肠道菌群介导GPR35-PI3K/AKT信号通路在肠外营养相关肝脏脂肪变中的作用机制研究

Parenteral nutrition saves thousands lives for patients with intestinal failure, however, parenteral nutrition associated fatty liver disease (PNAFLD) limits the long-term use of PN. The potential mechanism of PNAFLD is not clear. Our previous studies showed that PNAFLD mice were accompanied with disturbed gut microbiota (GM), reduced KYNA level and its receptor GPR35. Interestingly, the similar results were also observed after germ-free mice were transplanted the GM from PNAFLD mice. The bioinformatics results for liver tissue suggested that the AKT signaling pathway might be involved in the development of PNAFLD. Therefore, we hypothesize that PN causes GM disturbance, reduces the level of KYNA, inhibits the activation of GPR35-PI3K/AKT signaling pathway in liver, and promotes the occurrence of PNAFLD. This project plans to establish a mouse PNAFLD model, to explore the role of GM in PNAFLD through GM transplantation from PNAFLD mice to Abx or GF mice; to investigate the mechanism of GM-mediated GPR35-PI3K/AKT signaling pathway in PNAFLD through cell experiments, animal experiments involving gene knockdown or protein inhibition and molecular biology techniques. The results of this project will provide a new theoretical basis for reducing PNAFLD and promoting the long-term effective implementation of PN in patients with intestinal failure.

肠外营养挽救大量肠衰竭病人生命，但PNAFLD的发生限制PN的长期使用，目前PNAFLD的发生机制并不明确。我们前期研究表明：PN可诱发小鼠PNAFLD伴随肠道菌群紊乱；KYNA及其受体GPR35水平降低；无菌小鼠移植PNAFLD小鼠肠菌后出现相似结果；肝组织生物信息学结果提示AKT信号通路可能参与PNAFLD发生。因此我们提出假设PN导致肠道菌群紊乱，引起KYNA降低，抑制肝组织GPR35-PI3K/AKT信号通路活化，促进PNAFLD发生。本项目拟建立PNAFLD小鼠模型，利用Abx、GF小鼠，通过肠道菌群移植探讨GM在PNAFLD中的作用；通过细胞实验，动物实验，利用基因敲减或蛋白抑制，运用分子生物学技术等方法验证，探讨GM介导GPR35-PI3K/AKT信号通路在PNAFLD中的作用机制。这一结果将为临床减少PNAFLD的发生发展及推动PN治疗的长期有效实施，提供新的理论基础。

背景与目的:肠外营养(PN)是一种挽救肠衰竭患者生命的治疗方法。肝脏脂肪变性是长期PN的潜在致命并发症，但其病理机制尚不完全清楚。在此，我们确定了蛋白磷酸酶2A (PP2A)在肠外营养相关肝脂肪变性(PNAHS)发病机制中的作用。..方法:对PNAHS患者肝脏样本进行蛋白质组学/磷蛋白质组学分析，以确定PNAHS的机制。采用全肠外营养(TPN)小鼠模型、体内、体外实验评价PP2A-Ca对肝脏脂肪酸代谢的影响。..结果:PP2A-Ca(催化亚基)表达降低，丝氨酸/苏氨酸激酶Akt2激活增强，磷酸腺苷活化蛋白激酶(AMPK)激活降低与PNAHS患者肝脏脂肪变性相关。给予PN 14天的小鼠出现肝脏脂肪变性，PP2A-Ca下调，Akt2激活，AMPK抑制。在给予PN的小鼠中，肝细胞特异性的PP2A-Ca缺失通过对脂肪酸降解的影响加剧了Akt2的激活、AMPK的抑制和肝脏脂肪变性，而肝细胞特异性的PP2A-Ca过表达显著改善了伴随Akt2抑制和AMPK激活的肝脏脂肪变性。此外，PP2A-Ca过表达小鼠中Akt2的药理激活导致肝脏脂肪变性的加重。..结论:我们的研究结果表明，肝脏PP2A-Ca凭借其改善肝脏脂肪变性和改善肝功能的作用而成为PNAHS的保护因素。我们的研究为PP2A-Ca可能参与PNAHS的发病机制提供了强有力的理论依据。