目的 基于miR-146a-5p/Notch1信号通路探究补肾壮筋汤（BZD）对骨质疏松小鼠骨密度及成骨分化的影响。方法 采用地塞米松诱导的方法构建小鼠骨质疏松症模型，造模成功60只小鼠，将其随机分为模型组，BZD低、高剂量（5.4、10.8 g·kg^-1）组，BZD＋agomir NC（10.8 g·kg^-1＋8 mg·kg^-1）组，BZD＋miR-146a-5p agomir（10.8 g·kg^-1＋8 mg·kg^-1）组，每组12只。另取12只小鼠正常饲养，记为对照组。造模成功后开始ig BZD，每天1次，连续8周；agomir NC、miR-146a-5p agomir采用尾iv给药，隔天给药，共8周。给药结束后测定小鼠骨密度和骨结构参数：骨矿物质含量（BMC）、骨矿物质密度（BMD）、总体积（TV）、骨体积（BV）、骨体积分数（BV/TV）、骨小梁数量（Tb.N）、骨小梁厚度（Tb.Th）和骨小梁分离度（Tb.Sp）；酶联免疫吸附试验（ELISA）检查小鼠血清骨代谢指标：骨钙素（OCN）、骨源性碱性磷酸酶（BALP）、Ⅰ型前胶原羧基端前肽（PINP）、抗酒石酸酸性磷酸酶（Trap）；HE染色观察小鼠骨组织的结构变化；实时荧光定量PCR（qRT-PCR）法测定小鼠股骨组织miR-146a-5p、Notch1 mRNA的表达水平。Western blotting检查小鼠股骨组织Notch1蛋白表达水平。从Balb/c小鼠中分离骨髓间充质干细胞（BMSCs）并诱导成骨分化，分为对照组，模型组（1×10^-5 mol·L^-1地塞米松处理48 h），BZD低、高浓度组（造模后用100、200 μg·mL^-1 BZD处理48 h），BZD＋agomir NC组（造模后用200 μg·mL^-1 BZD和转染agomir NC处理48 h），BZD＋miR-146a-5p agomir组（造模后用200 μg·mL^-1 BZD和转染miR-146a-5p agomir处理48 h）。CCK-8实验检测细胞增殖，ALP染色和茜素红染色分析细胞成骨分化，qRT-PCR测定细胞miR-146a-5p及Notch1 mRNA表达，Western blotting检查细胞成骨标志蛋白及Notch1蛋白表达，双荧光素酶实验检测miR-146a-5p及Notch1的相互作用。结果 与对照组比较，模型组小鼠BMC、BMD、BV/TV、Tb.N、Tb.Th水平，血清BALP、PINP水平，股骨组织Notch1 mRNA和蛋白表达水平显著降低（P＜0.05）；Tb.Sp水平，血清OCN、Trap水平，股骨组织miR-146a-5p水平显著升高（P＜0.05）；骨小梁数量减少，间隙变宽，出现断裂，骨陷窝增大。与模型组比较，BZD高剂量组小鼠相关指标变化与上述相反（P＜0.05），且过表达miR-146a-5p抑制了BZD对骨质疏松小鼠的上述改善作用。体外研究结果显示，与对照组比较，模型组BMSCs的活力，相对ALP活力，茜素红阳性染色面积比例，ALP、OCN、Runx2、Osterix蛋白及Notch1 mRNA和蛋白表达显著降低，miR-146a-5p表达显著增高（P＜0.05）；与模型组比较，BZD高剂量组细胞中的上述指标均呈相反变化（P＜0.05），且过表达miR-146a-5p逆转了高剂量BZD对小鼠BMSCs的上述作用。荧光素酶实验发现，Notch1是miR-146a-5p的直接靶点，且被miR-146a-5p负调控。结论 BZD可能通过下调miR-146a-5p/Notch1信号通路促进BMSCs成骨分化，提高骨质疏松小鼠的骨密度。

Objective To investigate the effect of Bushen Zhuangjin decoction (BZD) on bone density and osteogenic differentiation in osteoporosis mice based on the miR-146a-5p/Notch1 signaling pathway. Methods A mouse model of osteoporosis was constructed using dexamethasone induction, and 60 mice were successfully modeled. They were randomly divided into model groups, BZD low and high dose (5.4, 10.8 g·kg^-1) groups, BZD+agomir NC (10.8 g·kg^-1+8 mg·kg^-1) group, and BZD+miR-146a-5p agomir (10.8 g·kg^-1+8 mg·kg^-1) group, with 12 mice in each group. Another 12 mice were taken from normal feeding and designated as the control group. After successful modeling, start ig BZD once a day for 8 consecutive weeks; agomir NC and miR-146a-5p agomir were administered via tail vein injection every other day for a total of eight weeks. After administration, the bone density and structural parameters of the mice were measured, including bone mineral content (BMC), bone mineral density (BMD), total volume (TV), bone volume (BV), bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation/spacing (Tb.SP). Enzyme-linked immunosorbent assay (ELISA) was applied to examine serum bone metabolism indicators in mice, including osteocalcin (OCN), bone alkaline phosphatase (BALP), N-terminal propeptide of type I procollagen (PINP), and tartrate acid-resistant phosphatase (Trap). HE staining was applied to observe