Effect Of Raloxifene On Vitamin D Receptor Of Cadmium Contamination Rat Osteoblasts

Objective: The study of rat osteoblast cells in vitro, establishment of cadmium exposure model, use raloxifene intervention, observation of cadmium and raloxifene intervention into osteoblast cells survival rate and ERα and VDR expression, to investigate the effect of raloxifene on cadmium exposure rat osteoblasts, which provides a new method for clinical treatment of chronic cadmium poisoning osteoporosis.Methods: Select 5 neonatal rats, by trypsin and collagenase Ⅰ digestion extract the osteoblast isolated from the cranial bone primary culture. Select the 4 generation cells for experiment. The survival rate of osteoblast was detection by CCK-8; select the appropriate concentration of Cd and raloxifene for subsequent experiments.The experiment was divided into 3 groups: Control group, Cadmium Chloride group, Raloxifene group. The composition of osteoblasts cells total RNA extracted by Trizol method, reverse transcription into c DNA, the technology of RT-PCR amplified fragment, expression of ERα and VDR gene in osteoblast. The composition of osteoblasts cells total protein, expression of VDR protein by Western blot. The experimental data were analyzed by SPSS 17.0 statistical software, with P﹤0.05 for the difference had statistical significance.Results: Primary osteoblasts adherent ago showed homogeneous spherical, about 24 h began to paste the wall, 48~96h completely adherent, cells were fusiform or polygon. In control group, the osteoblast cells grow well, closely with the culture bottle; compared with the control group, cadmium chloride group a large number of cell morphological changes, not closely with culture bottle, and a large number of cell death; compared with cadmium chloride group, morphological changes occur raloxifene group fewer cells, more closely and culture bottle wall joint. CCK-8 experiments show: compared with control group, cadmium chloride group significantly decreased cell viability(P﹤0.01), cell survival rate and is inversely proportional to the cadmium concentration(24h:r=﹣0.956,P﹤0.01;48h:r=﹣0.992,P﹤0.01), 1μmol/L Cd Cl2 function of 24 h can make the osteogenic cell survival rate decreased from 100.00% to 85.49%, 8μmol/L Cd Cl2 function of 24 h osteogenic cell survival rate decreased to 50.22%. Compared with cadmium chloride group, the composition of osteogenic cell activity of raloxifene significantly increased(P﹤0.01), cell survival rate was proportional to the concentration of raloxifene(24h:r=0.877,P﹤0.01;48h:r=0.965,P﹤0.01). 1mg/L RAL intervention cells of 24 h the survival rate had no obvious change, but the cell survival rate can be as high as 152.67% after 8mg/L RAL intervention 24 h. RT-PCR experiments show: compared with control group, ERα expression significantly increased(P﹤0.05)and VDR gene expression significantly decreased(P﹤0.05)in cadmium chloride group. Compared with cadmium chloride group, ERα expression significantly decreased(P﹤0.05)and VDR gene expression significantly increased in raloxifene group(P﹤0.05). WB experiments show: compared with control group, VDR protein expression significantly decreased in cadmium chloride group(P﹤0.05). Compared with cadmium chloride group, VDR protein expression significantly increased in raloxifene group(P﹤0.05).Conclusions: Cadmium can produce direct toxicity on osteoblasts through its estrogenic effect; it maybe combines with ERα to decreased VDR expression cause bone damage. Raloxifene can interfere with cadmium estrogenic effect, the protective effect through antagonist cadmium down regulated expression of VDR in osteoblast; improve osteoporosis in chronic cadmium poisoning.