青蒿素抗性疟原虫改变宿主肠道菌群-肠-肝代谢轴功能及潜在机制研究

The artemisinin resistance monitoring is deeply limited because of its nubilous resistant mechanism and the absence of effective biomarkers. It, therefore, is very important to find the artemisinin resistant biomarkers. It was reported that malaria parasites might pathologically altered the host gut barrier function which leads to dysbiosis and microbiota translocation. After then, endotoxin enters the blood through portal vein and hepatic metabolism was disordered. The metabolic signals in host gut microbiota-gut-liver axis are changed eventually. Our previous study also revealed a significant difference on the expression level of phaseⅠand Ⅱmetabolic enzymes among infected mice by sensitive or resistant parasites and control group. Hence, it is feasible to find the resistant biomarkers from the gut-liver metabolic axis. This project, therefore, constructed the relationships between resistant index and the functional alteration of gut microbiota-gut-liver axis and varied metabolic signals, by combining with analysis of gut barrier function, NMR and LC-MS/MS based metabolomics, LC-MS based protein absolute quatification proteomics, and high-through 16S rRNA sequence technology based microbiomics in established mice models infected by artemisinin sensitive and resistant malaria parasites. We aimed at searching the common and specific potential altered metabolites, metabolic enzymes, and gut microbiota induced by artemisinin senstive and resistant malaria parasites in host. Signals related to artemisinin resistance and potential mechanism are certified to lay the basis for a systematic elucidation of artemisinin resistance. Meanwhile, it will provide a new view for clinical malaria therapy to delay the arteminsin resistance assisted by fecal transplantation treatment.

青蒿素耐药机制不明，缺乏有效标志物，抗性监控受限，故其抗性标志物的发现十分重要。研究表明疟原虫可病理性改变宿主肠道屏障功能，引起菌群失调与易位，内毒素由门静脉入血，肝代谢紊乱，导致肠道菌群-肠-肝轴代谢信号改变；项目前期也发现敏感/抗性疟原虫感染及正常小鼠在青蒿素处理前后肝脏一相、二相代谢酶表达存在显著差异，表明从肠-肝代谢轴寻找抗性标志物可行。故本课题在已建立青蒿素敏感/抗性小鼠模型上，结合肠道病理分析、基于NMR/LC-MS代谢组学、基于质谱技术绝对定量蛋白质组学及高通量16SrRNA标签测序技术的微生物组学方法，构建抗性指数与肠道菌群-肠-肝轴屏障功能改变及差异代谢信号的相关性，以寻找青蒿素敏感及抗性疟原虫引起宿主共有/特异潜在差异代谢物、代谢酶及肠道菌群为目标，明确与青蒿素抗性相关的生物标志物及其潜在机制，为深入研究青蒿素耐药机制及临床上引入粪便移植方案，延缓青蒿素耐药奠定基础。

项目发现青蒿素抗性疟原虫为了逃避药物压力，延缓其生长发育，致病力下降，宿主死亡率降低，但较敏感疟感染小鼠小肠NK-κB mRNA水平显著上调近4倍，肠上皮紧密连接蛋白Claudin-1表达较敏感组下调，但Occuludin、ZO-1表达上调，青蒿素给药对敏感/抗性疟原虫感染小鼠肠紧密连接蛋白表达调控亦存在显著差异；青蒿素对抗性疟原虫活性下降，提示存在耐药风险，猫眼草黄素作为潜在P-gp抑制剂，与阳性药维拉帕米对敏感疟原虫无效，但直接杀灭抗性疟原虫，青蒿素-猫眼草黄素复配组对敏感疟原虫活性显著低于抗性疟原虫； 1H-NMR非靶向代谢组学发现敏感/抗性疟原虫感染小鼠血浆和不同组织存在代谢表型差异，筛选到共有/特异差异代谢物及代谢通路，其中糖代谢、氨基酸代谢/能量代谢可能是耐药性关键代谢途径，且猫眼草黄素可能对这些差异代谢物和代谢通路进行双向调控；敏感/抗性疟原虫感染后一相代谢酶CYP3a11、2C29及二相代谢酶Sult1d1、1a1绝对含量均显著下调，但各给药组间无显著差异；但二者感染后小鼠粪便肠道菌群多样性存在明显差异，与代谢组学结果一致，既有共有菌类似的变化（如拟杆菌属均下降，抗性组下降更多；乳酸杆菌敏感组下降更多，双歧杆菌二者均下降至1%以下，埃希氏菌属二者均增加，敏感组更高），也有各自不同的差异菌（敏感独有norank_c__1-20、Lautropia、红蝽杆菌属，抗性独有芽球菌属、微小杆菌属、树源生孢杆菌属及Solibacillus等）；猫眼草黄素亦可对相关菌群产生调控作用，如与免疫应答有关的埃希氏菌属增加，而青蒿素单独给药后埃希氏菌群明显减少。项目证明敏感伯氏疟原虫K173与以青蒿素等量倍增法筛选得到中等抗性的耐药疟原虫在宿主体内存在显著的表型差异，可能与青蒿素耐药密切相关，但肝代谢酶表达水平对耐药性影响不大；猫眼草黄素有望成为青蒿素耐药抑制或逆转剂，然而其上游调控机制仍不十分明确。