structural changes in bone tissue. Real-time fluorescence quantitative PCR (RT-qPCR) method was applied to determine the expression levels of miR-146a-5p and Notch1 mRNA in femoral tissue. Western Blot was applied to examine the expression level of Notch1 protein in mouse femoral tissue. Bone marrow mesenchymal stem cells (BMSCs) were isolated from Balb/c mice, induced osteogenic differentiation, and then divided into control group, model group (treated with 1×10^-5 mol·L^-1 dexamethasone for 48 h), BZD low and high concentration groups (treated with 100 and 200 μg·mL^-1 BZD after modeling for 48 h), BZD+agomir NC group (treated with 200 μg·mL^-1 BZD and transfected agomir NC after modeling for 48 h), BZD+miR-146a-5p agomir group (treated with 200 μg·mL^-1 BZD and transfected miR-146a-5p agomir after modeling for 48 h). Cell proliferation was detected by CCK-8 assay, cell osteoblastic differentiation was analyzed by ALP and alizarin red staining, the expression of miR-146a-5p and Notch1 mRNA were determined by RT-qPCR, and the expression of osteoblastic marker protein and Notch1 protein were detected by Western Blot. The interaction between miR-146a-5p and Notch1 was detected by dual luciferase assay. Results Compared with the control group, the BMC, BMD, BV/TV, Tb.N, Tb.Th levels, serum BALP, PINP levels, and Notch1 mRNA and protein expression levels in the femur tissue of mice in Model group were obviously lower (P < 0.05), the Tb.SP level, serum OCN, Trap levels, and miR-146a-5p level in femoral tissue were obviously higher (P < 0.05), the number of bone trabeculae decreased, gaps widen, fractures occurred, and bone lacunae increased. Compared with the Model group, the changes in relevant indicators of mice in the BZD high dose group were opposite to the above (P < 0.05). Overexpression of miR-146a-5p inhibited the increase of bone density in osteoporotic mice induced by BZD. In vitro study results showed that compared with control group, cell vitality, relative ALP activity, alizarin red positive staining area ratio, the expression of ALP, OCN, Runx2, Osterix protein and Notch1 mRNA and protein of BMSCs in model group were significantly decreased the expression of miR-146a-5p was significantly increased (P < 0.05). Compared with model group, the above indexes were inversely expressed in BZD high dose group (P < 0.05). Overexpression of miR-146a-5p reversed the promoting effect of high dose BZD on proliferation and osteogenic differentiation of BMSCs in mice (P < 0.05). The luciferase assay found that Notch1 was a direct target of miR-146a-5p and was negatively regulated by miR-146a-5p. Conclusion BZD may promote osteogenic differentiation of BMSCs by down-regulating miR-146a-5p/Notch1 signaling pathway and increase bone density in osteoporosis mice.

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河南省自然科学基金青年项目（232300420269）；2023年度河南省中医药科学研究专项课题重大专项（2023ZYZD06）；河南省高等学校重点科研项目（24A360002）