| 许 勇,谢贤斐,薛 彬,熊焱昊,江 敏.川续断提取皂甙促进骨质疏松模型中骨髓基质细胞成骨分化的作用机制研究[J].,2022,(3):401-406 |
| 川续断提取皂甙促进骨质疏松模型中骨髓基质细胞成骨分化的作用机制研究 |
| Study on the Mechanism of Asperosaponin in Promoting Osteogenic Differentiation of Rat Bone Marrow Stromal Cells in Osteoporosis Model |
| 投稿时间：2021-06-05 修订日期：2021-06-28 |
| DOI：10.13241/j.cnki.pmb.2022.03.001 |
| 中文关键词: 川续断提取皂甙 骨质疏松 骨髓基质细胞 PI3K AKT 成骨分化 |
| 英文关键词: Asperosaponin Osteoporosis Rat bone marrow stromal cells PI3K AKT Osteogenic differentiation |
| 基金项目:上海市卫计委科研项目（20134211） |
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| 中文摘要: |
| 摘要 目的：探讨川续断提取皂甙（ASA）促进骨质疏松模型中骨髓基质细胞（rBMSCs）成骨分化的作用机制。方法：选取3月龄的雌性SD大鼠60只，随机分为卵巢切除组和假手术组，每组30只。采取卵巢切除法构建骨质疏松模型。建模成功后采用微型计算机断层扫描获得其松质骨微观结构的三维图像并进行分析。采用CCK-8法测定ASA对卵巢切除组rBMSCs增殖的影响。分析碱性磷酸酶（ALP）活性；荧光定量PCR检测成骨基因ALP、骨桥蛋白（OPN）、Runt相关转录因子2（RUNX2）的表达情况；蛋白免疫印迹试验检测磷脂酰肌醇3激酶（PI3K）、磷酸化蛋白激酶B（p-AKT）蛋白表达情况。结果：与假手术组比较，卵巢切除组骨小梁数量、骨小梁厚度和骨体积分数下降，但骨小梁分离度升高，差异有统计学意义（P＜0.05）。第4天和第7天，ASA(10-5 mol/L)组、ASA(10-6 mol/L) 组、ASA(10-7 mol/L) 组和ASA(10-8 mol/L) 组rBMSC增殖均显著高于ASA(0学艺术mol/L)组，以ASA(10-5 mol/L)最为显著；而ASA(10-1 mol/L) 组、ASA(10-2 mol/L) 组、ASA(10-3 mol/L) 组和ASA(10-4 mol/L) 组rBMSC增殖显著低于ASA(0 mol/L)组，以ASA(10-4 mol/L)最为显著，差异均有统计学意义（P＜0.05）。第7天和第14天，ASA(10-5 mol/L)组、ASA(10-6 mol/L) 组、ASA(10 -7 mol/L) 组和ASA(10-8 mol/L) 组ALP活性均显著高于ASA(0 mol/L)组，且第14天ALP活性高于第7天，差异均有统计学意义（P＜0.05）。与对照组比较，ASA组成骨相关基因ALP、OPN和RUNX2相对mRNA表达水平显著升高，差异有统计学意义（P＜0.05）；与ASA组比较，wortmannin组成骨相关基因ALP、OPN和RUNX2相对mRNA表达水平均显著降低，差异有统计学意义（P＜0.05）。与对照组比较，ASA组PI3K、p-AKT蛋白表达水平显著升高，差异有统计学意义（P＜0.05）；与ASA组比较，wortmannin组PI3K、p-AKT蛋白表达水平显著降低，差异均有统计学意义（P＜0.05）。结论：ASA能促进骨质疏松模型rBMSCs增殖，增强ALP活性，增强ALP、OPN和RUNX2的表达，而PI3K通路抑制剂wortmannin降低了这些成骨作用，并降低了ASA诱导的PI3K、p-AKT水平，表明ASA通过PI3K/AKT信号通路促进骨质疏松模型rBMSCs成骨分化。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the mechanism of asperosaponin(ASA) in promoting osteogenic differentiation of rat bone marrow stromal cells (rBMSCs) in osteoporosis model. Methods: 60 female SD rats aged 3 months were selected and randomly divided into ovariectomized group and sham operation group, with 30 rats in each group. The ovariectomy method was used to construct an osteoporosis model. After the modeling was successful, the microcomputer tomography was used to obtain the three-dimensional image of the microstructure of the cancellous bone and analyzed. The CCK-8 method was used to determine the effect of ASA on the proliferation of rBMSCs in the ovariectomized group.Analyzed the alkaline phosphatase (ALP) activity, and fluorescent quantitative PCR to detect the expression of osteogenic genes ALP, osteopontin (OPN), Runt-related transcription factor 2(RUNX2). Western blotting was used to detect the expression of phosphatidylinositol 3-kinase (PI3K) and phosphorylated protein kinase B (p-AKT). Results: Compared with the sham operation group, the trabeculae number, trabecular thickness and bone volume fraction in the ovariectomized group decreased, but the trabecular separation increased, and the difference were statistically significant(P<0.05). On 4 d and 7 d, the proliferation of rBMSCs in the ASA (10-5 mol/L) group, ASA (10-6 mol/L) group, ASA (10-7 mol/L) group and ASA (10-8 mol/L) group were all significantly higher than the ASA (0 mol/L) group, with ASA (10-5 mol/L) being the most significant;while the ASA (10-1 mol/L) group, ASA (10-2 mol/L) group, and ASA (10-3 mol/L) group and the ASA (10-4 mol/L) group was significantly lower than that in the ASA (0 mol/L) group, with ASA (10-4 mol/L) being the most significant, and the differences were statistically significant (P<0.05). On 7d and 14d, the ALP activity of ASA (10-5 mol/L) group, ASA (10-6 mol/L) group, ASA (10-7 mol/L) group and ASA (10-8 mol/L) group were all significantly higher than the ASA (0 mol/L) group, and the ALP activity on 14 d was higher than that on 7 d, the difference were statistically significant (P<0.05). Compared with the control group, the relative mRNA expression levels of the bone-related genes ALP, OPN and RUNX2 of ASA group increased significantly,and the difference were statistically significant(P＜0.05); compared with the ASA group, the relative mRNA expression levels of the bone-related genes ALP, OPN and RUNX2 of wortmannin group were significantly reduced, and the difference were statistically significant(P<0.05). Compared with the control group, the expression levels of PI3K and p-AKT protein in the ASA group were significantly increased, and the difference were statistically significant(P<0.05); compared with the ASA group, the expression levels of PI3K and p-AKT protein in the wortmannin group were significantly reduced, and the differences were statistically significant(P<0.05). Conclusion: ASA can promote the proliferation of osteoporosis model rBMSCs, enhance the activity of ALP, and enhance the expression of ALP, OPN and RUNX2.The PI3K pathway inhibitor wortmannin reduce these osteogenic effects and reduce the levels of PI3K and p-AKT induced by ASA. ASA promotes osteogenic differentiation of osteoporosis model rBMSCs through PI3K/AKT signaling pathway. |
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