissue-protective effects in glucocortico id-induced osteonecrosis in rats.Methods:1. The osteoblast of fetal rats were harvested and cultured in vitro. ALP testing and Alizarin red staining were applied to identify the osteoblast. The apoptosis of osteoblast was induced by10-6mol/L dexamethasone solution. EPO was administrated at4different concentration (10U/mL,20U/mL,40U/mL,80U/mL) to treat the dexamethasone-induced osteoblast. Flow cytometry and TUNEL assay were used to detect the apoptosis of the osteoblast. The expression of caspase-3protein was tested by western blotting assay.2. Osteonecrosis was induced by low-dose lipopolysaccharide and subsequent pulsed high-dose methylprednisolone. Rats in preventive group were treated with500U/kg/d recombinant human erythropoietin (rhuEPO) for one week. Then hematological and histomorphometric methods were used to determine the effects of rhuEPO administration. Trabecular bone architecture analysis was applied to evaluate bone mass change in the osteonecrosis zone. The terminal deoxynucl eotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay assay was performed to test the apoptotic index of osteoblasts and osteocytes. Immunoblot was used to assess the caspase-3and VEGF expression of the femoral head.Results:1. Osteoblasts grow well. Alizarin red staining showed it could form a calcified nodule. The alkaline phosphatase test was positive, while dexamethasone could inhibit the expression of alkaline phosphatase. The apoptosis of osteoblast was successfully induced by dexamethasone while it could be abolished by erythropoietin.40U/ml exhibits excellent anti-apoptotic effects on glucocorticoid-induced apoptosis of osteoblast of rats in vitro. Western blot showed that the caspase-3expression level of the dexamethasone group was the highest, and the difference was statistically significant (P<0.05). And the caspase-3expression level in the EPO group was significantly lower than in the dexamethasone group (P<0.05).2. The EPO treatment significantly improved the histological performance. Additionally, the incidence of osteonecrosis was significantly decreased in the EPO group (22.2%) than in the control group (66.7%). Furthermore, the expression of caspase-3was significantly reduced in the EPO group. Consistently, apoptosis of osteoblast and osteocyte cells determined by TUNEL assays was inhibited upon EPO administration. In contrast, the expression of vascular endothelial growth factor (VEGF) increased in the osteonecrosis zone in rats treated with EPO.Conclusions:1. Erythropoietin can abolish the glucocorticoid-induced apoptosis of osteoblast of rats in vitro. It may exerts its anti-apoptotic effects by inhibit the expression of caspase-3in the osteoblast.2. In this study, we demonstrate that EPO exerts prominent protective effects against glucocorticoid induced osteonecrosis of the femoral head in rats by inhibiting apoptosis of osteoblasts and osteocytes and elevating the expression of VEGF.