Effects And Mechanisms Of BGS3,An Extractive Of Fructus Psoraleae In Rats With Glucocorticoid-induced Osteoporosis

Objective:Osteoporosis(OP)is a systemic bone disease,characterized by reduction of bone tissue content and damage of bone microstructure,resulting in the decrease of the bone strength,the increase of bone fragility,and thus susceptibility to fracture.Osteoporosis can be divided into primary osteoporosis and secondary osteoporosis.With the wide clinical application of glucocorticoids(GC),the incidence of glucocorticoid induced osteoporosis(GIOP),an adverse reaction of glucocorticoid,is continually rising.Its incidence ranks third after postmenopausal osteoporosis and senile osteoporosis and it ranks first place in the secondary osteoporosis.Owing to the complicated pathogenesis of GIOP,at present there are no specific agents,and GIOP is generally treated with the treatment of primary osteoporosis.But in terms of some drugs to treat primary osteoporosis,there is no convincing evidence to prove that some drugs have curative effect on GIOP.and there are different degrees of security problems in drugs for GIOP treatment,such as Bi-phosphate,calci-tonon,parathyroid hormone,etc.Therefore,the research and development of safe and effective new agents to prevent and treat glucocorticoid-induced osteoporosis is of great importance.Phytoestrogens(PE)is a kind of natural nonsteroidal compound which exists in plants,fruit and vegetables.Both its structure and biological activity are similar to those of estrogen:they have both estrogen activity and anti-estrogen activity.Three categories of PE(isoflavones,lignans and coumarins)were the most concerned.Fructus Psoraleae is the dry and mature fruit of the leguminous plant Psoralea corylifolia L.and its function is to invigorate the kidney and strengthen yang.Its main ingredients are psoralen and isop-soralen.BGS3,an extractive of fructus psoraleae which belongs to coumarins phytoestrogens.The preliminary studies of the research group have indicated that BGS3 could promote the proliferation and differentiation of rat osteoblast in vitro and prevent rat osteoporosis induced by ovariectomy,slow down the rate of bone turnover and enhance the bone mineral density in vivo.But the curative effects of BGS3 on low conversion type osteoporosis remain to be seen.The study,by establishing osteoporosis models of rats with glucocorticoid induced osteoporosis,investigates the antiosteoporotic activity of BGS3 on rats with glucocorticoid induced osteoporosis and explores its possible mechanisms,in order to provide theoretical and scientific foundation for the research of BGS3 on prevention and cure of glucocorticoid induced osteoporosis.Methods:A total of 60 female Sprague-Dawley(SD)rats were randomly divided into Control group(Con),Dexamethasone group(Dex group),Hong Lilai group(HLL group),low dose BGS3 group(BGS3-L group),middle dose BGS3 group(BGS3-M group)and high dose BGS3 group(BGS3-H group),with 10 rats in each group.Con group was given equal normal saline by intramuscular injection,and other groups were given dexamethasone(Dex)2 mg/kg,intramuscular injected two times each week.HLL group was given 0.06 mg/kg,Hong Lilai conjugated estrogens,BGS3-L group 3 mg/kg BGS3,BGS3-M group 6 mg/kg BGS3 and BGS3-H group 12 mg/kg BGS3.The agents were intragastrically administrated 6 days per week for 25 weeks.After a day of fasting,the rats in all the groups were each put into metabolic cages.The fasting continued,but drinking water was offered freely.24h urine was collected and urine volume was recorded.Urine samples were analyzed to measure urinary calcium(Ca),urine phosphorus(P),creatinine(Cre)and hydroxyproline(Hop).After a night of fasting,dioestrous rats were anatomized.Blood was drawn from abdominal aorta,and then its uterine wet weight was measured.The right femurs,tibias and fifth lumbar vertebraes were collected to measure bone mineral density(BMD).Serum samples were isolated to measure acid phosphatase(ACP),tartrate-resistant acid phosphatase(StrACP),total alkaline phosphatase(ALP),bone alkaline phosphatase(b-ALP),bone gla protein(BGP),calcium(Ca),phosphorus(P),parathyroid prime(PTH),calcitonin(CT),estradiol(E2)and transforming growth factor ?(TGF-?).Results:1 Body and uterine weightThe body weights of each group were not significantly different at the start of the study.After intramuscular injection of dexamethasone,in the first two weeks body weight of rats in Dex group and each treatment group decreased;and in the third week,the weight began to recover,then in the fourth week,the weight recovered to the initial level;since then the weight showed a gradually increasing trend.While body weight of rats in Con group continued to grow.After two weeks of administration,there was a statistically significant difference in body weight between rats in Dex group and in each treatment group and rats in Con group(P<0.01),and the difference remained until the end of the experiment.Compared with the Con group,Dex group had a significantly lower uterine weight level(P<0.01).Compared with the Dex group,uterine weight of each treatment group was higher.The uterine weight increase of BGS3-L group and BGS3-H group was statistically significant(P<0.05).Compared