| Objective:Osteoporosis is a skeletal disorder characterized by a decrease in bone mass,micro-architectural deterioration of bone tissue and compromised bone strength leading to bone fragility and an increased risk of fracture.The most common complication resulted from osteoporosis is fracture,particularly those of the hip and spine.Osteoporosis has become a worldwide health problem.In 2000,the global incidence of osteoporotic fractures among women and men was estimated at 9.0 million,and the number of new osteoporotic fractures (especially of the hip) increases with aging.Notably,nearly 75% of all hip fractures occur in women.Worldwide, the lifetime risk of a woman dying from a hip fracture is higher than the total risks of death from breast cancer, endometrial carcioma and ovarian cancer,making postmenopausal osteoporosis a major public hazard.Estrogens deficiency,through natural or surgical menopause,plays a key role in the occurrence of postmenopaus- al osteoporosis in women.Menopause stimulates an increase in the remodeling rate of bone tissue,leading to an imbalance between the resorption and formation of bone,and results in accelerated bone loss.It is still not clear exactly how these processes are mediated,but it is known that estrogen deficiency is associated with the production of a variety of pro-resorptive cytokines that favor osteoclastogenesis and inhibit osteoclast apoptosis.In bone,The inducible prostaglandin synthesis enzyme,cyclooxygenase-2(COX-2),produced mainly by osteob- lasts and osteoclast precursors,which is involved in bone resorption and osteoclastogenesis,and functions indirectly through prostaglandinE2(PGE2) produced by osteoblastic cells.PGE2 acts as a potent stimulator of bone resorption.The withdrawal of estrogen,through natural or surgical menopause, appears to lead to a PGE2-dependent pro-inflammatory state characterized by bone loss in vivo.Nuclear factor-kappa B is a pleiotropic transcription factor,which regulates osteoclast formation,function and survival.The research indicates that NF-κB can activate the transcription of COX-2 in vitro and stimulate the differentiation of osteoclast,which will increase the number of osteoclast and accelerate bone absorption.We presume that NF-κB–dependent COX-2 expression plays a key role in osteoclast differentiation via PGE2 production in vivo, therefore inhibition of the COX-2 enzyme by suppression of NF-κB activation in postmenopausal women may prevent menopausal bone loss.We used overiectomized adult rats in our study to establish exprimental postmenopausal osteoporosis.The goal of the present study was to investigate the molecular mechanism of selective cyclooxygenase-2 inhibitor–Celecoxib and whether it could be able to reduce bone loss.We examine the expression level of NF-κB p65 and COX-2 and their dependence in rat bone tissue in each experimental group,explore the molecular mechanism of postmenopausal osteoporosis and develop specific medical intervention strategies to treat postmenopausal osteoporosis.Methods:1 The grouping and duplication of model of postmenopausal osteoporosis40 female Sprague/Dawley rats at the age of 3 month were randomly divided into 4 groups(10 in each group):①S ham- operated(Sham);②O VX;③O VX treated with Nilesteriol(OVX+ E);④OVX treated with Celecoxib(OVX+C).Rats were given an intraperitoneal injection of 10% Chloral Hydrate (100mg/kg) before surgery.Under aseptic condition,rats were open from the mid-line of back into the backside of the abdominal cavity,then remove ovaries for rats in group 2-group 4 and an equivalent weight of adipose tissue for rats in group 1.One week after surgery,rats in group 3 were treated with Nilesteriol at 1.0mg/kg per week,rats in group 4 were treated with Celecoxib at 4.0 mg/kg per day,while rats in group 1 and group 2 were treated with an equivalent volume of saline per day.The dosages of drugs were proofread per week in accordance with their body weights.2 The establishment of postmenopausal osteoporosis model through dual-energy X-ray absorption method12 weeks after surgery,the bone density of the femurs were measured by the equipment of dual-energy X-ray bone density. 3 Hematoxylin-eosin stain and immunohistochemistry stainThe NF-κB p65 and COX-2 levels in bone tissue were determined by immunohistochemistry techniques.After sacrificing the rats,we obtained the inferior extremity of femurs by sterile operation and performed hematoxylin-eosin stain and immunohistochemistry stain.4 Result and image analysisWe applied One-Way ANOVA using Statistical Product and Service Solutons 13.0(SPSS13.0) for data analysis.All values were demonstrated by average±standard deviation(X-±S).And two-two comparison among the means were done by Student-Neuman-Keuls.P < 0.05 was considered statistically significant.Results:1 The body weightCompared with the sham-operated group,the body weight were gained in OVX group and in OVX+C group(P<0.05;P<0.05),while there were no significant differences in OVX+E group(P>0.05);Compared with OVX group,the body weight were lost in OVX+E group and in OVX+C group(P<0.05,P<0.05);Compared with OVX+E group,there were no significant differences in OVX+C group(P>0.05)2 The bone densityCompared with the sham-operated group,the BMD of the femur were reduced in OVX group and in OVX+C group(P<0.05,P<0.05),while there were no significant differences in OVX+E group(P>0.05);Compared with OVX group,the BMD of the femur were increased in OVX+E group and in OVX+C group(P<0.05,P<0.05).Compared with OVX+E group,there were no significant differences in OVX+C group(P>0.05).3 Histology observationCompared with sham-operated group,a few changes were found in the OVX group:rarefaction of trabecular,the narrowing width of trabecula and the broadening interval among trabecula. The OVX+E group share similar changes with sham-operated group, and the changes in OVX+C group is between sham-operated group and the OVX group.4 The immunohistochemistry stain and observationThe immunohistochemistry results showed that the positive expression of NF-κB p65 and COX-2 were found in all four groups.NF-κB p65 mainly expresses in the cytoplasm of osteoblast,osteocytes and stroma cell,while COX-2 mainly in the cytoplasm of osteoblast.5 The result of immunohistochemistry stainCompared with the sham-operated group,the content of protein of NF-κB p65 and COX-2 were higher in OVX group and OVX+C group(P < 0.05,P < 0.05) and (P < 0.05,P <0.05),while there were no significant differences in OVX+E group(P > 0.05,P < 0.05);Compared with OVX group,the content of protein of NF-κB p65 and COX-2 were lower in OVX+E group and in OVX+C group(P<0.05,P<0.05) and (P<0.05,P<0.05);Compared with OVX+E group,the content of protein of NF-κB p65 and COX-2 were higher in OVX+C group(P<0.05,P<0.05).6 The correlation analysis resultThe NF-κB p65 and COX-2 levels are negatively correlated with the bone mineral density in OVX group and in OVX+C group. At the same time,there are a positive correlation between NF-κBp65 and COX-2 in OVX group and in OVX+C group;There is no correlation between the expression of NF-κB p65 or/and COX-2 with bone mineral density in Sham group and in OVX+E group.Conclusions:1 Ovariectomized rats successfully established model of postmenopausal osteoporosis;2 OVX could increase the body weight and it may partly compensate the bone loss ;3 NF-κB–dependent COX-2 expression plays a key role in ovariectomy–induced bone loss via PGE2 production in rats;4 Celecoxib could be a useful therapeutic agent for the prevention of ovariectomy–induced bone loss;5 The mechanism of preventing bone loss by Celecoxib is to inhibit the COX-2 enzyme by suppression of NF-κB activation in ovariectomized rats.
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