Effect Of Bushenhuoxue Recipe United Denosumab Mediated OPG/RANKL Signaling Pathway On Vascular Calcification In Rats With Senile Osteoporosis

Objective: In this study,we established a model of osteoporosis in aged rats,observed the vascular calcification in rats with senile osteoporosis,and explored the possible mechanism of OPG/RANKL signaling pathway in rats with senile osteoporosis and vascular calcification.Through the treatment of Bushenhuoxue Recipe and DMAB alone and in combination,the possibility of synchronous prevention and treatment of senile osteoporosis and vascular calcification was explored.Methods: Seven-month-old male SD rats were divided into blank control group(n=16),sham-operated group(n=16)and castrated group(n=40)randomly.Blank control group,no treatment.In the sham-operated group,only part of the adipose tissue around the testis was removed.In the other groups,bilateral orchiectomy was used to build the senile osteoporosis model of rats.Eight rats in the blank control group,sham-operated group and castrated group were sacrificed at the 16 th week after operation.The bone mineral density of the right posterior femur of the rats was measured by Double Energy X-ray Absorpmeter to verify the success of the model.After successful modeling,the castrated group was randomly divided into osteoporosis group,Bushenhuoxue group,DMAB group and combined treatment group,with 8 rats in each group.In the Bushenhuoxue group,55g/kg Bushenhuoxue recipe was given orally once a day for 8 weeks.The DMAB group was given 10 mg/kg of DMAB subcutaneously twice a week for 4 weeks.The combined treatment group was treated with Bushenhuoxue recipe and DMAB in the same dosage and route.The other groups were subcutaneously injected with 10 mg/kg saline twice a week.All rats were killed after the intervention.Bone mineral density of femur in right hind limb was measured by Double Energy X-ray Absorpmeter,calcification degree of thoracic aorta was detected by alizarin red staining,serum levels of P?NP,BGP and CTX-?were measured by ELISA.Western-blot was used to detect the expression of OPG and RANKL proteins in thoracic aorta.Results:1.At the 16 th week after operation,compared with the blank control group,bone mineral density in castrated group decreased significantly(P<0.01).At the 24 th week after operation,bone mineral density in osteoporosis group decreased further than that in blank control group(P<0.01),thoracic aorta showed obvious calcification deposition,the concentration of serum P?NP and BGP decreased significantly(P<0.05),the concentration of serum CTX-? concentration increased(P<0.01),OPG protein expression decreased(P<0.01),and RANKL protein expression increased(P<0.01).There was no statistical difference between sham operation group and blank control group.2.Compared with osteoporosis group,bone mineral density of Bushenhuoxue group increased significantly(P<0.05),bone mineral density increased further in DMAB group and combined treatment group(P<0.01).Calcification deposits in Bushenhuoxue group,DMAB group and combined treatment group were significantly reduced.The serum levels of P?NP and BGP in Bushenhuoxue group and DMAB group were significantly increased(P<0.05).The serum CTX-? concentration of bone resorption markers in DMAB group decreased significantly(P<0.01).The expression of OPG in Bushenhuoxue group,DMAB group and combined treatment group increased significantly(P<0.01),while the expression of RANKL decreased significantly(P<0.01).3.The increase of bone mineral density in the combined treatment group was greater than that in the Bushenhuoxue group(P<0.01),and the reduction of vascular calcification was better than that in the Bushenhuoxue group and the DMAB group.The expression of OPG was higher than that of Bushenhuoxue group and DMAB group(P<0.01),and the expression of RANKL was lower than that of Bushenhuoxue group and DMAB group(P<0.01).Conclusion: 1.There were obvious calcification deposits in the thoracic aorta of senile osteoporotic rats.2.Bushenhuoxue recipe,DMAB and Bushenhuoxue recipe combined with DMAB can effectively inhibit bone resorption,improve bone formation and inhibit vascular calcification,and the combined treatment group was superior to Bushenhuoxue recipe in improving bone mineral density,and the effect of inhibiting calcification is better than that of Bushenhuoxue recipe and DMAB alone.3.The effects of Bushenhuoxue recipe and DMAB alone or in combination on anti-senile osteoporosis and anti-vascular calcification may be through OPG/RANKL signaling pathway.