with the Con group,the unterine index decrease of Dex group was not significant.Compared with the Dex group,the uterine index of each treatment group increased and uterine index increase of BGS3-L group and BGS3-H group was statistically significant(P<0.05).2 Bone mineral densityCompared with the Con group,Dex group right tibia BMD showed a decreasing tendency,and the right femoral and vertebral BMD decreased significantly(P<0.01),suggesting that glucocorticoid induced osteoporosis model of rats was successfully established.After 6 months of treatment,compared with the Dex group,the right femur,tibia and vertebral BMD of each treatment groupincreased.Among them,the vertebral BMD of HLL group increased significantly(P<0.01);femur and vertebral BMD of BGS3-L group increased significantly(P<0.01);tibial and vertebral BMD of BGS3-M group increased significantly(P<0.01);the femur and tibia BMD of BGS3-H group increased significantly(P<0.01);the vertebral BMD of BGS3-H group increased significantly(P<0.05).The results suggested that medications for each treatment group had a therapeutic effect on glucocorticoid induced osteoporosis in rats.3 Calcium and phosphorus metabolism3.1 Calcium regulating hormone3.1.1 Serum parathyroid hormoneCompared with the Con group,Dex group rats serum PTH decreased significantly(P<0.01);compared with the Dex group,serum PTH levels decreased in all treatment groups,but only the BGS3-H group serum PTH concentration decreased with statistical significance(P<0.01),while the decrease of serum PTH of the other treatment groups had no statistical significance.3.1.2 Serum calcitoninCompared with the Con group,serum CT of Dex group showed a decreasing tendency;compared with the Dex group,serum CT levels in each treatment group decreased in different degrees.The HLL group rats serum CT decreased significantly(P<0.01),and the serum CT decrease of the BGS3-L group also had statistical significance(P<0.05),while the change of CT in the other groups was not significant.3.2 Serum calcium and phosphorusCompared with the Con group,Dex group Ca concentration showed a increasing trend;serum Ca concentration in BGS3-L group and BGS3-H group was higher than the other groups,but there was no significant difference between the groups.Compared with the Con group,the serum P concentration of the Dex group showed an increasing trend.Compared with the Dex group,the serum P concentration in each treatment group showed an increasing tendancy,but there was no statistically significant difference in serum P concentration.3.3 Calcium and phosphorus in urineThere was no difference in the concentration of Ca and P in urine in each group.4 Biochemical markers of bone turnover4.1 Biochemical markers of bone formation4.1.1 Serum alkaline phosphataseCompared with the Con group,the serum ALP in the Dex group was significantly higher(P<0.05);compared with the Dex group,the ALP in the HLL group,BGS3-L group and BGS3-H group showed an increasing trend,and the ALP in the BGS3-M group showed a decreasing trend,but the change of ALP in each treatment group was not obvious.4.1.2 Serum bone alkaline phosphataseCompared with the Con group,Dex group rats serum b-ALP increased;compared with the Dex group,HLL group and BGS3-M group b-ALP significantly decreased(P<0.05),and BGS3-L group and BGS3-H group serum b-ALP increased significantly(P<0.01).4.1.3 Serum osteocalcinCompared with the Con group,Dex group serum BGP concentration showed a decreasing trend.HLL group serum BGP concentration was slightly higher than the other groups,and the BGP concentration change of rat in each group was not obvious.4.2 Biochemical markers of bone resorption4.2.1 Serum acid phosphataseCompared with the Con group,the serum ACP of Dex group increased significantly(P<0.01);compared with the Dex group,the serum ACP concentration increase of BGS3-L group,BGS3-M group and BGS3-H group was statistically significant(P<0.01)4.2.2 Serum tartrate resistant acid phosphataseCompared with the Con group,the increase of serum StrACP in the Dex group was significant(P<0.01);compared with the Dex group,the serum StrACP of HLL group,BGS3-L group,BGS3-M group and BGS3-H group increased significantly(P<0.01)4.2.3 Urine hydroxyprolineThere was no significant difference in urine hydroxyproline concentration between the groups.5 EstrogenThe estrogen concentrations in the Con group and the HLL group were slightly higher than other groups,but there was no significant difference between all the groups.6 TGF-?Compared with the Con group,Dex group TGF-? showed an increasing trend;compared with the Dex group,the TGF-? of HLL group decreased significantly(P<0.05)and the TGF-? of BGS3-L group,BGS3-M group and BGS3-H group decreased significantly(P<0.01).Conclusions:1 BGS3 can prevent glucocorticoid induced osteoporosis in rats.2 The mechanism of action of osteoporosis prevention and treatment by BGS3 may be:through regulating bone resorption and bone formation,BGS3 can adjust the rate of bone turnover,and thus increase the bone mineral density.3 BGS3 has a regulatory effect on parathyroid hormone,calcitonin and TGF-? in rats.4 The mechanism of action of BGS3 prevention and treatment of osteoporosis is not same with that of estrogen.