幼鼠非酒精性脂肪肝病对胰岛素样生长因子-1表达的影响机制研究

The incidence of children with non-alcoholic fatty liver disease (NAFLD) has gradually increased over the years. Growth is an important measure of the health of children, and the insulin-like growth factor (IGF-1) is an important factor to promote children's growth. Preliminary studies confirmed that growth retardation in NAFLD pups is related to the decreased IGF-1 expression, and that berberine can improve fatty liver intracellular lipid deposition and significantly increase the IGF-1 expression levels. This study intends to build pups NAFLD animal (organism) models and cell models based on fat diet, observe the effects of high-fat environment on the AMP-activated protein kinase (AMP-Activated Protein Kinase AMPK) signaling pathway, and make a preliminary investigation of the relationship between AMPK and IGF-1 low expression in organisms; build the NAFLD cell model, and through the AMPK inhibitor Compound C, AMPK agonist AICAR, adenovirus knock down or over-expression of AMPK, observe the expression levels of IGF-1, IGFBP3 mRNA and protein in liver cells; and through further use of AMPKa1 specific knockout mice, verify the functioning mechanism of the fatty liver in the lowering of IGF-1 expression levels as induced by AMPK.AMPK activity in the liver cells observed after berberine intervention, to explore new drugs to improve children's fatty liver of IGF-1 low expression.

儿童非酒精性脂肪肝病（NAFLD）发病率逐年提高，生长是儿童健康的重要指标，胰岛素样生长因子（IGF-1）是促进生长的重要因子，前期研究证实NAFLD幼鼠存在生长障碍与IGF-1低表达相关，小檗碱能改善脂肪肝细胞内脂质沉积，并上调IGF-1的表达。本研究拟通过高脂环境诱导幼鼠NAFLD模型和细胞模型，观察高脂环境对AMP激活的蛋白激酶(AMPK)信号通路的影响，探讨AMPK活性与IGF-1低表达的关系；通过AMPK抑制剂Compound C及AMPK激动剂AICAR、腺病毒敲降和过表达AMPK，观察肝细胞内IGF-1、IGFBP3的mRNA和蛋白表达水平；进一步利用AMPKa1特异性敲除小鼠，验证脂肪肝通过AMPK引起IGF-1低表达的作用机制；观察小檗碱的干预对肝细胞AMPK活性的影响，探索改善儿童脂肪肝IGF-1低表达的新药物。

儿童肥胖及非酒精性脂肪肝病（non-alcoholic fatty liver disease，NAFLD）发病率呈不断增高趋势，且发病年龄趋向年轻化。我们前期研究证实NAFLD 幼鼠存在生长障碍与IGF-1 表达下降有关。IGF-1 调节机体的糖脂代谢，在参与糖脂代谢的靶器官中AMPK 起着重要的作用, 激活 AMPK 活性具有改善胰岛素抵抗，进而促进肝细胞分泌胰岛素样生长因子表达的作用，据此，我们提出非酒精性脂肪肝病通过机体AMPK 活性影响IGF-1 上游信号通路的科学假说。本研究拟通过高脂饮食建立幼鼠NAFLD模型和油酸诱导人肝癌细胞，检测机体IGF-1 及其结合蛋白IGFBP-3 的表达水平，观察高脂环境对肝细胞磷酸化AMPK 活性及ACC 的蛋白和AMPK、ACC mRNA 的表达影响，初步探讨机体AMPK 活性与IGF-1 低表达的相关性。. 研究结果表明：（1）高脂饮食诱导的NAFLD幼鼠肝细胞内IGF-1、IGFBP-3、p-AMPK、p-ACC表达明显下调，IGF-1、IGFBP-3、p-AMPK、p-ACC蛋白表达明显下调。（2）脂肪变性肝细胞内IGF-1、IGFBP-3、p-AMPK、p-ACC表达明显下调，IGF-1、IGFBP-3、p-AMPK、p-ACC蛋白表达明显下调。（3）使用AMPK抑制剂处理动物模型和细胞模型后，IGF-1、IGFBP-3、p-AMPK、p-ACC表达明显下调，IGF-1、IGFBP-3、p-AMPK、p-ACC蛋白表达明显下调，且血中的IGF-1浓度明显减少；用激动剂后，其表达相反。（4）敲除肝细胞模型AMPK基因后，IGF-1、IGFBP-3、p-AMPK、p-ACC表达明显下调，IGF-1、IGFBP-3、p-AMPK、p-ACC蛋白表达明显下调。（5）小檗碱有抑制肝细胞内TG 堆积的作用，且浓度为1mmol/L 时效果最佳；小檗碱能激活肝细胞内FXR 的表达，抑制FAS 和SREBP-1c 的表达，提示小檗碱可能是通过作用于FXR/SREBP-1c/FAS 信号通路改善肝脏脂肪变性。（6）非酒精性脂肪肝病抑制肝内IGF-1的表达可能是通过调节AMPK/ACC信号通路实现的。