Therapeutic Effect And Mechanism Of Bushen Jianpi Recipe On Osteoporosis In Ovariectomized Rats

ObjectiveThe purpose of this study was to investigate the effect of Bushen Jianpi Recipe on ovariectomized rats and reveal its intervention mechanism partly,by observing the change of bone histomorphology,bone biomechanical properties and the expression of TRAF6,c-Src and Nfatc1 in tibial bone marrow of rats.This study will provide scientific evidence for clinical treatment of osteoporosis based on Spleen and Kidney Theory of traditional Chinese medicine(TCM).MethodsSixty-six SPF SD female rats of the same age(3 months)were randomly divided into three groups:the blank control group(n=12),the sham-operated group(n=12)and the model group(n=42).Anesthetized rats in the model group were treated with bilateral ovariectomized.The procedure was also conducted on the rats belonging to the sham-operated group,except that the ovaries of these rats were retracted and put back into their abdomen and some adipose tissues around their ovaries were removed.During the operation,five rats died in the model group.Three months after operation,the remaining thirty-seven rats in the model group were randomly divided into three groups:the OVX group(n=13),the E2 group(OVX+Estradiol Valerate,n=12)and the BSJP group(OVX+Bushen Jianpi Recipe,n=12).And then feeding drugs to rats in each group by gavage:BSJP group was given water decoction of the Bushen Jianpi Recipe at the dosage of 14.2 g/kg body weight,the volume of 10 ml/kg body weight and the concentration of 1.42g crude drug/ml.E2 group was given the Estradiol Valerate solution at the dosage of 0.184mg/kg body weight,the volume of 10ml/kg body weight,the concentration of 0.0184mg/ml body weight.Rats in the above groups were administered once a day,6d/wk for three months.The blank control group,sham-operated group and OVX group were given the equal volume of pure water.All rats were weighed once a week and the drug dosage was adjusted accordingly.The rats were killed after three months of administration.The bilateral femurs of rats were used to detect the histomorphological indexes of bone and bone biomechanics.Removed the soft tissue around the tibias of rats and fixed them with 4%paraformaldehyde.And then they ware decalcified with EDTA to make frozen tissue sections of bone,which were used to detect the expression of TRAF6,c-Src,Nfatc1 of tibial marrow in ovariectomized osteoporosis ratsMeasurement data is presented as Mean±Standard Deviation by adopting SPSS 20.0 statistical software.The data need to be tested for normality and homogeneity of variance.For normal distribution data,single factor analysis of variance(ANOVA)was used for comparison.The data of variance homogeneity was tested by LSD and the data with uneven variance was tested by Dunnett's T3 test for the comparison between groups.For non-normal distribution data,non-parametric test should be used.Results1 Effect of Bushen Jianpi Recipe on Bone Histomorphology in Ovariectomized Osteoporosis RatsCompared with sham-operated group,the femur BV/TV in OVX group decreased significantly.Compared with OVX group,the femur BV/TV in E2 group and BSJP group increased,but still significantly lower than that of the sham-operated group.Compared with sham-operated group,the femur Tb.Sp in OVX group increased significantly,while the femur Tb.N in OVX group decreased significantly.Compared with OVX group,the femur Tb.Sp in E2 group and BSJP group decreased,but still significantly higher than that of the sham-operated group,the femur Tb.N in E2 group and BSJP group increased,but still significantly lower than that of the sham-operated group.Compared with sham-operated group,the femur SMI in OVX group increased significantly.Compared with OVX group,the femur SMI in E2 group and BSJP group decreased,but still significantly higher than that of the sham-operated groupThere was no significant difference in Tb.Th among these groups.2 Effect of Bushen Jianpi Recipe on Bone Biomechanical Properties in Ovariectomized Osteoporosis Rats2.1 Effect of Bushen Jianpi Recipe on Maximum Load in Ovariectomized Osteoporosis RatsCompared with sham-operated group,the maximum load of the femur in OVX group