补肾活血方调控Nampt/NAD<sup>+</sup>/Sirt1信号通路促进氧化损伤ADSCs类髓核分化的研究

段嘉豪; 杨少锋; 聂颖; 徐诗嘉; 吕福丹; 李维; 李硕夫

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段嘉豪, 杨少锋, 聂颖, 徐诗嘉, 吕福丹, 李维, 李硕夫.补肾活血方调控Nampt/NAD⁺/Sirt1信号通路促进氧化损伤ADSCs类髓核分化的研究[J].湖南中医药大学学报,2026,46(9):1808-1815[点击复制]

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补肾活血方调控Nampt/NAD⁺/Sirt1信号通路促进氧化损伤ADSCs类髓核分化的研究

段嘉豪,杨少锋,聂颖,徐诗嘉,吕福丹,李维,李硕夫

(湖南中医药大学第一附属医院, 湖南长沙 410007)

摘要:

目的探讨补肾活血方通过烟酰胺磷酸核糖转移酶（Nampt）/烟酰胺腺嘌呤二核苷酸（NAD⁺）/沉默信息调节因子1（Sirt1）信号通路对氧化损伤脂肪间充质干细胞（ADSCs）类髓核分化的影响。方法采用Transwell小室建立大鼠ADSCs与髓核细胞（NPCs）共培养模型，以叔丁基过氧化氢（T-BHP）干预24 h建立氧化应激微环境模拟椎间盘退变（IDD）。将细胞分为空白对照组、模型对照组、补肾活血方低剂量组、补肾活血方高剂量组、烟酰胺单核苷酸（NMN）组和Nampt抑制剂（FK866）组。在T-BHP干预48 h后用显微镜观察细胞形态；甲苯胺蓝染色评价聚集蛋白聚糖（aggrecan）合成；qPCR检测Ⅱ型胶原蛋白（collagenⅡ）、aggrecan、细胞角蛋白19（KRT19）及配对盒基因1（Pax1） mRNA表达水平；Western blot检测collagenⅡ和肿瘤抑制蛋白（p53）表达水平；ELISA检测Sirt1含量，比色法检测NAD⁺/NADH比值。结果与空白对照组比较，模型对照组collagenⅡ、aggrecan、KRT19、Pax1 mRNA表达水平及collagen Ⅱ蛋白表达水平均显著降低（P<0.01），p53蛋白表达水平显著升高（P<0.01），Sirt1含量及NAD⁺/NADH比值显著降低（P<0.01）。与模型对照组比较，补肾活血方高剂量组collagenⅡ、aggrecan、KRT19、Pax1 mRNA表达水平及collagenⅡ蛋白表达水平显著升高（P<0.01），p53蛋白表达水平显著下降（P<0.01），Sirt1含量及NAD⁺/NADH比值显著升高（P<0.01）；补肾活血方低剂量组collagenⅡ和aggrecan mRNA表达水平升高（P<0.05），collagenⅡ蛋白表达水平升高（P<0.05）。与补肾活血方高剂量组比较，NMN组collagenⅡ和aggrecan mRNA表达水平及collagenⅡ蛋白表达水平显著升高（P<0.01），p53蛋白表达水平显著降低（P<0.01），Sirt1含量及NAD⁺/NADH比值显著升高（P<0.01）；FK866组collagenⅡ和aggrecan mRNA表达水平显著下降（P<0.01），KRT19和Pax1 mRNA表达水平降低（P<0.05），collagenⅡ蛋白表达水平显著升高（P<0.01），p53蛋白表达水平显著降低（P<0.01），Sirt1含量及NAD⁺/NADH比值下降（P<0.05）。结论补肾活血方可改善氧化应激损伤ADSCs的基质合成能力，促进其向类髓核表型分化，该作用可能与提高NAD⁺/NADH比值、抑制p53、激活Nampt/NAD⁺/Sirt1信号通路相关。

