基于调节酸性微环境与CD8+T细胞活性研究盐肤木三萜抗大肠癌作用机制

Wet poison accumulate and endogenous phlegm is the basic pathogenesis of colorectal cancer (CRC), and tumor acidic microenvironment that induced by phlegm is one of the main factors that restricted CD8 + T cell activity and limited its response to killing the tumor. In our previous studies, we have found that triterpenoids of Rhus chinensis Mill. could inhibit acidic metabolites and increase CD8 + T cell activity in CRC. As a breakthrough point in this project, we put forward the working hypothesis that the mechanism of triterpenoids of Rhus chinensis Mill. inhibiting the CRC maybe by adjusting the acidic environment and enhancing the vitality of CD8 + T cell and antitumor activity. The total triterpenoids and its active sites are separated for detecting the influence on the tumor glycolysis and the lactic acid metabolism, and its effect on the regulation of CD8 + T cell activity in CRC are analyzed. Moreover, we discuss the roles and mechanisms of triterpene components in the intervention of glucose metabolic signaling pathways in CRC cells. In addition, the influence of triterpene components on CD8 + T cell and antitumor activity are analyzed, furthermore, the regulatory mechanism in PD-L1/PD-1, Fas/Fas L pathways are investigated by the flow and the molecular biology technology. The spleen injection of liver metastasis model is used to investigate the relationship between the host immune function and antitumor activity. This project takes the acidic microenvironment and CD8 + T cell activity as the focal point, further to study the role of triterpenoids in Rhus chinensis Mill. in improving antitumor activity of CD8 + T cells, and mobilizing the body's own immune and killing tumor function. Furthermore, we discuss its molecular mechanisms on regulation of lactic acid metabolism, glycolysis in tumor cells and the enhanced antitumor immune response. The aim of this project is to clarify the scientific connotations and mechanisms of herbal medicine with the efficacy of phlegm and detoxification on inhibiting CRC, further to provide more effective therapeutic strategies, new methods and ideas for TCM prevention and treatment of colorectal cancer.

针对湿毒蕴结、痰浊内生为大肠癌的基本病机，结合痰浊所致的酸性微环境限制了CD8+T细胞对癌症控制的现代认识。在盐肤木三萜抑制大肠癌细胞酸性产物代谢及其上调CD8+T细胞活性的前期实验基础上，本项目提出“盐肤木三萜抗大肠癌作用机制可能通过调节酸性微环境进而增强CD8+ T细胞活性与抗肿瘤活力”的工作假说。本项目以调节酸性微环境平衡为切入点，观察盐肤木总三萜及其活性部位对大肠癌细胞糖酵解途径的酸性产物生成、CD8+ T细胞活性的影响并分析其信号调控机制；采用流式分析和分子生物学技术，研究盐肤木三萜类物质对CD8+ T细胞活性与抗肿瘤活力的影响及PD-L1/ PD-1、Fas/Fas L信号机制；采用脾脏注射肝转移模型进行宿主免疫机能与抗肿瘤活力关系的研究。本项目以调节酸性微环境与CD8+ T细胞活性为聚焦点，深入研究盐肤木三萜改善CD8+ T细胞的抗肿瘤活力，调动机体自身免疫和肿瘤杀伤功能的作用，进而揭示其改善肿瘤细胞糖酵解、乳酸代谢与增强机体抗肿瘤免疫反应的分子机制。以期阐明化痰解毒中草药抗癌作用机理的科学内涵，进而为中医药防治大肠癌提供更为有效的治疗策略、研究新方法和思路。

针对湿毒蕴结、痰浊内生为大肠癌的基本病机，结合痰浊所致的酸性微环境限制了CD8+T细胞对癌症控制的现代认识。本项目以调节肿瘤酸性微环境与CD8+T细胞活性为切入点，主要研究内容如下：（1）研究盐肤木防治大肠癌的成分与效应之间的关系，明确盐肤木三萜有效部位对大肠癌细胞生长与免疫功能的影响；（2）盐肤木三萜活性部位及其有效成分抑制大肠癌细胞中乳酸产生及其作用机制；（3）探讨其调节T细胞、NK细胞活性与抑制大肠癌细胞的作用关键靶点和信号调控机制；证实盐肤木三萜有效部位对大肠癌动物模型的体内效应及其抗癌机理，初步评价其安全性。.本研究通过实验验证盐肤木三萜活性部位及其两种主要的活性化合物桦木酸（BTA）或桦木酮酸（BOA）对大肠癌细胞糖酵解和乳酸转运、酸离子通道介导的乳酸代谢靶标（即ALDOA，PKM2和LDHA）和关键途径（即与代谢相关的AKT / mTOR途径）具有调控作用，为盐肤木三萜发挥多层次、多靶点抗肿瘤作用与机制研究提供了实验依据。本项目进而发现盐肤木三萜活性部位上调CD8 + T细胞中GLUT1和LDHA的表达，诱导了T细胞糖酵解活性，并促进CD8 + T细胞代谢和激活的多种代谢受体的表达增加。此外，通过盐肤木三萜活性部位增强了CT26荷瘤小鼠模型CD8 + T细胞的活性，下调CT26细胞的PD-L1表达，且调节肿瘤诱导的CD8 + T细胞中PD-1的表达。盐肤木三萜有效成分（部位）调节CD8+ T细胞活性、NK细胞活性，进而抑制大肠癌细胞作用与增强抗肿瘤活力通过PD-L1/PD-1、Fas/Fas L信号调控机制密切相关，揭示盐肤木三萜活性部位不仅增强了CD8+ T细胞介导的免疫反应，且改善肿瘤酸性微环境，这一发现有望成为中医药防治大肠癌提供新的参考。本项目研究成果初步揭示盐肤木三萜活性部位及其有效成分对大肠癌细胞糖酵解及其酸性代谢产物的干预模式，进而阐明盐肤木解毒化痰的功效与抗癌作用机理的科学内涵，进而为中医药防治大肠癌提供了更为有效的治疗策略、研究新方法和思路。