“干细胞巢细胞极性”视角下萸连干预结肠癌前病变功效方向的配伍控制研究

It is a key event for the initiation of precancerous changes of colorectal cancer that the loss of cell polarity in intestinal stem cells niche stimulates cancer stem cell niche and induces aberrant crypt foci. The previous studies have confirmed that the compatibility of Wuzhuyu and Huanglian can regulate the aberrant crypt foci to block and delay the precancerous changes of colorectal cancer, which may be related to that compatibility characteristics of Wuzhuyu and Huanglian — a combination of cold and hot drugs fits the key pathogenesis of precancerous changes of colorectal cancer — “depression of the liver resulting in obstructing of the spleen”. The latest research finds that Rspo3-Wnt/β-catenin pathway stimulates the loss of cell polarity of intestinal stem cells niche. Accordingly, a research hypothesis is proposed that: the reason of the effect display direction of Wuzhuyu and Huanglian’s intervention on the precancerous changes of colorectal cancer,is that specific proportion of compatibility structure regulates Rspo3, inhibits Wnt/β-catenin pathway, affects the number of cell population and dynamic behavior, adjusts cell polarity of stem cell niche. This topic intends to construct a model of the precancerous changes of colorectal cancer for monitoring Rspo3-Wnt/β-catenin pathway, revealing the biological effect, regulation mechanism and key target of intervention on cell polarity of stem cell niche by Wuzhuyu and Huanglian’s different proportion of compatibility, providing actual and reliable basis for the theory of science of Traditional Chinese Medicine(TCM) formula for reference — The TCM controls the effect display direction by compatibility, giving a new connotation to “depression of the liver resulting in obstructing of the spleen”, promote the research and development of the TCM on treatment and prescription of tumor.

结肠干细胞巢细胞极性丧失促肿瘤干细胞巢生成，诱发隐窝畸变，是结肠癌前病变启动关键事件。前期研究证实吴茱萸和黄连配伍可抑制畸变隐窝灶形成从而阻延结肠癌前病变，可能是萸连药对寒热并用之配伍特点契合了结肠癌前病变“木郁土壅”的关键病机。最新研究发现Rspo3-Wnt/β-catenin通路促结肠干细胞巢细胞极性丧失。据此提出研究假说：萸连药对干预结肠癌前病变之功效方向的发挥，与其特定比例的配伍结构调控Rspo3-Wnt/β-catenin通路，继而影响干细胞巢中细胞种群数量、动力学行为，调整干细胞巢细胞极性有关。本课题拟构建结肠癌前病变模型，监测Rspo3-Wnt/β-catenin通路，揭示萸连药对不同配伍比例干预干细胞巢细胞极性的不同生物学效应、调控机制和关键靶点，为中药通过配伍控制其功效发挥方向的方剂学理论提供实证依据，赋予“木郁土壅”新的内涵，推动中医肿瘤治法与方剂研究的进一步

畸变隐窝灶出现（小事件）的实质是结肠干细胞巢细胞极性丧失（微事件），最终导致了结肠肿瘤的发生发展（大事件）。课题组基于结肠癌前病变 “木郁土壅”之关键病机，结合前期研究证实吴茱萸黄连药对畸变隐窝灶（ACF）形成呈现效应差异，是萸连特定配伍比例控制其功效发挥方向的结果，据此提出萸连药对干预结肠癌前病变之功效方向的发挥，与其特定比例的配伍结构调控 Rspo3、抑制 Wnt/β-catenin 通路，继而影响干细胞巢中的细胞种群数量及其动力学行为，调整干细胞巢细胞极性有关的科学假说。. 研究发现，在AOM/DSS结肠癌前病变小鼠模型中：①萸连不同配比均可不同程度降低结肠组织炎症因子表达，减少ACF形成，有效阻延结肠癌前病变演进病程，其中以萸连1:1配比疗效最佳，为配伍比例控制功效发挥方向提供了实证依据。②并且萸连1:1配比通过抑制结肠癌前病变小鼠结肠癌干细胞异常增殖，调复结肠干细胞巢细胞极性从而减少ACF形成；③萸连1:1配比调控干细胞标记因子表达，与其降低结肠组织中Rspo3表达，抑制Wnt/β-catenin通路，调复结肠干细胞巢细胞极性减少ACF形成密切相关。在结肠癌细胞模型中，我们观察萸连药对主要药效成分小檗碱、吴茱萸碱联用效应，研究发现：①小檗碱、吴茱萸碱联用对结肠癌细胞活力明显优于两药单用，对细胞周期及迁移侵袭亦表现出相同效应；细胞周期的阻滞作用不但没有被消减，反而即可阻滞在G0/G1期，亦可阻滞在G2/M期，发挥双重保险的作用；②小檗碱和吴茱萸碱联合使用能显著抑制结肠癌细胞侵袭与转移，其作用机制与抑制P38/MAPK/MAPKAPK2/HSP27信号通路相关。. 以上研究探明了萸连不同配伍比例发挥调控ACF以及“ACF-不典型增生-腺瘤-腺癌”进程从而阻延结肠癌前病变功效方向的特定优势配比，揭示了萸连优势配比调控阻延结肠癌前病变的交联机制，为配伍比例控制方药功效发挥方向提供实证依据，有助于推动方剂配伍理论的创新发展。

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