基于能量代谢介导的Th17/Treg平衡研究大黄蛰虫丸逆转肝癌耐药免疫学机制

Regulatory T cells (Treg) infiltration is an important immunological feature of HCC tissue. The energy metabolism intermediate α-KG could regulate the balance between Treg and T helper cell 17 cells (Th17) and determine the body's immune surveillance of liver cancer. However, whether these two groups of T cells participate in resistance of HCC still remains unclear. Our previous study showed that Dahuang Zhechong Pills (DHZCP) reversed the drug resistance in liver cancer by intervening cellular energy metabolism. Furthermore, our studies showed that DHZCP could affect Treg and Th17 related cytokines in drug resistant HCC tissues. We anticipated that DHZCP inhibit Treg differentiation and activates tumor immunity by interfering with energy metabolism in immune cells. Our subject here focuses on the energy metabolism of T cell and the balance and function of Th17/Treg affected by DHZCP in vitro and in vivo liver cancer models. This study will clarify the immunological mechanism of DHZCP in reversing drug resistant of HCCs by energy metabolism, and reveal its scientific connotation of “slow mid supplement deficiency”; At the same time, combination of immunotherapy drug pembrolizumab was to explore the scientific rationality of the DHZCP combined with immunotherapy.

调节性T细胞（Treg）浸润是肝癌的重要免疫学特征，能量代谢中间产物α-KG能够调节Treg与辅助T 17细胞(Th17)的分化平衡，决定了机体的免疫应答状态及免疫治疗的成败，是否与肝癌耐药有关，尚未充分阐明。我们前期研究显示大黄蛰虫丸（DHZCP）通过干预细胞能量代谢，逆转肝癌耐药，并能调节Th17/Treg细胞因子水平。由此提出科研假说：DHZCP能够调节肝癌组织T细胞能量代谢及分化，激活机体免疫监视功能，以杀灭癌细胞、逆转耐药。本课题拟采用免疫学和分子生物学方法，在阐述α-KG介导的T细胞代谢表型改变参与肝癌耐药的分子机制基础上，利用体内外模型，探究DHZCP对T细胞能量代谢、DNA甲基化、Th17/Treg平衡的影响及逆转肝癌耐药的免疫学机制，揭示其“缓中补虚” 的科学内涵；与免疫治疗药物pembrolizumab联用，探讨DHZCP配合免疫治疗方案的科学合理性。

调节性T细胞（Tregs）浸润是肝癌的重要免疫学特征，能量代谢中间产物α-KG参与调节Treg的分化，决定了机体的免疫应答状态及治疗成败。本研究发现肿瘤代谢产物乳酸能显著激活Naïve CD4+T细胞内LDHA及其辅酶NADH活性，促进α-KG向2HG转变，并通过ATP5B/mTOR/HIF-1α信号通路，诱导Treg细胞分化，形成抑制性免疫微环境；小剂量抗血管生成药索拉非尼虽然能抑制肝癌血管生成，但缺氧导致的乳酸堆积进一步加重免疫抑制，导致肿瘤耐药；大黄蛰虫丸（DHZCP）能显著增加DEN肝癌大鼠及H22肝癌小鼠CD8+T、Th1、Th17数量及细胞因子水平，减少Treg数量，调节其功能，恢复CD8+/CD4+T细胞平衡，改善抑制性免疫微环境。其中，大黄䗪虫丸正丁醇萃取（PB）部位通过激活Treg细胞PI3k-Akt的磷酸化可增加IFN-γ分泌；水提部位（PW）和乙酸乙酯萃取部位（PE）可抑制乳酸脱氢酶活力以及乳酸分泌，抑制Treg分化。谱效相关研究发现：大黄䗪虫丸中的大黄素、大黄酚、芦荟大黄素、汉黄芩素、黄芩素、去甲黄芩素、没食子酸、柚皮素、芹菜素、白杨素、甘草酸、刺芒柄花素、棕榈酸等成分变化与Treg细胞变化密切相关，是本方抑制CD4+T分化为Treg的主要物质基础。进一步利用体内外模型研究发现，有效成分汉黄芩素能够通过影响LDHA的活性及表达，调节α-KG向2HG转化，干预Naïve CD4+T细胞乳酸代谢，抑制Treg细胞分化；汉黄芩素与索拉菲尼联用能够改善缺氧和乳酸堆积造成的Treg比例上升及免疫抑制，作用与PD-1抗体Pembrolizumab相当，逆转索拉非尼耐药。总之，本研究在阐述α-KG/2HG介导的Treg分化机制基础上，利用体内外模型，探究DHZCP对T细胞乳酸、α-KG代谢、Th1/Treg平衡的影响及逆转肝癌耐药的免疫学机制，揭示其“缓中补虚”的科学内涵。相关研究成果可为中西药联用抗肝癌提供理论依据。