Experimental Research On The Expression Of The BMP6/BMP7,Smurf1/Smurf2 MRNA And Protein In The Femur, Kidney, Hypothalamus Of The Osteoporosis Rats Using Ovariectomy With Kidney-deficiency

Purpose : In order to explore the etiological factors and pathologh of the osteoporosis due to kidney deficiency,We study the expression of BMP6/BMP7 and smurf1/smurf2 mRNA and proteins in the femur, kidney, hypothalamus of the rats of osteoporosis due to kidney deficiency by using ovariectomy.To explore the adjustable function of Chinese medicine and its regulating mechanism.Materials and Method:By using ovariectomy, we established the rat model of osteoporosis due to Kidney-deficiency. We treated the osteoporosis rat with tonifying kidney Chinese herbs (high dose, small dosage) for 12 weeks. Simultaneously,used GuShuKang granule and GaiTianLi tablets, tonifying spleen Chinese herbs, as positive control groups. And rats in the normal group and model group were not given any treatment. We used dual energy X-ray bone density instrument to detect the bone mineral density of the femur of the rats. We used the RT–PCR and Western Blotting method to investigate the expression of BMP6/BMP7 and smurf1/smurf2 mRNA and proteins in the femur, kidney,hypothalamus of the rats.Result:(1)The comparation of uterus exponent of the experimental rats: Comparing with the uterus exponent of the normal group and fake operational group , the uterus exponent of the model group is lower evidently. Comparing with the uterus exponent of the model group,the experimental drug groups(high, small dosage),the Gushukang granule group and the Gaitianli tablets group can increase the uterus exponent,and tonifying spleen Chinese herbs can decrease the uterus exponent,but all of them have no significance difference in statistics.(2)The comparison of the BMD of the femur of the experimental rats:After 12 weeks,comparing with the normal group and fake operational group, the BMD of the femur has evident tendency to reduce;comparing with the model group,the BMD of femur increased evidently in the experimental drug groups(high dosage) and the Gaitianli tablets group. (3)RT-PCR and Western blotting detection: the BMP6 existed in the normal femur, kidney, hypothalamus tissue. Comparing with the normal group and fake operational group,the level of the expression of the gene and protein of BMP6 in the femur and kidney tissue of model group decreased evidently,and the level of the expression of the gene and protein of BMP6 in the hypothalamus tissue of model group increased evidently.After the precaution treatment with the experimental drug groups(high,small dosage),Gushukang granule,Gaitianli tablets group and tonifying spleen Chinese herbs,the experimental drug groups(high,small dosage) can increase evidently the mRNA and protein expression level of BMP6 in the femur and kidney tissue;and the experimental drug groups(high,small dosage),Gushukang granule,Gaitianli tablets group and tonifying spleen Chinese herbs can decrease evidently the mRNA and protein expression level of BMP6 in the hypothalamus tissue.(4)RT-PCR and Western blotting detection: the BMP7 existed in the normal femur, kidney,hypothalamus tissue. Comparing with the normal group and fake operational group,the level of the expression of the gene and protein of BMP7 in the femur and kidney tissue of model group decreased evidently,and the level of the expression of the gene and protein of BMP7 in the hypothalamus tissue of model group increased evidently.After the precaution treatment with the experimental drug groups(high,small dosage),Gushukang granule , Gaitianli tablets group and tonifying spleen Chinese herbs,the experimental drug groups(high,small dosage) can increase evidently the mRNA and protein expression level of BMP7 in the femur and kidney tissue;and the experimental drug groups(high,small dosage),Gushukang granule , Gaitianli tablets group and tonifying spleen Chinese herbs can decrease evidently the mRNA and protein expression level of BMP7 in the hypothalamus tissue.(5)RT-PCR and Western blotting detection: the Smurf1 existed in the normal femur, kidney, hypothalamus tissue. Comparing with the normal group and fake operational group,the level of the expression of the gene and protein of Smurf1 in the femur and kidney tissue of model group decreased evidently,and the level of the expression of the gene and protein of Smurf1 in the hypothalamus tissue of model group increased evidently.After the precaution treatment with the experimental drug groups(high,small dosage),Gushukang granule , Gaitianli tablets group and tonifying spleen Chinese herbs can increase the mRNA and protein expression level of Smurf1 in the femur and kidney tissue in different degrees,and decreased the mRNA and protein expression level of Smurf1 in the hypothalamus tissue in different degrees.(6) RT-PCR and Western blotting detection: the Smurf2 existed in the normal femur, kidney, hypothalamus tissue. Comparing with the normal group and fake operational group,the level of the expression of the gene and protein of Smurf2 in the femur and kidney tissue of model group decreased evidently,and the level of the expression of the gene and protein of Smurf2 in the hypothalamus tissue of model group increased evidently.After the precaution treatment with the experimental drug groups(high,small dosage),Gushukang granule , Gaitianli tablets group and tonifying spleen Chinese herbs can increase the mRNA and protein expression level of Smurf2 in the femur and kidney tissue in different degrees,and decreased the mRNA and protein expression level of Smurf2 in the hypothalamus tissue in different degrees.Conclusion:(1).Using the method of ovariectomy, the rat model of osteoporosis due to Kidney-deficiency successfully can be established.(2). In normal rats,the tissue of the femur ,the kidney and the hypothalamus can express the BMP6/BMP7 and smurf1/smurf2 in the gene and protein level. That means normal the tissue of the femur ,the kidney and the hypothalamus exist BMP6/BMP7 and smurf1/smurf2,and it may have a very important contribution on adjusting osteal- metabolism.(3). The decreasing lever of BMP6/BMP7 and smurf1/smurf2 mRNA and protein expression in the femur and kidney tissue,and the increasing lever of BMP6/BMP7 and smurf1/smurf2 mRNA and protein expression in the hypothalamus tissue,may be one of the important occurring mechanism of the osteoporosis due to kidney deficiency.(4). The experimental Chinese herbs,which have the function of tonifying kidney Qi and strengthening essence ,can increase the level of the expression of the gene and protein of BMP6/BMP7 and smurf1/smurf2 in the femur and kidney tissue,and can decrease the level of the expression of the gene and protein of BMP6/BMP7 and smurf1/smurf2 in the hypothalamus tissue. It would be preventing and treating to the osteoporosis due to kidney deficiency.(5). The function of BMP6/BMP7 and smurf1/smurf2 to adjusting osteal metabolism would be one of the aspects of the kidney controlling bone in TCM.