The Study On Effect Of Yigukeli In Osteoporosis And Its Mechanism

ObjectiveTo investigate the healing effect of Yigukeli on ovariectomized (OVX) rats. And to investigate the mechanism by observing the effect of Yigukeli on proliferation and differentiation as well as the ability of alkaline phosphatase (ALP) synthesis of osteoblast (OB).Methods1. The effect of Yigukeli on OVX-induced osteoporosisThe female SD rats were randomized into sham group, model group, E2 group (0.0003 g · kg-1 · d-1) and Yigukeli at low, moderate, high dosage (0.4,0.8, 1.6 g · kg-1 · d-1). Ovariectomy were performed on all rats, except the sham group. Instead of ovary, fat (about 1g) around the ovary was cut in the sham group. lweek after the surgery, drugs were orally administrated 1 time daily. And the administration was last for 16 weeks.12,16 weeks later, bone mineral density (BMD) of rats’whole body were detected.14 days before rats’sacrifices, tetracycline hydrochloride was administrated (i.h.25mg/kg); 4 days before rats’sacrifices, calclein was administrated (i.h. 10mg/kg).1 hour after the last administration, the rats were anaethetized (phenobarbital i. p.30mg/kg) and blood samples were collected from abdominal aorta for analysis of serum calcium (Ca), phosphorus (P), ALP, osteocalcin (OCN) and tartrate resistant acid phosphatase 5b (TRACP5b). After blood samples collection, rats were sacrificed. Uterus, lumbar vertebra, femurs and left tibia were collected. Uterus histological slices were made to observe the effect of Yigukeli on uterus. Lumbar vertebra and right femur were used to measure BMD. Left femur was used to measure the biomechanical indexes (stain elastic modules, maximum bending stress, maximum strain, bone rigidity coefficient). Left tibia was used to observed the effect of Yigukeli on bone histomorphometric indexes.2. The effect of Yigukeli on OB proliferation rate and ALP synthesis abilityThe skulls were taken from the 2-day-old fetal rats. Then the skulls were digested with trypsin and collagenase I to get the primary OB. ALP staining was used to identify the culctured cells. The third generation of OB was treated with estradiol or Yigukeli drug-contained serum at different concentration. 48h and 72h after the treatment, the cell proliferation rate was detected with CCK-8 assay, and cellular ALP level was detected with P-nitrophenyl phosphate assay.Results1. The effect of Yigukeli on OVX-induced osteoporosis(1) Compared with model group, in the third month after administration, Yigukeli at high dose can increase the BMD of whole body significantly. In the forth month, Yigukeli at different doses have the same effect. Besides, Yigukeli at moderate and high dose can increase the BMD of femur and lumbar.(2) Compared with model group, Yigukeli at moderate dose can increase the serum Ca concentration. In addition, the moderate and high dose can lower the serum concentration of TRACP5b and OCN.(3) In the biomechanical test, Yigukeli at moderate and high dose can increase the maximum bending stress, maxium strain, bone rigidity coefficient.(4) In the tibia histomorphometric test, compared with the model group, Yigukeli at high dose can increase the percentage of trabecular, the moderate and high dose can decrease the trabecular separation. Besides, Yigukeli at the moderate and high dose can lower the percentage of periosteum double labeled perimeter, and each dose can also lower the periosteum formation rate.2. The effect of Yigukeli on osteoblast proliferation rate and ALP synthesis ability(1) Compared to the blank control group, OB proliferation rate increased significantly 72h after the treatment with Yigukeli-contained serum.(2) Compared to the blank control group, the ability of ALP synthesis of OB increased significantly 48h after the treatment with Yigukeli-contained serum.ConclusionBilateral ovariotomy is a classic way to build the osteoporosis model. Based on this model, we found that Yigukeli has an obvious therapeutic effect on osteoporosis. According to this study, Yigukeli have the ability to stimulate osteoblast proliferation and differentiation, to restore the bone resorption-formation balance, to decrease calcium excretion. Also, Yigukeli can keep trabecular in normal morphology and improve the bone biomechanical. These may be how Yigukeli treat osteoporosis.