基于炎症微环境探讨鳖龙软肝汤通过CXCL12/CXCR4轴抑制肝纤维化-肝癌进程的机制研究

Liver fibrosis is an important link in the evolution of chronic liver disease to hepatocellular carcinoma. Bielong Ruangan Decoction has the effect of anti-hepatic fibrosis and anti-hepatocellular carcinoma, however, the mechanism remains unclear. Our pilot study found hepatic Stellate cells (HSCs) were continuously activated in the progression of liver fibrosis to hepatocellular carcinoma, leading to up-regulation of CXCL12/CXCR4 expression, and activation of Wnt/β-catenin signaling pathway. Our results also showed macrophages were therefore recruited to liver inflammatory microenvironment, and Bielong Ruangan Decoction inhibited the serum level of CXCL12 in various stages of liver fibrosis-hepatocellular carcinoma process. Based on our pilot study, we hypothesize that Bielong Ruangan Decoction inhibits liver fibrosis-hepatocellular carcinoma process through CXCL12/CXCR4 axis. In this project, we will use serum pharmacology method and cell co-culture technique to study the effect of Bielong Ruangan Decoction on the activation of HSCs, Wnt/β-catenin signaling pathway and the expression of CXCL12/CXCR4, to study the interaction between HSCs and macrophages, as well as the impact of macrophages on the biological behaviors of hepatocellular carcinoma cells. Results will be further confirmed by data obtained in tissues from Hepatitis-fibrosis-hepatocellular carcinoma rats model and patients with liver fibrosis, liver cirrhosis and hepatocellular carcinoma. The purposes of this study is to elucidate the inhibitory effect and the mechanism of Bielong Ruangan Decoction on liver fibrosis-hepatocellular carcinoma process, and by which will provide new ideas for the prevention and treatment of liver fibrosis and hepatocellular carcinoma with traditional Chinese medicine.

肝纤维化是慢性肝病向肝癌演变的重要环节，鳖龙软肝汤具有抗肝纤维化及抗肝癌作用，机制不明。前期研究发现，“肝纤维化-肝癌”演进过程中肝星状细胞（HSCs）持续活化， CXCL12/CXCR4表达上调，Wnt/β-catenin通路活化，肝脏炎症微环境内巨噬细胞聚集，鳖龙软肝汤抑制“肝纤维化-肝癌”各期患者血清CXCL12水平。据此，我们提出科学假设：鳖龙软肝汤通过CXCL12/CXCR4轴抑制肝纤维化-肝癌进程。本研究将应用中药血清药理学方法及细胞共培养策略，研究鳖龙软肝汤对HSCs活化、Wnt/β-catenin通路活性及CXCL12/CXCR4表达的影响，HSCs与巨噬细胞的相互调控作用及巨噬细胞对肝癌细胞生物学行为的影响，并在“肝纤维化-肝癌”大鼠模型及肝炎、肝硬化、肝癌患者组织中验证。研究将阐明鳖龙软肝汤抑制“肝纤维化-肝癌”演进的作用及机制，为中医药防治肝纤维化-肝癌提供新思路。

肝纤维化是慢性肝病向肝癌演变的重要环节，鳖龙软肝汤具有抗肝纤维化及抗肝癌的作用，机制不明。本研究首先通过回顾性研究，观察鳖龙软肝片对乙型肝炎肝硬化患者肝癌发生率的影响，评价鳖龙软肝片降低乙型肝炎肝硬化相关肝癌发生率的效果；随后从网络药理学及炎症微环境角度探讨鳖龙软肝汤抗肝纤维化-肝硬化-肝癌的物质基础与作用机制。结果发现鳖龙软肝片能抑制乙型肝炎肝硬化患者肝癌的发生率，鳖龙软肝汤能抑制肝纤维化-肝癌进程。网络药理学分析发现鳖龙软肝汤与肝纤维化-肝癌相关度较大的核心成分是paeoniflorgenone（芍药苷），albiflorin（芍药内酯苷），hederagenin（常春藤皂苷元），bisdemethoxycurcumin（双去甲氧基姜黄素）等，核心靶点是IL-6、MAPK8、CCND1、RELA、ICAM1等，核心通路包括 Metabolic pathways、TNF、pathways in cancer等。成功构建小鼠肝纤维化模型，发现纤维化小鼠模型中TGF-β1、CXCL12和IL-6等促炎因子以及IL-10等抗炎因子与肝纤维化密切相关。鳖龙软肝汤的活性成分芍药苷能通过抑制Wnt信号通路抑制CXCL12/CXCR4轴抑制 TGF-β1 诱导的肝星状细胞活化及自噬。鳖龙软肝汤的活性成分双去氧甲基姜黄素能通过CXCL12/CXCR4抑制巨噬细胞M2极化抑制肝纤维化-肝癌，提示鳖龙软肝汤可能通过抑制CXCL12/CXCR4轴调节炎症微环境抑制肝纤维化-肝癌进程。本研究为中医药防治纤维化及肝癌提供新的思路，同时为开发药效物质基础明确、作用机理清楚的创新药物提供前期基础。