BACKGROUND: Studies have shown that bone marrow mesenchymal stem cells (BMSCs) can differentiate into nucleus pulposus-like cells, but the mechanism of differentiation is not clear. OBJECTIVE: To induce bone BMSCs into nucleus pulposus-like cells using Achyranthes bidentata Bl. saponins (ABS) and to explore the role of Nampt/NAD/Sirt1 axis in the differentiation of BMSCs. METHODS: BMSCs were collected from Sprague-Dawley rats. Cells at passage 3 were divided into four groups: BMSCs group, BMSCs+ABS group, BMSCs+ABS+nicotinamide mononucleotide (an exogenous small molecule substance promoting NAD synthesis) group, BMSCs+ABS+FK866 (nicotinamide phosphoribosyltransferase inhibitor) group, in which the cells were induced for 14 days. Alcian blue staining was used to show the changes of glycosaminoglycan in the cells. RT-PCR was used to detect the mRNA expression of COL2, Aggrecan, KRT19, Pax1. The protein expression level of COL2 was detected by western blot. The activity of Sirt1 was detected by Sirt1 assay kit and the content of NAD+ was measured by NAD+/NADH kit. RESULTS AND CONCLUSION: (1) Compared with the BMSCs group, BMSCs+ABS group showed significant increases in the expression levels of glycosaminoglycan, Aggrecan, KRT19, and Pax1 (P < 0.05), and the protein expression levels of COL2, activity of Sirt1, and content of NAD+ were also significantly increased (all P < 0.05). (2) Compared with the BMSCs+ABS group, the above-mentioned indicators were significantly increased in the BMSCs+ABS+nicotinamide mononucleotide (P < 0.05); on the contrary, these indicators were all decreased significantly in the BMSCs+ABS+FK866 group (P < 0.05). To conclude, ABS could induce the differentiation of rat BMSCs into nucleus pulposus-like cells, in which the Nampt/NAD/Sirt1 axis might play a promotion role.%背景:研究表明,骨髓间充质干细胞可向髓核样细胞分化,但分化机制尚不明确.目的:利用牛膝总皂苷诱导大鼠骨髓间充质干细胞向髓核样细胞分化,并探讨Nampt/NAD/Sirt1 轴在其中的作用.方法:取第3 代SD 大鼠骨髓间充质干细胞,随机分成4 组:①骨髓间充质干细胞组;②骨髓间充质干细胞+牛膝总皂苷组;③骨髓间充质干细胞+牛膝总皂苷+烟酰胺单核苷酸组(烟酰胺单核苷酸为促NAD 合成的外源性小分子物质);④骨髓间充质干细胞+牛膝总皂苷+FK866 组(FK866 为烟酰胺磷酸核糖转移酶抑制剂).连续诱导培养14 d,阿尔新蓝染色法检测细胞内糖胺多糖含量,RT-PCR 检测髓核样细胞相关基因COL2、Aggrecan、KRT19 和PAX1 mRNA 表达,Western blot 检测COL2的蛋白表达,Sirt1测定试剂盒检测Sirt1活性,NAD+/NADH试剂盒检测NAD+含量.结果与结论:①与骨髓间充质干细胞组相比,骨髓间充质干细胞+牛膝总皂苷组能够使糖胺多糖、髓核样细胞相关基因表达、COL2 蛋白表达、Sirt1 活性、NAD+含量明显增加(P < 0.05);②与骨髓间充质干细胞+牛膝总皂苷组相比,骨髓间充质干细胞+牛膝总皂苷+烟酰胺单核苷酸组糖胺多糖、髓核样细胞相关基因表达、COL2蛋白表达、Sirt1活性和NAD+含量增加(P < 0.05);相反,骨髓间充质干细胞+牛膝总皂苷+FK866组上述指标均降低(P < 0.05);③结果表明,牛膝总皂苷可诱导大鼠骨髓间充质干细胞向髓核样细胞分化,Nampt/NAD/Sirt1 轴在其中发挥促进作用.
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