Abstract: Objective To explore the mechanism of miR-494 on the pathogenesis of osteoporosis in rats by targeting TLR-4 pathway. Method Thirty 8-week-old female Sprague Dawley rats were selected to construct the rat model of osteoporosis. Bone mineral density was measured by dual energy X-ray absorptiometry. The contents of osteocalcin (BGP) and total alkaline phosphatase ( TALP) in rat serum were detected by ELISA. Rat bone marrow mesenchymal stem cells (BMSCs) were cultured. qRT-PCR was used to detect the expression of mir-494 and TLR4 mRNA. The expression of TLR4 protein was detected by Western blot. Double luciferase reporter gene verified the targeting relationship between miR494 and TLR-4. The proliferation ability of BMSCs in each group was detected by CCK8 experiment. Alizarin red S staining was used to detect the osteogenic differentiation of BMSCs in each group. The adipogenic differentiation of BMSCs in each group was detected by oil red O staining. Result Compared with NC group, bone mineral density, BGP content, cell proliferation ability and the number of mineralized nodules in OP group were significantly decreased, while the content of TALP, the expression of miR-494 and TLR4 and the number of adipocytes were significantly increased ( P<0. 05) . Compared with OP group, the bone mineral density, BGP content, cell proliferation ability and the number of mineralized nodules in OP + miR-494 inhibitor group were significantly increased, while the content of TALP, the expression of miR-494 and TLR4 and the number of adipocytes were significantly decreased (P<0. 05) . Compared with OP + miR-494 inhibitor group, the bone mineral density, BGP content, cell proliferation ability and the number of mineralized nodules in OP + miR-494 inhibitor + TLR-4 vector group were significantly decreased, while the content of TALP, the expression of miR-494 and TLR4 and the number of adipocytes were significantly increased; In OP + miR-494 inhibitor + sh TLR-4 group, bone mineral density, BGP content, cell proliferation ability and the number of mineralized nodules increased significantly, while the content of TALP, the expression of mir-494 and TLR4 and the number of adipocytes decreased significantly ( P < 0. 05) . Conclusion MiR-494 can accelerate the progress of osteoporosis in rats by promoting the expression of TLR-4 pathway. To provide a new theoretical basis for the pathogenesis of osteoporosis.

Key words: miR-494, TLR-4 pathway, osteoporosis, pathogenesis

摘要： 目的 探究 miR-494 在骨质疏松大鼠中的表达及其调控机制。 方法 选取 30 只 8 周龄雌性 Sprague-Dawley大鼠,构建骨质疏松大鼠模型。 双能 X 线吸收仪测定骨密度。 ELISA 检测大鼠血清中骨钙素( BGP) 和总碱性磷酸酶( TALP)含量。 分类培养大鼠骨髓间充质干细胞 ( BMSCs) 。 qRT-PCR 检测 miR-494 和 TLR4 mRNA 的表达。Western blot 检测 TLR4 蛋白的表达。 双荧光素酶报告基因验证 miR-494 与 TLR-4 的靶向关系。 CCK8 实验检测各组 BMSCs 的增殖能力。 茜素红 S 染色检测各组 BMSCs 成骨分化情况。 油红 O 染色检测各组 BMSCs 成脂分化情况。 结果 和 NC 组相比,OP 组大鼠骨密度、BGP 含量、细胞增殖能力、矿化结节数均显著降低,TALP 含量miR494 和 TLR4 的表达、脂肪细胞数均显著升高( P< 0. 05) 。 和 OP 组相比,OP +miR-494 inhibitor 组大鼠骨密度、BGP含量、细胞增殖能力、矿化结节数均显著升高,TALP 含量、miR-494 和 TLR4 的表达、脂肪细胞数均显著降低( P <0. 05) 。 和 OP+miR-494 inhibitor 组相比,OP+miR-494 inhibitor+TLR-4 vector 组大鼠骨密度、BGP 含量、细胞增殖能力、矿化结节数均显著降低,TALP 含量、miR-494 和 TLR4 的表达、脂肪细胞数均显著升高;OP+miR-494 inhibitor+shTLR-4 组大鼠骨密度、BGP 含量、细胞增殖能力、矿化结节数均显著升高,TALP 含量、miR-494 和 TLR4 的表达、脂肪细胞数均显著降低(P<0. 05) 。 结论 miR-494 通过促进 TLR-4 通路的表达,加快大鼠骨质疏松的进展,为骨质疏松的发病机制提供新的理论依据。

关键词: miR-494, TLR-4 通路, 骨质疏松, 发病机制