基于乏氧介导的STAT3/NF-κB/PD-L1信号轴解析化痰祛瘀方调控肝癌细胞免疫逃逸的分子机制

Immune escape is an important cause of tumor development, and enhancing the anti-tumor immunity of body as the core treatment strategy of Traditional Chinese Medicine (TCM) is the hot issue in present cancer research, and it is not clear for the effects of TCM on immune escape of liver cancer. Recent studies have shown that PD-L1 overexpression in tumor hypoxic microenvironment is the key factor for immune escape of cancer cell and may be related to the regulation of STAT3/NF-κB pathway and HIF-1α. Our previous researches found that PD-L1 expression was up-regulated in hypoxic liver cancer cell model in vitro, and Huatanquyu herbs intervention reduced PD-L1 expression. The mechanism of these effects may be related to the decrease of VEGF expression, inhibition of STAT3 pathway activation, and down-regulation of TWIST expression in hepatocellular carcinoma(HCC) cells. This project attempts to establish hypoxic liver cancer cell model in vitro, and orthotopic liver cancer transplantation and DEN-induced HCC mouse models in vivo. The techniques of gene cloning, Co-IP, ChIP and flow cytometry are applied to explore the mechanism of Huatanquyu herbs' influence on immune escape of HCC. The regulating effects of STAT3/NF-κB/PD-L1 signal axis on the expression of PD-L1 induced by hypoxia in HCC will be emphatically observed. This study will reveal the new drug targets of Huatanquyu herbs treatment of HCC and also provide a new scientific basis for the TCM treatment of HCC through phlegm and blood-stasis.

免疫逃逸是肿瘤发生发展的重要原因，而以增强抗肿瘤免疫为核心的中医治疗策略是当前肿瘤研究的热点，中医治疗对肝癌免疫逃逸作用尚不明确。最新研究显示，肿瘤缺氧微环境中PD-L1过表达是免疫逃逸发生的关键，可能与STAT3/NF-κB通路和HIF-1α调控有关。我们前期研究发现，体外缺氧肝癌细胞模型中PD-L1表达上调，而应用化痰祛瘀方干预后，PD-L1表达减少。其机制可能与降低肝癌细胞VEGF表达，抑制STAT3通路激活，下调TWIST表达有关。本课题尝试应用基因克隆、Co-IP、ChIP、流式细胞仪等技术，在体外缺氧肝癌细胞模型、体内小鼠原位移植肝癌和DEN诱发肝癌模型中探索化痰祛瘀方影响肝癌免疫逃逸的作用机制，重点观察STAT3/NF-κB/PD-L1信号轴对缺氧诱导的肝癌PD-L1表达的调控作用，揭示化痰祛瘀方治疗肝癌的药物新靶点，为从“痰瘀”论治肝癌提供新的科学依据。

免疫逃逸是肿瘤发生发展的重要原因，而以增强抗肿瘤免疫为核心的中医治疗策略是当前肿瘤研究的热点，中医治疗对肝癌免疫逃逸作用尚不明确。既往研究显示，肿瘤缺氧微环境中PD-L1过表达是免疫逃逸发生的关键，本项目基于前期化痰祛瘀方治疗肝癌取得良好疗效的基础上，探讨了化痰祛瘀方调控缺氧微环境抑制肝癌免疫逃逸的分子机制。通过注射二乙基亚硝胺(DEN) 120d，建立肝癌SD大鼠模型以及肝癌129S2/SvPasCrl小鼠模型；经过化痰祛瘀方治疗后，采用流式细胞术分析外周血、脾脏和肿瘤的免疫细胞群；ELISA检测血清细胞因子浓度；免疫组化检测肿瘤组织中的关键蛋白表达；采用HPLC/MS对化痰祛瘀含药血清进行化学成分鉴定；通过Western blot检测CoCl2处理后肝癌细胞株（HCCLM3、Hep-3B和Huh-7）HIF-1α、PD-L1、p-STAT3以及p-p65蛋白的表达水平；利用STAT3和NF-κB抑制剂和激活剂进一步分析野生型和敲除肝癌细胞系中上述蛋白的表达；采用CCK-8、免疫荧光和Western blot检测化痰祛瘀含药血清对缺氧状态下肝癌细胞株的影响。研究结果显示，化痰祛瘀方改善了肝脏的病理损伤，并抑制肝癌进展；化痰祛瘀方重塑肝癌肿瘤微环境，包括增加NK1.1+（NK）细胞和CD3+ CD8+ T (CTL) 细胞的比例，降低Ly-6G+CD11b+（MDSC)、F4/80+CD11b+（TAM）、FOXP3+CD25+ T细胞(Treg)和炎症因子IL-4、IL-6、IL-10、TNF-a的表达水平，抑制PD-L1介导的免疫逃逸；在缺氧状态下，经STAT3和NF-κB激活剂处理后，肝癌细胞系中HIF-1α、PD-L1、p-STAT3和p-p65表达上调；而STAT3和NF-κB阻断剂则可降低p-STAT3、p-p65、HIF-1α和PD-L1的表达；化痰祛瘀含药血清以浓度依赖的方式抑制缺氧肝癌细胞系中HIF-1α、PD-L1、p-STAT3和p-p65的表达。以上结果提示，化痰祛瘀方通过抑制STAT3/NF-κB信号通路降低HCC中PD-L1的表达发挥抗肝癌作用。本研究揭示了化痰祛瘀方治疗肝癌的药物新靶点，为从“痰瘀”论治肝癌提供了新的科学依据。