SIRT1-SREBP-1c/PGC-1α通路在氧化应激介导的奶牛肝细胞脂沉积中的作用机制

The main pathogens of fatty liver in dairy cows are lipidosis and tissue damage in liver. Our previous research has demonstrated that high concentrations nonesterified fatty acids (NEFAs) triggered hepatocytes oxidative stress via ROS-MAPK-p53/Nrf2 pathway. Whether lipidosis is further promoted by oxidative stress is still unknown. Based on the fact that SIRT1 is activated by oxidative stress and plays a key role in the regulation of hepatotic fat metabolism, in this project, we hypothesis that oxidative stress which exists in fatty liver may disturb fat metabolism and promotes hepatic lipidosis in dairy cows. Fatty liver dairy cows and fatty liver cell model in vitro will be used for elucidating the correlation between SIRT1 expression and oxidative stress, hepatic lipidosis in dairy cows. Meanwhile, the hepatocytes will be transfected with SIRT1 overexpression and/or silencing adennvirus for studying the expression of SIRT1 genes and proteins, the expression and enzyme activity of downstream lipid metabolism related proteins and key enzymes. Finally, the mechanism of SIRT1 on the development of dairy cows fatty liver will be revealed. This study will provide valuable information for clarifying the mechanism and searching for therapeutic target of fatty liver in dairy cows.

奶牛脂肪肝的主要临床病理学特征是肝脂沉积和肝细胞损伤。本课题组先期研究证明脂肪动员所引起的高NEFA血症可通过R0S-MAPK-p53/Nrf2通路触发肝细胞氧化应激，引起肝细胞凋亡，但尚不知肝细胞氧化应激是否会进一步促进肝脂沉积。鉴于SIRT1既可在氧化应激中被激活，又在肝脂代谢调节过程中起着关键作用，本项目以“脂肪肝奶牛存在的氧化应激可能干扰肝脂代谢，促进肝脂沉积”为理论假设，以脂肪肝奶牛和体外脂肪肝细胞模型为研究对象，观测 SIRT1基因和蛋白表达水平与氧化应激和脂沉积的关系；同时，运用腺病毒转染技术过表达和/或沉默奶牛肝细胞SIRT1，研究肝脂沉积对肝细胞SIRT1基因和蛋白表达水平，以及对其下游脂代谢相关蛋白和关键酶表达水平和活性及肝脂沉积的影响，以明确SIRT1在奶牛脂肪肝病发生中的作用机制，为阐明奶牛脂肪肝的发病机制和寻找治疗靶点奠定理论和实验基础。

本试验以围产期脂肪肝奶牛和体外奶牛脂肪肝细胞模型为研究对象，运用腺病毒转染技术，研究SIRT1在奶牛脂肪肝病发生中的作用机制。体内试验表明，脂肪肝奶牛血清中ALT、ALP、TP升高，GLU显著降低，ROS浓度显著升高、CAT、Cu/Zn-SOD浓度显著降低；肝脏中TG、LDLC浓度显著升高，ROS浓度显著升高、LPO的浓度显著降低、血清以及肝脏中Mn-SOD、GSH、GSH-Px浓度显著降低。脂肪肝奶牛肝脏和血清中SIRT1和PGC-1α酶活性显著降低，血清中SREBP-1c酶活性显著升高；脂氧化相关的SIRT1、PGC-1α、ACO、PPARα、RXRα、CPT1、CPT2、LCAD、Nrf1、TFAM基因mRNA和蛋白的表达量均显著降低；脂合成SREBP-1c、ACSL、ACCα和FAS的蛋白和mRNA的表达量均显著升高。体外试验结果表明，OA造模组肝细胞内TG、TC的含量显著升高。SIRT1、PGC-1α、SREBP-1c的蛋白和mRNA的表达量显著降低。NEFAs可显著升高TG、TC的含量，降低SIRT1的mRNA和蛋白的表达，显著升高SREBP-1c的蛋白表达；添加抗氧化剂NAC可缓解以上的各种变化。转染重组过表达SIRT1腺病毒后，脂质转运的基因ApoE、LDLR的mRNA表达显著降低，脂氧化的SIRT1、PGC-1α、RXRα、PPARα mRNA和脂质合成的基因SREBP-1c、ACCα、FAS mRNA表达显著降低；SIRT1基因及其下游调控的PGC-1α通路的PPARα、RXRα基因蛋白表达显著升高，SREBP-1c、ApoE、LDLR、FAS基因蛋白表达显著降低，p-ACC基因蛋白表达显著升高。转染重组沉默SIRT1腺病毒后，SIRT1、PGC-1α、PPARα、RXRα mRNA表达水平显著降低，脂质合成的SREBP-1c、FAS、ACCα mRNA表达水平显著升高，调节脂质转运的基因ApoE、LDLR的mRNA表达水平显著升高。SIRT1及其下游调控的脂氧化PGC-1α、PPARα、RXRα蛋白的表达量显著降低，SREBP-1c、ApoE、LDLR蛋白的表达显著升高，FAS、ACCα蛋白的表达量显著升高。总之，围产期脂肪肝奶牛体内存在氧化应激干扰脂质代谢，促进肝脂沉积，SIRT1基因可通过调控SREBP-1c/PGC-1α通路影响肝脂蓄积。