苹果多酚基于SIRT1去乙酰化活性改善肝脏脂肪变性的研究

Non-alcoholic fatty liver disease (NAFLD) now affects up to one-third of adults and a growing number of children in developed countries, and the prevalence of NAFLD is increasing in China. NAFLD is now becoming a major health issue throughout the world. However, therapeutic options are limited, and there had no drugs that could effectively prevent and treat NAFLD, thus, alternative treatment options are therefore warranted and intensively sought. Recent studies focused on the relationship between autophagy and lipid metabolism. SIRT1 is a member of the sirtuin family and a NAD+-dependent deacetylase that acts as a master metabolic sensor of NAD+. SIRT1 could activate autophagy by deacetylating the autophagy (Atg) proteins, while the ubiquitination of SIRT1 could affect its nuclear location and deaceylating activity. Our primary experiments demonstrated that apple polyphenol (AP) could eliminate the lipid droplets (LDs) deposited in liver induced by high fat diet (HFD) treatment, which accompanied with the up-regulation of SIRT1, NAMPT and LC3-II, especially the expression of SIRT1 is positively related to the role of apple polyphenol in eliminating LDs. Apple polyphenol treatment also could down-regulate the protein level of UHRF1. UHRF1 is an nuclear protein, which had the E3 ligase activity and could mediate the ubiquitination of proteins. Whether apple polyphenol treatment could inhibit and treat the deposition of LDs in liver is unclear, and its underlying mechanisms are also need to be explored, especially the effects of the ubiquitination of SIRT1 mediated by UHRF1on its deacetylase activity and the activation of autophagy. The current project plans to elucidate the beneficial role of apple polyphenol in the prevention and treatment of steatosis and the underlying mechanisms. HepG2 cells with different expression level of SIRT1, Atg7 and UHRF1, and SvJ129 mouse with different genotype of SIRT1 (Wild type: SIRT1+/+; Mutant type: SIRT1 y/y, namely ablation of the catalytic activity of SIRT1) are to be employed. The effect of apple polyphenol on hepatic steatosis would be determined by histopathological analysis after being treated with apple polyphenol for different time and at different concentration, and the role of apple polyphenol in the regulation of SIRT1 deacetylase activity, lipophagy and lipid metabolisms would be determined by RT-PCR, western blot, Co-IP, deaceylated analysis and ubiquitination analysis respectively. By determining the ubiquitination of SIRT1, the nuclear location and the deacelyated activity, we aims to manifest the facts that apple polyphenol could prevent and treat steatosis by regulating the ubiquitination of SIRT1,which would affect its deaceylated activity, the activation of lipophagy, then subsequently resulted in the improvement of steatosis. The project is to provide clues for the prevention and treatment of steatosis by using apple polyphenol or by targeting deaceylases SIRT1.

非酒精性脂肪性肝病是备受关注的公共卫生问题，而临床并无有效的防治药物。自噬参与调控脂代谢。SIRT1可介导自噬蛋白的去乙酰化而激活自噬，泛素化修饰可调控SIRT1的核定位及去乙酰化活性。前期研究发现：苹果多酚（AP）清除肝细胞沉积脂质的效应与SIRT1的表达水平呈正相关，并上调NAMPT、LC3-II的表达，下调E3泛素连接酶UHRF1的水平。AP是否通过调控SIRT1的去乙酰化活性激活自噬，进而改善和逆转脂质沉积还需明确。本项目以不同SIRT1、Atg7和UHRF1表达水平的肝细胞，以及SIRT1去乙酰化功能缺失小鼠为研究对象，采用免疫组化、免疫共沉淀、乙酰化和泛素化分析等方法，旨在明确AP抑制和逆转脂质沉积的作用及机制，尤其是UHRF1的泛素化修饰对SIRT1去乙酰化活性、核定位的调控作用，以及对自噬诱导和脂代谢的影响，研究结果可为将AP、SIRT1用于防治肝脏脂肪变性提供数据支持。

非酒精性脂肪性肝病是全球性的公共卫生问题，而临床并无有效的防治药物。苹果多酚(APE)介导的肝细胞脂质清除伴有SIRT1及NAMPT的表达上调，SIRT1参与代谢调控和自噬调节，我们以不同SIRT1、Atg7表达水平的HepG2细胞和SIRT1肝脏特异性突变的小鼠为研究对象，探讨APE是否可以改善肝脏脂肪变性及其作用机制，特别是SIRT1的作用。.体外实验：APE通过激活自噬、恢复溶酶体的酸度、抑制脂类的合成和促进脂肪酸的氧化，缓解棕榈酸和油酸诱导的HepG2细胞内的脂滴沉积。APE通过诱导SIRT1的表达激活 LKB1/AMPK通路，抑制mTOR通路。过表达或敲低SIRT1正调控AMPK和ATG7表达，敲除SIRT1和ATG7抑制APE诱导的脂质清除。.体内实验：雄性C57BL/6小鼠，给与HFD+125或500mg/(kg·bw·d)APE(LAP或HAP)或无菌水(CON)灌胃12周。与CON组相比，APE显著抑制小鼠体重增加，升高血清白蛋白/球蛋白比值，降低肝重、TG和TC含量。APE通过激活SIRT1-AMPK通路，抑制脂类生成，改善脂肪变性。APE降低粪便总胆汁酸、初级胆汁酸、结合胆汁酸含量和初/次级胆汁酸比值，上调FXR、MAFG蛋白水平，抑制胆汁酸合成，上调回肠中胆固醇逆转运基因的mRNA表达，下调胆汁酸重吸收基因的mRNA水平。APE增加肠道菌群的多样性，增加Akkermansiaceae和Faecalibaculun，降低Enterococcaceae和Lactobacillus的相对丰度。.对于SIRT1肝脏特异性突变的杂合雌、雄性小鼠，给与HFD+HAP干预8周后，APE显著逆转SIRT1+/−小鼠的体重、肝重、皮下脂肪重量、4和8周的糖耐量、脂肪变性评分显著升高，降低肠道菌群的多样性，增加Akkermansiaceae，抑制Bilophila和 Eisenbergiella的丰度，且除脂肪变性评分外，APE对雌性小鼠的作用较雄性小鼠更显著。.体外APE诱导自噬改善细胞脂质沉积的作用依赖于SIRT1的正常表达，体内通过SIRT1-AMPK通路调控肝脏脂代谢、促进回肠胆固醇逆转运、抑制胆汁酸合成、影响肠道菌群的多样性后改善C57BL/6小鼠和SIRT1+/−小鼠的脂肪变性，总之，体、外的实验均证实SIRT1在APE改善脂代谢过程中的关键作用。