目的 基于miR-146a-5p/Notch1信號通路探究補腎壯筋湯（BZD）對骨質(zhì)疏松小鼠骨密度及成骨分化的影響。方法 采用地塞米松誘導(dǎo)的方法構(gòu)建小鼠骨質(zhì)疏松癥模型，造模成功60只小鼠，將其隨機分為模型組，BZD低、高劑量（5.4、10.8 g·kg^-1）組，BZD＋agomir NC（10.8 g·kg^-1＋8 mg·kg^-1）組，BZD＋miR-146a-5p agomir（10.8 g·kg^-1＋8 mg·kg^-1）組，每組12只。另取12只小鼠正常飼養(yǎng)，記為對照組。造模成功后開始ig BZD，每天1次，連續(xù)8周；agomir NC、miR-146a-5p agomir采用尾iv給藥，隔天給藥，共8周。給藥結(jié)束后測定小鼠骨密度和骨結(jié)構(gòu)參數(shù)：骨礦物質(zhì)含量（BMC）、骨礦物質(zhì)密度（BMD）、總體積（TV）、骨體積（BV）、骨體積分?jǐn)?shù)（BV/TV）、骨小梁數(shù)量（Tb.N）、骨小梁厚度（Tb.Th）和骨小梁分離度（Tb.Sp）；酶聯(lián)免疫吸附試驗（ELISA）檢查小鼠血清骨代謝指標(biāo)：骨鈣素（OCN）、骨源性堿性磷酸酶（BALP）、Ⅰ型前膠原羧基端前肽（PINP）、抗酒石酸酸性磷酸酶（Trap）；HE染色觀察小鼠骨組織的結(jié)構(gòu)變化；實時熒光定量PCR（qRT-PCR）法測定小鼠股骨組織miR-146a-5p、Notch1 mRNA的表達(dá)水平。Western blotting檢查小鼠股骨組織Notch1蛋白表達(dá)水平。從Balb/c小鼠中分離骨髓間充質(zhì)干細(xì)胞（BMSCs）并誘導(dǎo)成骨分化，分為對照組，模型組（1×10^-5 mol·L^-1地塞米松處理48 h），BZD低、高濃度組（造模后用100、200 μg·mL^-1 BZD處理48 h），BZD＋agomir NC組（造模后用200 μg·mL^-1 BZD和轉(zhuǎn)染agomir NC處理48 h），BZD＋miR-146a-5p agomir組（造模后用200 μg·mL^-1 BZD和轉(zhuǎn)染miR-146a-5p agomir處理48 h）。CCK-8實驗檢測細(xì)胞增殖，ALP染色和茜素紅染色分析細(xì)胞成骨分化，qRT-PCR測定細(xì)胞miR-146a-5p及Notch1 mRNA表達(dá)，Western blotting檢查細(xì)胞成骨標(biāo)志蛋白及Notch1蛋白表達(dá)，雙熒光素酶實驗檢測miR-146a-5p及Notch1的相互作用。結(jié)果 與對照組比較，模型組小鼠BMC、BMD、BV/TV、Tb.N、Tb.Th水平，血清BALP、PINP水平，股骨組織Notch1 mRNA和蛋白表達(dá)水平顯著降低（P＜0.05）；Tb.Sp水平，血清OCN、Trap水平，股骨組織miR-146a-5p水平顯著升高（P＜0.05）；骨小梁數(shù)量減少，間隙變寬，出現(xiàn)斷裂，骨陷窩增大。與模型組比較，BZD高劑量組小鼠相關(guān)指標(biāo)變化與上述相反（P＜0.05），且過表達(dá)miR-146a-5p抑制了BZD對骨質(zhì)疏松小鼠的上述改善作用。體外研究結(jié)果顯示，與對照組比較，模型組BMSCs的活力，相對ALP活力，茜素紅陽性染色面積比例，ALP、OCN、Runx2、Osterix蛋白及Notch1 mRNA和蛋白表達(dá)顯著降低，miR-146a-5p表達(dá)顯著增高（P＜0.05）；與模型組比較，BZD高劑量組細(xì)胞中的上述指標(biāo)均呈相反變化（P＜0.05），且過表達(dá)miR-146a-5p逆轉(zhuǎn)了高劑量BZD對小鼠BMSCs的上述作用。熒光素酶實驗發(fā)現(xiàn)，Notch1是miR-146a-5p的直接靶點，且被miR-146a-5p負(fù)調(diào)控。結(jié)論 BZD可能通過下調(diào)miR-146a-5p/Notch1信號通路促進BMSCs成骨分化，提高骨質(zhì)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.

R285.5

河南省自然科學(xué)基金青年項目（232300420269）；2023年度河南省中醫(yī)藥科學(xué)研究專項課題重大專項（2023ZYZD06）；河南省高等學(xué)校重點科研項目（24A360002）