大豆苷元干预Hedgehog信号通路调控去卵巢骨质大鼠基质金属蛋白酶代谢

Daidzein interferes with Hedgehog signaling pathway to regulate matrix metalloproteinase metabolism in ovariectomized rats

目的 探究大豆苷元对骨质疏松(osteoporosis, OP)模型大鼠Hedgehog信号通路的调控作用,分析大豆苷元对OP大鼠基质金属蛋白酶代谢的影响。方法 随机将60只雌性SPF级Wistar大鼠分为假手术组、模型组、大豆苷元低、中、高剂量治疗组5组,每组12只。除假手术组,其他4组均采用去卵巢法建立OP模型。造模成功后开始干预治疗,假手术组、模型组等剂量生理盐水灌胃,其他给予大豆苷元低、中、高剂量治疗,连续干预12周。酶联免疫吸附法测定各组大鼠血清雌激素(E2)、骨钙素(BGP)、血清碱性磷酸酶(ALP)含量水平;免疫组化法检测各组大鼠股骨组织中基质金属蛋白酶组织抑制剂-1(TIMP-1)和基质金属蛋白酶-7(MMP-7)免疫蛋白的表达情况;TUNEL染色法检测各组骨细胞凋亡情况;RT-PCR法和WB法检测各组骨组织中Shh、PTC1、Gli1 mRNA及蛋白表达水平。结果 大豆苷元能显著提高OP大鼠大鼠E2、TIMP-1水平,降低细胞凋亡及BGP、ALP、MMP-7水平;显著改善股骨组织中Shh、PTC1、Gli1 mRNA及蛋白表达水平(P<0.05),且大豆苷元不同组具有剂量依赖性(P<0.01)。结论 大豆苷元可有效调控OP模型大鼠基质金属蛋白酶代谢水平,这可能与大豆苷元能够有效调节Hedgehog信号通路表达相关。

Objective To explore the regulatory effect of daidzein on Hedgehog signal pathway in osteoporosis(OP) model rats, and to analyze the effect of daidzein on matrix metalloproteinase metabolism in OP rats. Methods Sixty female SPF Wistar rats were randomly divided into blank group, model group, and daidzein low, medium, and high dose treatment groups, with 12 rats in each group. Except for rats in the blank group, rats in the other four groups were ovariectomized to establish OP model. Intervention treatment was started after successful modeling. Rats in the blank group and model group received equal doses of normal saline by gavage. Rats in the others received low, medium, or high doses of daidzein for 12 weeks. The serum contents of estrogen(E2), osteocalcin(BGP), and alkaline phosphatase(ALP) were detected. Matrix metalloproteinase-7(MMP-7) and matrix metalloproteinase tissue inhibitor-1(TIMP-1) protein expression were detected with immunohistochemistry. TUNEL method was used to detect the apoptosis of osteocytes in the distal femur of rats in each group. Western blotting and real-time fluorescence quantitative polymerase chain reaction were used to detect the mRNA and protein expression levels of Shh, PTC1, and Gli1 in the femur of rats in each group. Results Daidzein significantly increased the levels of E2 and TIMP-1, reduced apoptosis and the levels of BGP, ALP, and MMP-7 in OP rats. The expression levels of Shh, PTC1, and Gli1 mRNA and protein in the femur improved significantly(P<0.05), and the increase in daidzein treatment groups was dose-dependent(P<0.01). Conclusion Daidzein effectively regulates the metabolic level of matrix metalloproteinases in OP model rats, which may be related to the effective regulation of hedgehog signal pathway.