葛根总皂苷调控NAMPT-Sirt1轴诱导BMSCs向髓核样细胞分化#br#

摘要：目的　研究葛根总皂苷（TSPL）对大鼠骨髓间充质干细胞（BMSCs）向髓核样细胞分化的影响及其作用机制。方法　体外建立大鼠腰椎间盘退变模型，分为假手术组、模型组、30 mg/kg TSPL治疗组和50 mg/kg TSPL治疗组。原代培养的BMSCs分为BMSCs组，BMSCs-TSPL（10、20、30、40、50 μmol/L）组，BMSCs-TSPL（30 μmol/L）-FK866［烟酰胺磷酸核糖转移酶（NAMPT）特异性抑制剂，2.5 μmol/L］组，BMSCs-TSPL（30 μmol/L）-SRT2104（Sirt1激活剂，10 μmol/L）组。二甲基亚甲基蓝比色法检测细胞和组织中糖胺聚糖含量，qPCR和Western blot检测髓核样细胞COL2A1、Aggrecan、Sox9、Tie2以及Sirt1的mRNA和蛋白表达量。WST-8法检测细胞中烟酰胺腺嘌呤二核苷酸（NAD+）水平，酶联免疫吸附试验（ELISA）检测腺苷三磷酸（ATP）和NAMPT水平。结果　与假手术组相比，模型组大鼠椎间盘L5~S1组织中糖胺聚糖含量减少（P＜0.05）；与模型组相比，TSPL治疗组大鼠糖胺聚糖含量增加，且50 mg/kg TSPL效果优于30 mg/kg TSPL（P＜0.05）；与BMSCs组相比，TSPL单独或联合SRT2104处理增加BMSCs中糖胺聚糖、COL2A1、Aggrecan、Sox9和Sirt1的mRNA和蛋白表达量及NAD+、ATP和NAMPT含量，而降低Tie2的mRNA和蛋白表达量（P＜0.05）；与BMSCs-TSPL组相比，BMSCs-TSPL-FK866组COL2A1、Aggrecan、Sox9和Sirt1的mRNA和蛋白表达量及糖胺聚糖、NAD+、ATP和NAMPT含量降低，而BMSCs-TSPL-SRT2104组上述指标水平增加（P＜0.05）。结论　TSPL可通过NAMPT-Sirt1轴诱导大鼠BMSCs向髓核样细胞分化。

Puerarin saponins induce the differentiation of BMSCs into nucleus pulposus-likecells via NAMPT-Sirt1 axis#br#

Abstract: Objective　To study the effect of total saponins of pueraria lobata (TSPL) on the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into nucleus pulposus-like cells and its mechanism. Methods　An animal model of lumbar intervertebral disc degeneration in rats was established in vitro. The rats were divided into sham group, model group, 30 mg/kg TSPL treatment group and 50 mg/kg TSPL treatment group. The primary cultured rat BMSCs were divided into four groups: BMSCs group, BMSCs-TSPL (10, 20, 30, 40, 50 μmol/L) group, BMSCs-TSPL (30 μmol/L) -FK866 ［a specific inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), 2.5 μmol/L］ group and BMSCs -TSPL (30 μmol/L) -SRT2104 (a Sirt1 activator, 10 μmol/L) group. The contents of glycosaminoglycan in cells and tissues were detected by dimethylmethylene blue method, and the mRNA and protein expression levels of nucleus pulposus-like cells related genes COL2A1, Aggrecan, Sox9, Tie2 and Sirt1 were measured with RT-qPCR and Western blot assay. WST-8 method was used to detect the level of nicotinamide adenine dinucleotide (NAD +) in cells. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of adenosine triphosphate (ATP) and NAMPT, respectively. Results　Compared with the sham operation group, the content of glycosaminoglycan in the intervertebral disc L5-S1 tissue was decreased in the model group (P＜0.05). Compared with the model group the content of glycosaminoglycan increased in TSPL treatment group, and the effect of 50 mg/kg TSPL treatment was better than that of 30 mg/kg treatment (P＜0.05). Compared with BMSCs group, themRNA and protein expression levels of glycosaminoglycan, COL2A1, Aggrecan, Sox9 and Sirt1 and the contents of NAD+, ATP and NAMPT increased in TSPL group and TSPL+SRT2104 group, but the expression level of Tie2 decreased (P＜0.05). Compared with BMSCs-TSPL group, the mRNA and protein levels of COL2A1, aggrecan, Sox9 and Sirt1 and the contents of glycosaminoglycan, NAD+, ATP and NAMPT decreased after treatment with FK866, while the above indexes increased after treatment with SRT2104 (P＜0.05). Conclusion　TSPL could induce BMSCs to differentiate into nucleus pulposus-like cells through the NAMPT/Sirt1 axis.