基于“肠道菌-短链脂肪酸-肝NRF2”途径的人参缓解环磷酰胺肝毒性机制研究

To illuminate the protective mechanisms of Traditional Chinese Medicine on chemotherapeutic drugs induced toxicity are very important in clinical tumor treatment. Our previous researches, funded by the Youth Project of National Natural Science Foundation of China, showed that ginseng could alleviate cyclophosphamide induced hepatotoxicity through inducing the expression of hepatic NRF2 with the presence of intestinal bacteria. However, the intestinal bacteria involved mechanism of how ginseng up-regulates the level of hepatic NRF2 is still unknown. Mountain evidences exhibited that butyrate, one of short chain fatty acids (SCFAs) metabolites of intestinal bacteria, could elevate the expression of NRF2, and ginseng could regulate the diversities of intestinal bacteria. Therefore, in this project, a hypothesis is proposed: ginseng will modulate the characterization of intestinal bacteria, alter the absorbed compositions and/or contents of SCFAs from intestine to liver, activate the relative pathways to elevate the hepatic NRF2 level, and finally alleviate cyclophosphamide induced hepatotoxicity. Several experiments will execute both in cells and animals to verify the hypothesis, such as using metagenomics technique to characterize the profile of intestinal bacteria, utilizing metabolomics method to investigate the compositions and contents of SCFAs in intestinal tract and liver, and employing integrated methods based on iTRAQ proteomics and molecular biology to analyze the expression of NRF2 and its upstream regulatory proteins. The aim of this project is to illuminate intestinal bacteria-SCFAs-hepatic NRF2 pathway involved protective mechanism of ginseng on cyclophosphamide induced hepatotoxicity, which could provide scientific basis for the combination of ginseng and cyclophosphamide for the clinical treatment, and novel idea for the anti-tumor research of integrated Chinese Medicine and Western Medicine.

阐明中药与化疗药联用减毒机制对肿瘤治疗具有重要意义。申请人在国自然青年项目的资助下证实，人参通过上调肝NRF2水平缓解环磷酰胺所致的肝毒性，该调控作用与肠道菌密切相关，但具体机制有待深入研究。业已证明丁酸（肠道菌的短链脂肪酸类代谢产物）可上调NRF2水平，人参可改变肠道菌组成。本研究拟采用宏基因组方法表征肠道菌组成、采用代谢组学方法分析肠内容物和肝脏中丁酸等短链脂肪酸组成和含量、采用蛋白组学iTRAQ技术和分子生物学手段评价肝NRF2和上游调控蛋白水平，在动物和细胞水平验证如下科学假说：人参通过调节肠道菌组成，改变由肠道吸收入肝的短链脂肪酸的组成或/和含量，提高肝NRF2和上游调控蛋白水平，从而缓解环磷酰胺所致的肝毒性。通过假说的验证，揭示肠道菌-短链脂肪酸-肝NRF2途径的人参缓解环磷酰胺肝毒性机制，阐明联用的科学内涵，为临床应用提供理论依据，同时也为中西医结合抗肿瘤创新研究提供新思路。

临床研究表明人参有效缓解环磷酰胺所致的肝损伤，课题组前期研究提示该作用与肠道菌密切相关，但具体机制有待深入研究。本项目采用宏基因组方法表征肠道菌组成、采用代谢组学方法分析肠和肝内短链脂肪酸等代谢产物组成和含量、结合蛋白组学等分子生物学技术手段，在动物和细胞水平探讨肠道菌介导的人参缓解环磷酰胺肝毒性机制。主要研究结果如下：（1）动物水平证实人参有效缓解环磷酰胺所致的肝损伤。具体机制包括：人参改善环磷酰胺所致的肠道菌失调，提高菌群多样性以及拟杆菌、双歧杆菌和粪杆菌等短链脂肪酸生成菌的丰度，并调节菌群参与的能量代谢和脂质代谢等；人参改善环磷酰胺所致肠内代谢紊乱，显著提高丙酸和丁酸等短链脂肪酸水平，回调肠内α-二吗啡酸等25个潜在生物标志物的相对丰度，调节亚油酸代谢等生物学过程；人参改善环磷酰胺所致肝内代谢紊乱，特异性提高丙酸和丁酸等短链脂肪酸水平，回调肝内牛磺脱氧胆酸等17个潜在生物标志物的相对含量，调节甘油磷脂代谢等生物学过程；人参改善环磷酰胺所致的肝内蛋白表达失衡，提高肝内GST和COX等蛋白表达水平，改善亚油酸代谢、NF-κB信号通路和胆固醇生物合成等生物学过程。（2）在细胞水平证实肠菌代谢产物——丁酸等短链脂肪酸通过IL-17信号通路和MAPK信号通路缓解环磷酰胺所致的肝细胞损伤，进一步研究表明，丁酸等短链脂肪酸通过促进PI3KR3的表达，提高核内NRF2的水平，继而通过JAK-STAT等信号通路，促进GST等基因表达。总之，人参通过促进拟杆菌、双歧杆菌和粪杆菌等肠道菌增殖，改善肠内和肝内丙酸、丁酸等代谢物的丰度，进而通过NRF2-JAK-STAT等信号通路，促进肝脏中GST和COX等蛋白表达，缓解环磷酰胺的肝毒性。本研究为人参与环磷酰胺联用提供理论依据，也为中西医结合抗肿瘤研究提供新思路。本项目已发表学术论文9篇，申请发明专利1项，培养研究生5名、省级人才1人。