The Effects Of Naringin On Prevention Of Disuse Osteoporosis And The Mechanisms Involved In

Objective:1. To investigate whether naringin has a role in the prevention of disuse osteoporosis.2. To investigate whether up-regulation of periostin and semaphorin3 A and subsequent activation of Wnt/β-catenin signaling pathway is involved in the osteoprotective effect of naringin.3. To investigate whether periostin-induced activation of Wnt/β-catenin signaling pathway is implicated in the role of naringin in promoting osteoblast differentiation.Methods:1. Ninety six-month-old male Sprague-Dawley rats were randomly divided into five groups: the control group, the hindlimbs suspension group(HS), 30mg·kg-1·d-1, 100mg·kg-1·d-1 and 300mg·kg-1·d-1 naringin-treating group. The disuse osteoporosis models of the latter four groups were induced by tail suspension. Naringin was administered four weeks before and four weeks after operation. Rats in the control group and HS group received equivalent saline as vehicle. Four weeks following operation, all rats were sacrificed, with femurs tested for biomechanical properties, bone mineral density(BMD), scanned by Micro-CT, stained by haematoxylin adenosine(HE), osteocalcin(OCN) and tartrate-resistant acid phosphatase(TRAP). The harvested serum were tested for Cross-linked C-terminal telopeptides of type I collagen(CTX-1) and amino-terminal propeptide of type 1 procollagen(P1NP).2. The m RNA and protein level of semaphorin3 A, periostin, sclerostin and β-catenin in tibias were analyzed by q RT-PCR and Western-blot respectively. Harvested femurs were immunohistochemical stained for Semaphorin3 A, periostin and sclerostin.3. MC3T3-E1 cells were divided into five groups: the control group, 0.2ng/ml、2ng/ml、20ng/ml and 100ng/ml naringin-treating groups. The ALP activities were measured to determine optimal concentration of naringin in promoting osteoblastic differentiation. The l of osteoblastic differentiation markers BMP-2、OCN and Runx2 were analyzed by q RT-PCR. Expression of Semaphorin 3A, periostin and β-catenin were analyzed by q RT-PCR and Western-blot. Periostin expression by osteoblasts was inhibited by the periostin affinity purified polyclonal antibody to further confirm periostin was involved in effect of naringin on enhancing osteoblast differentiation.Results:1. Compared with the control group, femoral mechanical properties, BMD and trabecular microstructure in HS group decreased significantly, the serum level of CTX-1 increased while P1 NP decreased markedly, and the immunohistochemical reactivity of OCN decreased while the number of TRAP-positive osteoclasts increased. Naringin at 100mg/kg and 300mg/kg significantly alleviated immobilization-induced deterioration of femoral mechanical properties, BMD and trabecular microstructure, significantly decreased CTX-1 level and increased P1 NP in serum, and increased immunohistochemical reactivity of OCN and decreased the number of TRAP-positive osteoclasts compared to the HS group.2. Results from PCR, Western-blot and immunohistochemistry indicated that semaphorin3 A, periostin and nuclear β-catenin in tibias of HS group decreased while sclerostin increased compared to the control group. Semaphorin3 A, periostin and nuclear β-catenin in tibias increased while sclerostin decreased in the 100mg/kg and 300mg/kg naringin-treating group compared to the HS group.3. Compared to the control group, naringin at 20ng/ml showed the optical effect on promoting osteoblast differentiation, which significantly increased the m RNA level of BMP-2, OCN, Runx2, semaphorin3 A, Nrp1, periostin and β-catenin. Western-blot indicated that naringin at 20ng/ml increased the expression of Semaphorin3 A, periostin and nuclear β-catenin. The periostin polyclonal antibody could inhibit naringin-induced osteoblastic differentiation.Conclusion:1. Naringin could prevent the progress of disuse osteoporosis in rats.2. The osteoprotective role of naringin may be mediated by the increased periostin and semaphorin3 A expression, and subsequent activation of Wnt/β-catenin signaling.3. Naringin could promote the production of periostin, which then stimulate the activation of Wnt/β-catenin signaling to promote osteoblastific differentiation.