基于IDO调控免疫微环境探讨保肝宁防治肝纤维化的分子机制

Imbalance of hepatic immune microenvironment is an important mechanism for the development of liver fibrosis. Traditional Chinese Medicine and drugs have a unique advantage in the fields of the immune regulation and liver fibrosis prevention. It will provide safe and effective new tools for prevention hepatic fibrosis by Identification of key hepatic immunomodulatory signaling protein and development of natural drugs with clear targets. Clinical trials have indicated that Baoganning could block and reverse hepatic fibrosis, but its efficacy targets and molecular mechanisms are still not fully understood. Our previous study found that hepatic fibrosis induced the significantly up-regulation of indole amine 2,3-dioxygenase (IDO), with the elevated of dendritic cells expression and inflammatory cytokines releasing in the hepatic microenvironment. Interestingly, Baoganning could effectively reduce the liver fibrosis and the dendritic cells (DCs) expression and distribution in hepatic sinusoid, as well as the down-regulation of inflammatory cytokines. Based on the critical roles of IDO in tryptophan metabolism and DCs immune transmitting functions, we hypothesis that Baoganning could prevent hepatic fibrosis by improving hepatic immune microenvironment through IDO signal pathway. In this project, three-dimensional cell culture, gene recombination technology and current serum pharmacology will be used to investigate the mechanisms of Baoganning intervention immune microenvironment and the IDO-targeted regulation in liver fibrosis. This study will provide new ideas and new tools for further investigating of the mechanisms of liver fibrosis and related drug discoveries.

免疫微环境失衡是肝纤维化发生发展的重要因素，中医药在调节免疫与防治肝纤维化领域具有独特优势，鉴定肝脏免疫调节靶标及开发天然靶向制剂将对肝纤维化防治提供安全有效的崭新手段。临床试验证实保肝宁对肝纤维化具有阻断及逆转作用，但其药效靶标与分子机制尚不完全清楚。基于哚胺2, 3-双加氧酶（IDO）干预色氨酸代谢以及调控树突状细胞（DCs）免疫呈递功能，我们前期研究发现肝纤维化诱导IDO显著上调，同时伴随DCs增多与富集以及微环境中炎症因子释放增加；而保肝宁在显著改善纤维化的同时，降低肝脏DCs富集并减少微环境中炎症因子的释放。由此我们设想保肝宁通过介导IDO调控免疫微环境的动态平衡，从而达到延缓或逆转肝纤维化发展的作用。本项目拟通过基因重组、细胞三维培养及现代血清药理学等技术探讨保肝宁干预肝纤维化的免疫微环境机制内涵及IDO靶向调控机制。本研究的开展将为肝纤维化机制探索及药物开发提供新思路。

肝硬化是世界范围内危害健康的重要问题之一，进一步加快其前期病理进程“肝纤维化” 的发病机制和创新药物研发，显得刻不容缓。本课题组前期研究提示：IDO1可能在肝纤维化中起重要作用，抑制IDO1的表达可能是治疗肝纤维化的潜在靶点。保肝宁从清热祛湿解毒、活血化瘀软坚、益气疏肝健脾治疗临床上肝纤维化患者取得良好疗效。依据课题组前期对保肝宁防治肝纤维化机制的研究结果，我们推测保肝宁作用机制可能和抑制IDO1有关。.本项目主要在体内外探讨了IDO1干预色氨酸代谢及DCs免疫呈递参与调控肝纤维化的作用机制并验证了保肝宁及其有效成分-人参皂苷Rg1介导IDO1干预肝纤维化免疫微环境的调控作用和分子机制。.首先，我们利用WT小鼠建立了CCL4及胆管结扎诱导的肝纤维化模型，发现模型小鼠肝脏中IDO1表达水平、KYN/TRP均显著升高且伴随着DCs成熟表型的降低，表现为CD11C+CD80+、CD11C+CD86+、CD11C+CD40+、CD11C+MHCII+DCs细胞、CD4+T细胞比例及CD4+/CD8a+比值的降低。在LPS干预下，IDO1+/+BMDCs细胞的成熟表型较IDO1-/-BMDCs明显降低且IDO1抑制剂的浓度与BMDCs细胞成熟表型比例值之间存在显著的正性相关系。我们进一步利用IDO1缺失及过表达小鼠建立肝纤维化模型，发现IDO1的缺失能够抵抗CCL4及胆管结扎所诱导的肝纤维化损伤，可能是由于IDO1的缺失可诱导肝纤维化小鼠肝脏中DCs免疫表型的成熟，抑制肝星状细胞的活化；而肝脏特异性过表达IDO1可加重CCL4及胆管结扎所诱导的肝纤维化病理程度，可能是因为肝脏特异性过表达IDO1抑制了肝纤维化小鼠肝脏中DCs的免疫表型成熟，促进了胶原纤维的沉积。另外，我们发现保肝宁复方和其有效成分-人参皂苷Rg1均具有护肝、减轻肝纤维化程度的作用。其潜在机制可能是通过有效降低肝纤维化小鼠肝脏中IDO1表达水平，同时改变DCs的成熟表型，促进DCs功能的恢复。.本项研究通过探索保肝宁逆转肝纤维化发展的作用，明确了IDO1参与的TRP-KYN代谢及DCs功能和免疫呈递对肝纤维化调控的关键作用和分子机制，为肝纤维化有效预防及调控寻找了新的分子靶点，同时为肝纤维化预防药物开发提供新的思路。