Preventive Effect Of Puerarin On Disuse Osteoporosis Induced By Tail-suspension In Rats

Objective: To investigate the effect of puerarin on osteoporosis in tail-suspended rats,and to lay the foundation for the development of new anti-disuse osteoporosis drugs from Chinese medicine Pueraria.Methods: Thirty 2-month-old Wistar female rats were randomly fed for one week and randomly divided into control group(Con group,n=10),tail-suspension group(TS group,n=10)and tail-suspension + puerarin group(TS+Pur group,n = 10)3 groups.The TS group and the TS+Pur group were treated with a suspended tail to cause a disuse osteoporosis model.The TS+Pur group was administered with 15.4 mg /(kg·d)puerarin,and the Con group and the TS group were filled with the same volume.Distilled water.Rats in each group were treated separately in cages,diet and water were not restricted,and body mass was measured once a week.On the 13 th day,tetracycline was injected subcutaneously from the neck,30 mg/kg,and the next day was added.On the 23 rd day,calcein was injected at 8 mg/kg,and the next day was added.After 4 weeks,blood was collected from the abdominal aorta,centrifuged to obtain serum and frozen in a refrigerator at-80 °C.The main organs of the rats were removed,and the organ index was calculated after weighing.The formaldehyde solution was fixed and left for pathological observation.Lateral femur,humerus and vertebrae were measured by double-energy X-ray absorptiometry.Bone biomechanical analysis was performed by universal material testing machine.Bone morphometry analysis was performed with bone-grinding tablets.Biochemical indicators of serum bone metabolism were detected by ELISA.Results:1.Rat body weight results:During the experiment,the body weight of each group maintained a steady growth.No specific body mass changes were found in the rats.There was no statistically significant difference between the groups(P>0.05).2.Organ pathology test results:There was no significant difference in organ index between the groups(P>0.05),and no obvious abnormalities were found in the pathological examination of organs.3.Bone mineral density test results:Compared with Con,the bone mineral density of the femur,tibia and vertebrae in the TS group was significantly decreased(P<0.01).Compared with the TS group,the bone mineral density of the femur,tibia and vertebrae in the TS+Pur group was significantly increased(P<0.01).4.Bone biomechanical parameters test results:Three-point bending test results: Compared with Con,the maximum load and elastic modulus of the femur and tibia of the TS group decreased significantly(P<0.01);compared with the TS group,the maximum load of the femur and tibia of the TS+Pur group The elastic modulus values were all significantly increased(P<0.01).Vertebral compression test results: Compared with Con,the maximum load and elastic modulus of the fourth and fifth lumbar vertebrae(L4,L5)in the TS group were significantly decreased(P<0.01);compared with the TS group,the TS+Pur group The maximum load and elastic modulus of lumbar vertebrae in the same site were significantly increased(P<0.01).5.Bone morphology static metrology observation and analysis results:The results of VG staining and analysis of cancellous bone of tibial metaphysis: Compared with Con,the number of trabecular bone in the TS group was significantly reduced,the thickness of the trabecular bone was significantly thinner,and the bone separation was significantly larger,which was significantly different(P<0.01);Compared with the TS group,the number of trabecular bone of TS+Pur increased significantly,the thickness of trabecular bone became thicker,and the bone separation degree became significantly smaller(P<0.01).The results of VG staining and analysis of cancellous bone of vertebral vertebra : Compared with Con,the number of trabecular bone in the TS group was significantly reduced,the thickness of the trabecular bone was significantly narrowed,and the bone separation was significantly larger(P<0.01).Compared with the TS group,the number of trabecular bone of TS+Pur increased significantly,the thickness of trabecular bone was significantly widened,and there were statistical differences(P<0.01),while the bone separation degree was significantly smaller,with statistical difference.(P<0.05).6.Bone morphology Dynamic metrology observation and analysis results:The results of double fluorescence labeling and analysis of the middle tibial cortical bone: Compared with Con,the fluorescence interval of the TS group was significantly smaller,Mineral apposition rate is significantly reduced(P<0.01).Compared with the TS group,the fluorescence interval of the TS+Pur group was significantly larger,Mineral apposition rate is significantly increased(P<0.01).The results of double fluorescence labeling and analysis of vertebral cortical bone : Compared with Con,the fluorescence interval of TS group was significantly smaller,Mineral apposition rate is significantly reduced(P<0.01).Compared with TS group,the fluorescence interval of TS+Pur group was significantly larger,Mineral apposition rate is significantly increased(P<0.01).7.Serum biochemical indicators test results:Compared with Con,the TS group showed osteocalcin(Osteocalcin,OC)and(Procollagen I N-terminal peptide,PINP),and the bone absorption index was resistant to tartrate acid phosphatase 5b.(Tartrate-resistant acid phosphatase 5b,TRACP-5b)and collagen type C-terminal peptide(Collagn I C-terminal peptide,CTX-I)increased significantly,the difference was statistically significant(P<0.01);compared with TS group The contents of OC and PINP in TS+Pur group increased significantly(P<0.05),and the contents of TRACP 5b and CTX-I decreased significantly(P<0.01).Conclusion:1.The bone metabolism indexes of the tail-suspension rats model established in this experiment are consistent with the disuse osteoporosis,indicating that the model successfully simulated bone loss caused by prolonged bed rest or space flight;2.From the experimental results,the bone density of the femur,tibia and vertebrae decreased by 13.19%,19.03% and 11.17%,respectively,after the tailsuspension of the experimental rats,while the bone mineral density of the rats in the puerarin was only decreased by 7.90%.13.11%,6.23%,indicating that puerarin can partially prevent bone loss in rats with disuse osteoporosis,and has potential value for developing new anti-disuse osteoporosis drugs;3.Analysis of serum biochemical markers of bone metabolism showed that the bone resorption index of puerarin group was lower than that of the tail-suspension group and the bone formation index was higher than the latter,indicating that puerarin is the duality of inhibiting bone resorption and promoting bone formation.The activity exerts an anti-osteoporosis effect.