The Experimental Study Of Crocin On The Treatment Of Postmenopausal Osteoporosis

BackgroundOsteoporosis is defined as a systemic skeletal disease characterised by reduction in bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fractures. Osteoporosis has been a major public health problem for all over the world because of its high morbidity, serious complications, and weighty economy burden. In addition, woman is more susceptible to Osteoporosis, named Postmenopausal Osteoporosis(PMO), in the result of her lower peak bone mass and decreasing estrogen level after menopause. Although universal anti-Osteoporosis drugs, belonging to replacement therapy, may prevent progression of bone loss in established Osteoporosis, their costs are too high to fit a large population in the world, especially in developing countries, not to mention their side effects and poor compliance for long time application. Consequently, there are efforts to search for new drugs available for a modest price that not only improve therapeutic efficacy, but also have less undesirable side effects. The Chinese traditional herb medicine has raised concerns of researchers for its comprehensive management, little side effects, and reasonable price. Studies have indicated that Crocin has potential anti-Osteoporosis effects by improved inflammatory and oxidative status. ObjectiveThe purpose of this study is to examine the effects of Crocin on OVX-induced osteoporosis in rats,proliferation and apoptosis of osteoblast in vitro, and furthermore attempt to explore its possible mechanisms. Methods1. Animal experiment. Sixty female sprague-dawley(SD) rats were randomly assigned into sham-operated group(Sham) and five ovariectomy(OVX) subgroups, i.e. OVX with saline(OVX); OVX with 17β-estradiol(E2, 25μg/kg/day); OVX with Crocin of graded doses(5, 10, or 20 mg/kg/day). Each group consisted of 10 rats. Daily oral administration of E2 or Crocin started 4 weeks after OVX for 16 weeks. Bone mass, bone strength and bone turnover were evaluated by DEXA, three-point bending test(MTS 858 Mini Bionix II), and biochemical markers(Ciba-Corning 550, USA). The microarchitecture of trabecular bone was examined by Micro-CT(GE Healthcare, USA). Activity of SOD was detected by xanthine oxidase(OX) technique and content of MDA was detected by thiobarbituric acid(TBA) technique.2. Cell experiment. Cells were delievered and cultured from fetal SD rat calvaria by enzymes digesting and tissue block methods. Osteoblasts were identified with alkaline phosphatase(ALP) staining and Alizarin red staining of calcified nodules. The 3rd generation of osteoblasts were used in laboratory experiments.(1) Cell proliferative assay. After proliferation stabilized, the 3rd generation rat’s osteoblasts were grouped to receive the following four treatment separately:(1) Control;(2) Crocin at low dosage(1mmol/L);(3) Crocin at middle dosage(2mmol/L);(4) Crocin at high dosage(4mmol/L). MTT method was used to test the proliferation of osteoblasts. The activity of ALP was assayed by PNPP method.(2) Research of apoptosis. TNF-α was used as apoptosis inducer. The 3rd generation of osteoblasts were cultured and divided into five groups: Control group, TNF-α group, TNF-α with Crocin of graded doses(1, 2, or 4 mmol/L). The activity of Caspase-3 was examined by spectrophotometry. Flow cytometry was performed to analyze cell apoptosis rate. Activity of SOD in cell lysate was detected by xanthine oxidase(OX) technique and content of MDA was detected by thiobarbituric acid(TBA) technique. Results1. Animal experiment. Crocin prevented BMD decrease in the femurs and L4 vertebras induced by OVX(P<0.05), which was accompanied by a significant decrease in skeletal remodeling, as was evidenced by the decreased levels of bone turnover markers, such as serum alkaline phosphatese(ALP), osteocalcin(OC), and urinary Ca and P excretions(P<0.05). Higher dosage of Crocin treatments altered maximum load, maximum stress and Young’s modulus(P<0.05), and significantly reduce the deterioration of trabecular bone microarchitecture in L4 vertebras. Crocin also showed effects of enhancing SOD activity and decreasing MDA content(P<0.05).2. Cell experiment. The Osteoblasts grew well and its purity was completely satisfied for experiment.(1) Cell proliferative assay. Crocin at higher dosage could promote the proliferation and ALP activity compared to control group(P<0.05).(2) Research of apoptosis. Comparing with TNF-α group, Crocin at higher dosage could decrease Caspase-3 level(P<0.05), inhibit osteoblast apoptosis(P<0.05), stimulate activity of SOD(P<0.05), and decrease MDA content(P<0.05). ConclusionsThe results demonstrate that Crocin could prevented OVX-induced bone loss, benefit bone strength, reduce bone turnover rate, and improve bone microarchitecture. What is more, Crocin could promote osteoblast proliferation and protect osteoblast from TNF-α induced apoptosis. So Crocin may be an alternative medicine for the treatment of Postmenopausal Osteoporosis. Such effects can be explained partially by improved inflammatory and oxidative status. Further studies are required to explore its exact mechanisms of action.