基于Wnt/β-catenin信号通路探讨左归丸延缓骨髓间充质干细胞衰老机制

The Chinese Medicine believed that the kidney was the origin of congenital constitution and the deficiency of kidney was a fundamental internal cause of aging. The aging theory of stem cells supposed that the aging of stem cells determined the process of aging. Zuogui Wan had the effect of “tonifying the kidney essence”. Could Zuogui Wan also delay the senescence of stem cells? And what was the possible mechanism? In our past research, we found that Zuogui Wan could promote the differentiation of bone marrow mesenchymal stem cells (BMSCs) into neuron like cells and improve memory impairment with aging. It was also confirmed that Wnt signaling pathway was most closely related to stem cell senescence. Therefore, we hypothesized that Zuogui Wan could delay BMSCs senescence by regulating the expressions of some key molecules in Wnt/β-catenin pathway. In this study, the aging BMSCs model was made by D-galactose. The effects of Zuogui Wan on BMSCs proliferation, the expressions of stemness genes, the level of reactive oxygen species (ROS), the activity of β-galactosidase, the expressions of senescence related genes and the key molecules in Wnt signaling pathway were observed by MTT, flow cytometry, immunocytochemistry, Real-time quantitative PCR, Western blot and other molecular biology techniques. DKK1, the inhibitor of Wnt pathway, was also used as a positive control. The study might supply more evidences of Zuogui Wan on “tonifying the kidney essence” and provide experimental basis for clinical application.

祖国医学认为，肾为先天之本，肾虚为衰老之本；干细胞衰老学说认为，干细胞的老化决定了衰老的进程。左归丸具有“补肾填精”之功，该方是否可以延缓干细胞衰老？其机制又是什么？本项目组研究发现，左归丸可以促进骨髓间充质干细胞（BMSCs）向神经样细胞分化，改善增龄性记忆减退；亦有研究证实，Wnt信号通路与干细胞衰老关系最为密切。因此，我们提出假说：左归丸可通过调节Wnt/β-catenin信号通路从而延缓BMSCs衰老。本研究拟采用D-半乳糖制造衰老BMSCs模型，运用MTT、流式细胞术、免疫细胞化学法、Q-PCR及Western blot等分子生物学方法，观察左归丸对衰老BMSCs增殖、干性基因表达、活性氧水平、β-半乳糖苷酶活性、衰老相关基因及Wnt信号通路关键分子表达的影响，并以Wnt信号通路抑制剂DKK1作为阳性对照，以期进一步阐明左归丸“补肾益精填髓”的分子机制，为其临床应用提供实验依据。

祖国医学认为，肾为先天之本，肾精亏虚是机体衰老的根本所在。现代医学认为，干细胞是构成生命的基础，干细胞的老化决定了衰老的进程。由此可见，“肾精学说”与“干细胞学说”有着十分密切的联系。左归丸是明代著名医家张景岳创立的补肾经典方剂，具有滋阴补肾、益精填髓的功效。本研究采用D-半乳糖制造衰老骨髓间充质干细胞（BMSCs）模型，观察左归丸对衰老BMSCs增殖能力、衰老相关指标及干性基因表达的影响；结果表明：左归丸可以促进BMSCs增殖、改善衰老BMSCs的DNA损伤、下调衰老相关分泌表型（SASP）分子水平、降低衰老相关分子（p16、p21和p53）表达、提高衰老BMSCs干性基因表达，从而减少β-半乳糖苷酶染色阳性细胞，改善BMSCs衰老。另一方面，Wnt信号通路作为一条在进化上高度保守的通路，是无脊椎动物和脊椎动物发育过程中起关键作用的信号转导通路之一，参与细胞的增殖、分化、衰老和凋亡等过程，越来越多的研究证实Wnt通路与干细胞衰老的关系非常密切，直接参与了间充质干细胞（MSCs）衰老的调控。进而本研究从Wnt信号通路入手，观察左归丸对Wnt通路关键分子表达的影响，采用Wnt信号通路抑制剂XAV-939作为阳性对照，结果表明：D-gal诱导的BMSCs衰老可导致Wnt信号通路过度激活，左归丸可以通过调节Wnt信号通路关键分子的表达，从而延缓BMSCs的衰老，其作用与XAV-939类似。综合上述研究结果，得出以下结论：1）左归丸可以通过调节、延缓骨髓间充质干细胞衰老，从而维持机体平衡、延缓衰老；2）左归丸延缓骨髓间充质干细胞衰老的机制之一是通过调节Wnt信号通路关键分子表达而实现。本研究进一步阐明了左归丸“补肾益精填髓”的分子机制，为其临床应用提供实验依据。