目的 探讨当归多糖(ASP)对衰老小鼠造血干细胞(HSCs)Wnt/β-catenin 信号通路的影响。方法 6～8周雄性C57BL/6J小鼠30只,随机分为对照组、模型组和ASP组,每组10只。小鼠sc D-半乳糖(D-Gal,120 mg/kg),每天1次,连续42 d,复制衰老小鼠模型;ASP(200 mg/kg)组ip给药,连续35 d。给药结束后第2天,免疫磁珠分离HSCs,衰老β半乳糖苷酶(SA-β-Gal)染色观察衰老HSCs百分率;造血祖细胞混合集落(CFU-Mix)培养检测HSCs形成集落能力;流式细胞术和激光共聚焦检测HSCs中活性氧(ROS)水平;Western blotting检测HSCs胞质β-catenin、胞核β-catenin、GSK-3β、Phospho-GSK-3β和TCF-4蛋白表达。结果 与对照组比较,模型组HSCs的SA-β-Gal染色阳性百分率和ROS水平显著增加;胞质β-catenin、胞核β-catenin、Phospho-GSK-3β和TCF-4蛋白表达上调;CFU-Mix形成能力降低;GSK-3β蛋白表达下调。与模型组比较,ASP能显著降低衰老HSCs的SA-β-Gal染色阳性百分率和ROS水平;下调胞质β-catenin、胞核β-catenin、Phospho-GSK-3β和TCF-4蛋白表达;提高CFU-Mix形成能力;上调GSK-3β蛋白表达。结论 ASP能延缓或拮抗D-Gal致小鼠HSCs衰老,其机制可能与ASP抑制Wnt/β-catenin信号通路过度激活有关。

Objective To explore the effects of Angelica sinensis polysaccharide (ASP) on Wnt/β-catenin signaling pathway in hematopoietic stem cell (HSC) of aging model mice Methods Male Thirty C57BL/6J mice aging 6—8 weeks were randomly divided into normal group, aging model group, and ASP aging model group (ten in each group). After 2 d of finishing the treatment, magnetic activated cell sorting (MACS) were applied to the HSC from mouse bone marrow respectively. The ratio of the SA-β-Gal staining positive HSCs were counted; The capability of colony formation was examined by CFU-Mix cultivation; The distribution of ROS levels was analyzed by flow cytometry (FCM) and laser scanning confocal microscope assess; The content of advanced glycosylation end products (AGEs) was detected by Elisa; The proteins of β-catenin in cytoplasm, GSK-3β in nucleus, Phospho-GSK-3β, and TCF-4 were detected by Western blotting. Results Compared with the normal group, the percentage of SA-β-Gal, the product of ROS positive cells and AGEs in the aging model group were significantly increased; The expression of β-catenin in cytoplasm, β-catenin in nucleus, Phospho-GSK-3β, and TCF-4 were evidently up-regulated; The colony formation of CFU-Mix was markedly decreased; The expression of GSK-3β was evidently down-regulated. Compared with the aging model group, in ASP aging model group, the percentage of SA-β-Gal, the product of ROS positive cells, and AGEs were significantly decreased; The expression of β-catenin in cytoplasm and nucleus, Phospho-GSK-3β, and TCF-4 were evidently down-regulated; The colony formation of CFU-Mix was markedly increased; The expression of GSK-3β was evidently up-regulated. Conclusion ASP can protect HSC from aging by antagonizing D-galactose, and the mechanism may be ASP inhibiting the excessive activation of Wnt/β-catenin signaling pathway.

国家自然科学基金资助项目(81173398)