β肾上腺素能信号途径介导肝气郁结对结肠癌肝转移的影响及逍遥散的作用

Liver metastases is the most common cause of death in colorectal cancer patients. Stagnation of liver qi plays an important role in occurrence and development of colorectal cancer，but the mechanism is unknown. Our previous research found that stagnation of liver qi could promote liver metastases of colon cancer in mice, and increase peripheral blood norepinephrine, epinephrine levels. We put forward the hypothesis that stagnation of liver qi promote liver metastases of colon cancer throughβ-adrenergic signaling pathways.We will use the technology of RNA interference, flow cytometry, reverse transcriptase real-time fluorescent quantitative PCR, immunohistochemistry, Western Blot and high performance liquid chromatography (HPLC) to explore the relationship among liver depression, liver metastases and sympathetic function, confirm β-adrenergic signaling pathways as a bridge between liver depression and liver metastases, observe how liver depression affect tumor-associated macrophages and tumor metastases associated gene expression through β-adrenergic signaling pathways, and how representative prescription of dispersing stagnated liver qi for relieving qi stagnation(Xiaoyao Powder) affect tumor metastases. Combined with in vitro experiments, investigate the possible targets of Xiaoyao Powder affect β-adrenergic signaling pathways. The study is of great significance to clarify the essence of colon cancer caused by liver depression. It will lay the theoretical foundation for enriching the theories of TCM etiology and pathogenesis, and the connotation of dispersing stagnated liver qi for relieving qi stagnation, and improving the effect of colorectal cancer furtherly.

肝转移是结直肠癌最重要的死亡原因。情志不遂，久郁伤肝，肝气郁结在结直肠癌发生发展过程中发挥着重要作用，但其机制不明。我们前期工作发现，肝气郁结可促进小鼠结肠癌肝转移，使外周血去甲肾上腺素、肾上腺素水平升高。据此提出假设：肝气郁结通过β肾上腺素能信号途径促进结肠癌肝转移的发生。本课题用RNA干扰、流式细胞术、实时荧光定量PCR、免疫组化、Western Blot、高效液相色谱分析等技术，分析肝郁与肝转移、肝郁与交感神经之间的关系，验证β肾上腺素能信号途径在肝郁与肝转移之间的桥梁作用，观察肝郁通过β肾上腺素能信号途径对肿瘤相关巨噬细胞以及肿瘤转移相关基因表达等的影响，观察疏肝解郁代表方逍遥散对肿瘤转移的作用，并结合体外实验初步探讨逍遥散对β肾上腺素能信号途径影响的可能靶点。该研究对阐明结肠癌肝郁致病的本质有重要意义，为丰富中医病因病机学说，充实疏肝解郁内涵，进一步提高结直肠癌疗效奠定基础。

目的：.研究肝气郁结对结肠癌肝转移的影响及β-AR信号通路在其中的作用，并且采用逍遥散观察疏肝解郁法对防治肝气郁结证结肠癌肝转移的作用。.方法：.通过束缚制动法构建肝气郁结证模型，在肝气郁结证模型的基础上，通过脾内注射人结肠癌细胞株HT-29建立肝转移模型，BALB/C裸小鼠随机分为空白对照组、普萘洛尔对照组、ICI118,551对照组、空白肝郁组、普萘洛尔肝郁组、ICI118,551肝郁组,以了解肝气郁结对结肠癌肝转移的影响。并将BALB/C裸小鼠随机分为空白对照组、逍遥散对照组、空白肝郁组、逍遥散肝郁组，以观察逍遥散对肝气郁结证结肠癌肝转移的作用。.结果：.1.肝郁组与对照组相比，小鼠下丘脑组织中NE水平升高，DA和5-HT水平降低。肝郁组小鼠血清NE、E水平较对照组升高，脾脏种植瘤组织中NE 水平较对照组升高。肝郁组小鼠肝脏转移灶较对照组显著增多。肝气郁结能增强脾脏对TAMs 和MDSCs的募集作用，显著上调脾脏种植瘤组织TGF-β、IL-6、VEGF和MMP-9表达，显著增加肝转移瘤组织VEGF和CD31蛋白表达。.2. 逍遥散能显著抑制结肠癌肝转移，减少脾脏TAMs和MDSCs。逍遥散肝郁组与空白肝郁组相比，脾脏种植瘤组织TGF-β、IL-6、VEGF和MMP-9表达水平均显著降低，肝转移瘤组织VEGF和CD31蛋白表达均显著减少。.结论：.肝气郁结可通过β-AR信号通路促进结肠癌肝转移，具有疏肝健脾作用的逍遥散具有调节β-AR信号通路抑制结肠癌肝转移的作用。