Effects Of Ipriflavone On Osteoporosis In Ovariectomized Rats And Its Possible Mechanisms Through NO Pathway

Objective: To investigate the effect of Ipriflavone(7-isopropoxyisoflavone, IP), a synthetic phytoestrogen,on prevention and treatment of osteoporosis in ovariectomized rats;Isolation, expansion, and culture the new born rat calvarid osteoblastic cell in vitro,then evaluate the effect of Ipriflavone on the proliferation and osteogenic differentiation of cultured rat osteoblast in vitro;Investigate the effect of Ipriflavone on nitric oxide (NO ) synsthesis and expression of endothelium nitric oxide synathase (eNOS) mRNA in cultured rat osteoblast in vitro. Observe the effect of Ipriflavone on synthesis of nitric oxide (NO) and nitric oxide synathase (NOS) inovariectomized rats. Thereby ascertain the effects of Ipriflavone on osteoporosis in ovariectomized rats and its possible mechanisms through NO pathway,provides the theory basis to clinical practice much better.Methods: (1) 60 six-month-old Sprague-Dawley female rats were randomly assigned to four groups: sham,ovx, ovx+IP(50 mg/kg/d),ovx+IP(100 mg/kg/d), ovx+IP(200 mg/kg/d),and ovx+17β-estradiol (E2) (10ug/kg/d). After 12 weeks, bone mineral density, bone histomorphometry, biomechanics of bone and the markers of bone metabolism were measured. Observe the prevention and treatment effect of Ipriflavone on postmenopausal osteoporosis. (2) Isolation, expansion, and culture the new born rat calvarid osteoblastic cell in vitro, identified by alkaline phosphatase staining and mineralized nodules. The osteoblasts were cultured with the medium containing Ipriflavone of different concentration.The proliferation,the activity of alkaline phosphatase(ALP) and calcium node were determined and compared with control.Observe the effect of Ipriflavone on the proliferation and differentiation of cultured rat osteoblast in vitro. (3) New born rat calvarid osteoblastic cell were isolated and cultured with the medium containing Ipriflavone of different concentration.After 72hours, the levels of NO in the media were detectetd and the mRNA expression of eNOS were examined by reverse-transcriptase polymerase chain reaction (RT-PCR). (4) The model of postmenopausal osteoporosis was established and divided into different groups use the same methodsof the first part. After 12 weeks, the the level of serum NO and NOS were detected in grouped rats. nitric oxide (NO) and nitric oxide synthase (NOS). expression of eNOS in the bone tissue was detected by immunohistochemical staining.Results: (1) The bone mineral density (BMD) of OVX in ovariectomized rats decreased significantly . The mechanical properties and biomechanics markers of femur changed obviously. Ipriflavone and estrogen can increase the BMD(P<0.01)and demonstrate a dose-dependent manner. Meanwhile flexural strengh and flexural elastic module of femur increase, but lower than the estrogen group. (2)Compared with the negative control, Ipriflavone could increase the quantity of osteoblast(p<0.01). Ipriflavone could elevate the activity of ALP and ability of calcification(p<0.01). 10-8～10-5mol/L ipriflavone had more significant effect than other concentration. (3) In comparison to the negative control, Ipriflavone could increase the concentration of NO in the media and the expression levels of eNOS mRNA (P<0.01)。10-8～10-5mol/L ipriflavone had more significant effect than other concentration . (4) In comparison to sham group ,The level of NO and NOS in ovariectomized(OVX) rats decreased significantly. Ipriflavone could significantly increase the level of NO and NOS comparison to OVX . There was no statistical significance of the difference between Ipriflavone and sham group. Estrogen groups obtained higher level of NO and NOS than other groups.Conclusions : Ipriflavone demonstrated significant protective effect on bone in ovariectomized rats,also can promote the proliferation and osteogenic differentiation of rat osteoblast, and there is a definite dose-response relationship. It's effect was weaker than estrogen. The effect of Ipriflavone on prevention and treatment of postmenopausal osteoporosis(PMOP) probably regulated through NO-NOS system.