decreased significantlyCompared with OVX group,the maximum load of the femur in E2 and BSJP groups increased significantly.The maximum load of the femur in E2 group and BSJP group was still lower than that of sham-operated group significantly.2.2 Effect of Bushen Jianpi Recipe on Bending Strength in Ovariectomized Osteoporosis RatsCompared with sham-operated group,the bending strength of the femur in OVX group decreased significantly.Compared with OVX group,the bending strength of the femur in E2 and BSJP groups increased obviously.While the bending strength of the femur in E2 group and BSJP group was still lower than that of sham-operated group.2.3 Effect of Bushen Jianpi Recipe on Elastic Modulus in Ovariectomized Osteoporosis RatsCompared with sham-operated group,the elastic modulus of the femur in OVX group decreased obviously.Compared with OVX group,the elastic modulus of the femur in E2 and BSJP groups increased obviously.And the elastic modulus of the E2 group and BSJP group was not different from that of sham-operated group.3 Effect of Bushen Jianpi Recipe on Expression of TRAF6,c-Src and Nfatcl of Tibial Marrow in Ovariectomized Osteoporosis Rats3.1 Effect of Bushen Jianpi Recipe on Expression of TRAF6 of Tibial Marrow in Ovariectomized Osteoporosis RatsThe OVX group showed significantly higher positive density of TRAF6 protein expression in the tibial bone marrow of rats than that in the sham-operated group.The positive density of TRAF6 protein expression in the tibial bone marrow of E2 group was not different from that of the sham-operated group.Compared with OVX group,the positive density of TRAF6 protein expression in the tibial bone marrow of the rats in E2 group and BSJP group decreased significantly.While the positive density of TRAF6 protein expression in the tibial bone marrow of the rats in BSJP group was higher than that in sham-operated group significantly.3.2 Effect of Bushen Jianpi Recipe on Expression of c-Src of Tibial Marrow in Ovariectomized Osteoporosis RatsThe OVX group showed significantly higher positive density of c-Src protein expression in the tibial bone marrow of rats than that in the sham-operated group.The positive density of c-Src protein expression in the tibial bone marrow of E2 group was not different from that of the sham-operated group.Compared with OVX group,the positive density of c-Src protein expression in the tibial bone marrow of the rats in E2 group and BSJP group decreased obviously.While the positive density of c-Src protein expression in the tibial bone marrow of the rats in BSJP group was higher than that in sham-operated group significantly.3.3 Effect of Bushen Jianpi Recipe on Expression of Nfatcl of Tibial Marrow in Ovariectomized Osteoporosis RatsThe OVX group showed significantly higher positive density of Nfatcl protein expression in the tibial bone marrow of rats than that in the sham-operated group.Compared with OVX group,the positive density of Nfatcl protein expression in the tibial bone marrow of the rats in E2 group and BSJP group decreased significantly.The positive density of Nfatcl protein expression in the tibial bone marrow of E2 group was not different from those of the sham-operated group.While the positive density of Nfatcl protein expression in the tibial bone marrow of the rats in BSJP group was higher than that in sham-operated group obviously.There was no difference in the above indexes between the sham-operated group and the blank control group to exclude the influence of operation factors.Conclusions(1)Removing ovaries of rats can significantly reduce bone BV/TV,Tb.N and increase SMI,Tb.Sp,and reduce bone biomechanical properties,leading to osteoporosis.Bushen Jianpi Recipe can reverse the above indexes to a certain extent and has therapeutic effect on osteoporosis induced by ovariectomy in rats.(2)Removing ovaries of rats can significantly increase the expression of TRAF6,c-Src and Nfactl proteins in bone marrow,make osteoclasts abnormally active and lead to bone loss.However,Bushen Jianpi Recipe can inhibit osteoclast activity by inhibiting protein expression of TRAF6,c-Src and Nfactl,thus reducing bone loss.This is one of the mechanisms by which it can treat osteoporosis.