DOI：10.3969/j.issn.1674-070X.2026.09.008

投稿时间：2026-02-27

基金项目:湖南省中医药科研计划项目(B2023150);湖南省自然科学基金项目(2023JJ60342);湖南省教育厅优青项目(25B0358)。

Bushen Huoxue Formula promotes nucleus pulposus-like differentiation of oxidatively damaged ADSCs by regulating the Nampt/NAD⁺/Sirt1 signaling pathway

DUAN Jiahao, YANG Shaofeng, NIE Ying, XU Shijia, LYU Fudan, LI Wei, LI Shuofu

(The First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China)

Abstract:

Objective To investigate the effects of Bushen Huoxue Formula (BSHXF) on the nucleus pulposus-like differentiation of oxidatively damaged adipose-derived mesenchymal stem cells (ADSCs) through the nicotinamide phosphoribosyltransferase (Nampt)/nicotinamide adenine dinucleotide (NAD⁺)/sirtuin 1 (Sirt1) signaling pathway. Methods A co-culture model of rat ADSCs and nucleus pulposus cells (NPCs) was established using Transwell inserts. Tert-butyl hydroperoxide (T-BHP) was used to induce oxidative damage for 24 h to simulate the oxidative stress microenvironment associated with intervertebral disc degeneration (IDD). The cells were divided into blank control, model control, low-dose BSHXF, high-dose BSHXF, nicotinamide mononucleotide (NMN), and NAMPT inhibitor (FK866) groups. After 48 h of T-BHP treatment, cell morphology was observed under a microscope. Toluidine blue staining was performed to evaluate proteoglycan (aggrecan) synthesis. The mRNA expression levels of collagen Ⅱ, aggrecan, keratin 19 (KRT19), and paired box 1 (Pax1) were detected by quantitative real-time polymerase chain reaction (qPCR). The protein expression levels of collagen Ⅱ and tumor suppressor protein (p53) were checked by Western blot. Sirt1 content was measured by ELISA, and the NAD⁺/NADH ratio was determined using a colorimetric assay. Results Compared with the blank control group, mRNA expression levels of collagen Ⅱ, aggrecan, KRT19, and Pax1, as well as the protein expression level of collagen Ⅱ significantly decreased in the model control group (P<0.01), while p53 protein expression level significantly increased (P<0.01). The Sirt1 content and NAD⁺/NADH ratio significantly decreased (P<0.01). Compared with the model control group, high-dose BSHXF group showed significantly increased mRNA expression levels of collagen Ⅱ, aggrecan, KRT19, and Pax1, as well as increased protein expression level of collagen Ⅱ (P<0.01), together with significantly decreased p53 protein expression level (P<0.01) and significantly increased Sirt1 content and NAD⁺/NADH ratio (P<0.01). In the low-dose BSHXF group, mRNA expression levels of collagen Ⅱ and aggrecan, as well as collagen Ⅱ protein expression level significantly increased (P<0.05). Compared with the high-dose BSHXF group, NMN group showed significantly increased mRNA expression levels of collagen Ⅱ and aggrecan and protein expression level of collagen Ⅱ (P<0.01), significantly decreased p53 protein expression level (P<0.01), and significantly increased Sirt1 content and NAD⁺/NADH ratio (P<0.01). FK866 group showed significantly decreased mRNA expression levels of collagen Ⅱ and aggrecan (P<0.01), decreased KRT19 and Pax1 mRNA expression levels (P<0.05), significantly increased collagen Ⅱ protein expression level (P<0.01), significantly decreased p53 protein expression level (P<0.01), and decreased Sirt1 content and NAD⁺/NADH ratio (P<0.05). Conclusion BSHXF can improve the matrix synthesis capacity of oxidatively damaged ADSCs and promote their differentiation toward a nucleus pulposus-like phenotype. This effect may be associated with an increased NAD⁺/NADH ratio, inhibition of p53, and activation of the Nampt/NAD⁺/Sirt1 signaling pathway.

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