Study On Pathogenesis Of Postmenopausal Osteoporosis And Effect Of Jiangu Granule Based On OPG/RANKL/RANK System

ObjectiveTo clarify the relationships between osteoporosis of ovariectomized rats and OPG/RANK/RANKL system, and reveal the mechanism of Jiangu Granule preventing postmenopausal osteoporosis from the aspect of osteoclast differentiation and function.Methods1Dynamic changes of OPG/RANKL expression in ovariectomy-induced osteoporotic rats and effects of Jiangu Granule treatment.1.1Animal experiments:160female SD rats of6months old were randomly divided into4groups:sham operated, Ovariectomized, ovariectomized+estrogen and OVX+Jiangu Granule,40each group. Each group underwent bilateral ovariectomy exepted for the sham group, which were subjeted to sham surgery.3days after operation, OVX+Jiangu Granule group were given Jiangu Granule2g·kg-1·d-1, estradiol17p-estradiol group received100μg·kg-1·d-1, sham operation group and model group were given saline gavage. After2,6,12and24weeks,10rats of each group were sacrificed, the serum, femur, tibia and lumbar spine bone were harvested.1.2Detection of indexes:bone density was measured by dual-energy X-rays; three-point bending method were used in femur biomechanical testing; tibia were made paraffin setions, trabecular area and the amount of osteoclasts were measured after MAS SON staining and TRAP staining; serum TRAP, BALP, OC, RANKL and OPG levels were detected by ELISA; lumbar were extracted mRNA and protein, RANKL/OPG mRNA expression was detected by real-time PCR, and the protein was detectd by Western Blot assay.2The effect of Jiangu Granule on osteoclast differentiation and RANK signaling. pathway2.1Preparation of serum-containing drugs:20male SD rats of3months old were divided into Jiangu Granule group and control group,10each. Jiangu Granule group received medicine of2g·kg-1·d-1, the control group received saline2ml·d-1for7days.1h after the last gavage, abdominal aortic blood was harvested and centrifugated after solidification to obtain the serum.2.2Cell culture and index detection:osteoclast precursor cell line RAW264.7were cultured and separated into3groups:negative control group, blank serum and drug serum group. Negative control group without RANKL, blank serum and drug serum group containing with50ng/ml RANKL, and10%blank serum or drug serum. After6days culture, the cells were TRAP stained to calculate differentiated osteoclasts;24,48and96hours after the treatment, cells were extracted RNA and proteins, real-time quantitative PCR and Western Blot assay were applied to detect mRNA and protein expression of RANK and TRAF6, NF-κB (P50and P52subunits), c-Fos and NFATc1.Results1Compared with the sham-operated group, bone mass and biomechanical indicators of model group continued to decline, which indicates the success of osteoporosis model.2-12weeks post operation, bone osteoclasts, indicators of bone remodeling including serum TRAP, serum BALP and OC of the model group were significantly increased, and estrogen replacement therapy can reverse the decline of bone density and the increase of bone biomechanical indicators induced by ovariectomy. Compared with the sham operated group, bone RANKL in model group increased at2week post OVX, after which they fell to near the level of the sham group. OPG of bone model group decreased sharply at2week post OVX, and subsequently increased, but compared with the same period of sham control group, bone OPG in model group consistently lower; Serum OPG in OVX group was significantly lower than that in the sham group during6-12week post OVX. Estrogen can relieve bone and serum RANKL/OPG changes after ovariectomy. Correlation analysis showed a positive correlation between bone RANKL and osteoclast resorption indicators, but no correlation in RANKL levels between bone and serum. There was a positive correlation in OPG levels between bone and serum, while a negative correlation between bone OPG level and bone resorption markers.2Jiangu granule treatment can significantly increase bone density and bone strength in ovariectomized rats, and reduce osteoclasts volume and indicators of bone remodeling including serum TRAP, BALP and OC. Jiangu Granule can reduce bone RANKL expression, and increase OPG expression in bone and serum of ovariectomized rats.3RANKL stimulation increased mRNA and protein expression of NF-κB (P50), c-Fos and NFATc1, thus induced osteoclast differentiation; Jiangu Granule containing serum can reduce the rate of osteoclast differentiation induced by RANKL, and reduce mRNA and protein expression of RANK, c-Fos and NFATc1.Conclusions1RANKL and OPG showed nonlinear variations with time post ovariectomy in rats. Serum OPG level can be used as an indicator of postmenopausal osteoporosis disease.2Jiangu Granule may regulate the expression of RANKL and OPG to affect bone remodeling, so as to prevent and treat osteoporosis induced by ovariectomy.3Jiangu Granule may supress osteoclast differentiation in vitro by regulate RANK, c-Fos and NFATc1expression.