柚皮苷激活Wnt／β-catenin信号通路预防废用性骨质疏松

Stimulation of Wnt/β-catenin signaling pathway is involved in the effect of naringin on the prevention of disuse osteoporosis

目的探究柚皮苷对大鼠废用性骨质疏松的预防作用及其机制。方法采用后肢悬吊法制备废用性骨质疏松模型，分为单纯造模组和低、中、高剂量柚皮苷预防组（30mg·kg^-1·d^-1，100mg·kg^-1·d^-1和300mg·kg^-1·d^-1）；另取等量大鼠作为正常对照组。造模4周后处死各组大鼠取材，双能X线吸收法检测股骨远端骨密度（bonemineral density，BMD），Micro-CT分析股骨远端骨小梁微结构，骨生物力学性能测试检测股骨力学性能变化，ELISA法检测血清骨代谢指标，qRT—PCR检测胫骨骨膜素（Postn）、骨硬化蛋白（Sost）和β-catenin的mRNA水平，Western—blot检测胫骨骨膜素、骨硬化蛋白和细胞核β-catenin蛋白含量，并对股骨行骨膜素和骨硬化蛋白免疫组织化学染色。结果造模4周后，单纯造模组比正常对照组股骨远端BMD和股骨力学性能明显下降，股骨远端骨小梁微结构破坏明显，血清Ⅰ型前胶原氨基端肽（amino-terminal propeptide of type-1 procollagen，P1NP）明显下降，Ⅰ型胶原交联氨基末端肽cross—linked C—terminal telopeptides of callagen type-1，CTX-1）明显上升，qRT-PCR和Western-blot结果显示胫骨骨膜素表达水平下调，骨硬化蛋白表达上调，细胞核B—catenin含量减少，免疫组织化学染色结果显示股骨远端骨外膜成骨细胞分泌的骨膜素减少，而股骨干骨细胞骨硬化蛋白阳性程度上升。中、高剂量柚皮苷明显减弱悬吊导致的股骨远端BMD和股骨力学性能下降，以及骨小梁微结构破坏，上调血清P1NP水平，下调CTX-1含量，并明显增加骨组织中骨膜素和细胞核β-catenin的表达量，减少骨硬化蛋白的表达水平。结论柚皮苷能预防废用性骨质疏松发生，其机制可能是通过上调骨膜素，进而抑制骨硬化蛋白表达，最终激活Wnt／β-catenin信号通路所介导。

Objective To investigate whether naringin has a role in the prevention of disuse osteoporosis and the mechanisms that may be involved. Methods Disuse osteoporosis in male SD rats was induced by tail suspension, which were then randomly divided into four groups as following： the Hindlimb Suspension group （HLS）, 30 mg. kg^-1. d^-1, 100 mg. kg^-1. d^-1 and 300 mg. kg ^-1 . d^-1 Naringin-treating group. Rats in the control group and suspension only group received equivalent saline as vehicle. Another equal amount of rats was selected as the Control group. Four weeks following the operation, all rats were sacrificed. Dual energy X-ray absorptionmetry （DXA） was used to analyze the distal femoral BMD, Micro-CT was used to test the trabecular microstructure in distal femur, biomechanical tests were used to analyze femoral mechanical properties and bone tumover markers in serum were tested by ELISA. The expressions of periostin, sclerostin and nucleus β-catenin in tibias were evaluated by qRT-PCR and Western- blot. The femurs were immunohistochemical stained for periostin and sclerostin. Results Four weeks after operation, BMD of distal femurs in HLS group decreased significantly compared with the control group, together with the dramatical deterioration of distal femoral trabecular microstructure and femur mechanical properties. The serum level of CTX- 1 increased significantly while that of P1NP decreased. PCR and Western-blot indicated that the expression of periostin and nucleus β-catenin in tibias decreased dramatically, while sclerostin increased. The periostin secreted by periosteal osteoblasts decreased significantly while the intensity of positive immunostaining with sclerostin in osteocytes increased. Naringin at 100 mg. kg^-1. d ^-1 and 300 mg.kg^-1.d^-1 significantly inhibited hindlimb suspension-induced decreasing of BMD in distal femur, together with the deterioration of trabecular microstructure and femur mechanical properties, increased serum P1NP level and decreased CTX-1. Naringin increased the ex- pression of periostin and nucleus β-catenin and decreased the level of sclerositn. Conclusion Naringin could prevent the progress of disuse osteoporosis in rats, which may be mediated by the increased expression of periostin and subsequently inhibition of sclerostin and activation of Wnt/β-catenin signaling pathways.