壳寡糖基于miR-384-5p/LepRb通路调节肝脏糖代谢干预NAFLD的作用及机制研究

Glucose metabolic disorders play a key role in the development of non-alcoholic fatty liver disease (NAFLD), thus, seeking the possible treatment of glucose metabolic disorders is critical for the prevention and treatment of NAFLD. Leptin is involved in glucose metabolism regulation by LepRs which are on the surface of hepatocytes. Using transcriptome analysis, we previously found that chitosan oligosaccharide (COS) induced the upregulation of LepRb and the down-regulation of SOCS3. Also, COS changed the Jak-STAT pathways which were associated with Leptin receptor signal transmission to improve glucose metabolism disorder in NAFLD mice, while the specific mechanism is not clear. Combined with bioinformatics query and qRT-PCR analysis, we found that LepRb was the potential target gene of miR-384-5p, and COS could lower the expression of miR-384-5p in liver. Base on the above studies, the present project aims to induce miR-384-5p and LepRb overexpression, inhibition in vitro, as well as miR-384-5p overexpression in liver to study the detailed mechanism of COS in regulating hepatic glucose metabolism via miR-384-5p/LepRb, which will provide the evidence for the further development of COS, as well as the new strategy to prevent and cure NAFLD.

非酒精性脂肪性肝病（NAFLD）发生发展的中心环节是糖代谢紊乱，探寻糖代谢紊乱的有效控制手段对于防治NAFLD至关重要。瘦素通过肝细胞表面瘦素受体（LepRs）参与糖代谢调节。我们前期通过RNA-seq转录组测序发现，壳寡糖上调肝脏LepRb表达，下调SOCS3表达，诱导瘦素受体信号传递的Jak-STAT等糖代谢信号通路显著变化，改善肝脏糖代谢紊乱，但其具体机制尚不明确。申请人结合生物信息学查询和qRT-PCR检测，发现LepRb是miR-384-5p潜在靶基因，壳寡糖降低肝脏miR-384-5p表达。基于上述研究，本项目拟在细胞模型诱导miR-384-5p、LepRb过表达/抑制，动物模型诱导肝脏miR-384-5p过表达，研究miR-384-5p/LepRb通路在介导壳寡糖调节糖代谢，干预NAFLD中的作用机制。项目预期将为壳寡糖的功能开发提供理论依据，为NAFLD的防治提供新策略。

非酒精性脂肪肝已成为一个主要的公共卫生问题，给发达国家和发展中国家造成了巨大的医疗和经济负担。在动物实验和人体临床试验中，在对防治非酒精性脂肪肝的不同药物治疗效果的研究中都表明，许多药物存在着较大的副作用和持久稳定性较短的特点。壳寡糖是天然来源的低分子量氨糖聚合物，经研究表现出具有延缓肥胖和胰岛素抵抗发生的生物活性，作为自然食源性物质可以在预防脂质堆积，缓解系统炎症发挥积极作用。.本项目利用高脂饮食诱导非酒精性脂肪肝小鼠，RT-PCR和Western blotting检测GRP-78，PERK，NF-κB p85 mRNA和蛋白水平，结果显示壳寡糖预防组GRP-78，PERK，NF-κB p65表达低于高脂肥胖组。壳寡糖通过抑制脂肪组织内质网应激，缓解脂肪组织炎症和胰岛素抵抗，进而调节机体的代谢紊乱。检测小鼠皮下脂肪组织炎性因子IL-6，TNF-α的mRNA水平以及蛋白质表达。研究发现壳寡糖预防组较HF组皮下脂肪组织TNF-α，IL-6 mRNA和蛋白表达均显著降低。结果表明壳寡糖能有效预防皮下脂肪组织炎性因子的合成和分泌，缓解机体胰岛素抵抗。壳寡糖的干预可以有效降低小鼠体重、糖耐量、肝重、甘油三酯、总胆固醇含量和附睾脂肪重量，减轻肝脏脂质堆积，改善肝脏组织和附睾脂肪组织损伤。壳寡糖可以降低血清瘦素含量，提升肝脏瘦素受体水平，减轻由高脂饮食诱导的非酒精性脂肪肝小鼠的瘦素抵抗；降低空腹血清胰岛素值和空腹血糖值，减轻小鼠胰岛素抵抗；减轻血清内毒素累积，增强小鼠肠屏障功能。激活JAK2-STAT3信号通路中JAK2和STAT3蛋白的磷酸化，发挥生物学效应。壳寡糖可以提升肠道菌群的物种丰富度和群落多样性，调节肠道菌群的物种组成。增加肠道菌群中有益菌的丰度，调节肠道菌群平衡。增加短链脂肪酸含量，维持肠上皮屏障功能，改善葡萄糖和脂质代谢。壳寡糖主要通过调节与氨基酸代谢、碳水化合物代谢、脂质代谢、信号通路和人类疾病有关的肠道菌群，改善小鼠非酒精性脂肪肝症状。通过双荧光素酶报告基因检测miR-384-5p与LepRb靶标关系：发现LepRb是miR-384-5p潜在的靶基因，且壳寡糖能够降低肝脏miR-384-5p表达。