维生素B12缺乏致AGTRAP mRNA核-质穿梭障碍在NAFLD中的机制研究

The activation of AGT/AT1R pathway is essential to the pathogenesis of nonalcoholic fatty liver disease (NAFLD), the AGTRAP protein inhibits this pathway. Vitamin B12 deficiency is closely associated with non-alcoholic fatty liver disease (NAFLD), but there are few studies in its mechanism. Unpublished data of our laboratory found that vitamin B12 deficiency can induce NAFLD in high-fat diet rats and that was associated with AGTRAP down-regulation and AGT/AT1R pathway activation. In vitro experiments we found that vitamin B12 deficiency can induce the RNA binding protein HuR subcellular mislocalization (retention in nucleus), and the screening revealed that the AGTRAP mRNA has a binding site with the HuR, The comprehensive literature review and our unpublished data suggest that the posttranscriptional modification of HuR after vitamin B12 deficiency results in the decrease of nuclear export ability of AGTRAP mRNA and the down-regulation of AGTRAP protein, which weakens the antagonistic effect of AGTRAP on AT1R. This project intends to confirm that vitamin B12 deficiency can lead to impaired subcellular localization of HuR and down-regulation of AGTRAP in liver tissue and cell line level. Then using implication of cell experiments to confirm that vitamin B12 supplementation can promote HuR/AGTRAP mRNA nuclear export increase. This will give us a full understanding of the important role of vitamin B12 in the process of NAFLD and its regulatory mechanism, which will provide new targets for exploring NAFLD treatment.

AGT/AT1R通路激活是非酒精性脂肪性肝病（NAFLD）发病关键，AGTRAP蛋白可抑制该通路，维生素B12缺乏与NAFLD密切相关，但其机制未明。本室前期发现，B12缺乏可诱导大鼠NAFLD的发生并伴AGTRAP蛋白低表达和AGT/AT1R通路激活，体外实验发现B12缺乏可致RNA结合蛋白HuR定位发生胞核滞留，筛选发现AGTRAP mRNA与HuR有结合位点。综合文献和预实验可推测:B12缺乏导致HuR翻译后改变，使其携带AGTRAP mRNA出核能力降低，下调AGTRAP蛋白表达，从而弱化了该蛋白拮抗AT1R的促NAFLD作用。本项目拟先在肝组织和肝细胞水平证实B12缺乏可导致HuR亚细胞定位障碍和AGTRAP低表达，再利用细胞实验反证补充B12可以促进HuR携带AGTRAP mRNA出核增加。这将使我们充分认识B12在NAFLD中的重要地位及其调控机制，为探索其治疗提供新的靶点。

非酒精性脂肪性肝病（NAFLD）在全球发病率逐年增高，它的发生发展与炎症信号传导相关。近年来对NAFLD的研究越来越受到关注，但其潜在的机制仍然不明确。新近的研究发现AGT/AT1R通路激活是非酒精性脂肪性肝病（NAFLD）发病关键，ATRAP（又称AGTRAP）蛋白可抑制该通路。换言之，血管紧张素II（Ang II/AGT）可以通过作用于其主要受体AT1R（AGT的1型受体）引发脂质代谢障碍和胰岛素抵抗从而导致NAFLD进展，而ATRAP可以拮抗AT1R的促NAFLD作用。维生素B12缺乏与NAFLD密切相关，但其机制未明。本室前期发现，B12缺乏可诱导大鼠NAFLD的发生并伴ATRAP蛋白低表达和AGT/AT1R通路激活，体外实验发现B12缺乏可致RNA结合蛋白HuR定位发生胞核滞留，筛选发现ATRAP mRNA与HuR有结合位点。综合文献和预实验可推测:B12缺乏导致HuR翻译后改变，使其携带ATRAP mRNA出核能力降低，下调ATRAP蛋白表达，从而弱化了该蛋白拮抗AT1R的促NAFLD作用。因维生素B12可以调控机体内S-腺苷甲硫氨酸（SAM）的含量，SAM则是哺乳动物细胞中的主要生物甲基供体，故本项目进一步深入研究了补充SAM将如何调节ATRAP。我们发现SAM在NAFLD中被耗尽，而补充SAM可以改善脂肪变性。此外，在用油酸（OA）处理的L02细胞中和高脂饮食喂养C57BL/6小鼠体内ATRAP表达较低、SAM浓度较低。我们同时发现HuR的亚细胞定位由SAM浓度决定，而SAM可调控蛋白质甲基化。此外，我们证实了HuR可直接结合ATRAP mRNA并控制其核-质穿梭。因此，其中核心机制就是SAM可通过维持ATRAP mRNA的出核来上调ATRAP蛋白表达。我们的研究结果表明，SAM可以上调NAFLD中的ATRAP蛋白表达，抑制AGT/AT1R通路，从而对NAFLD的治疗带来益处。