SIRT3在调节围产期奶牛肝脏脂代谢及抗氧化防御中的作用

The transition period (or perinatal period) is the time when most of contagious or metabolic disorders of dairy cows take place.Negative energy balance (NEB) is the common pathological basis,and hepatic lipid metabolism disturbances and oxidation stress are pathological features of those diseases.It is known that SIRT3 (silent mating-type information regulation 2 homolog 3),a major deacetylase in mitochondria,activates during NEB and plays a pivotal role in the regulation of lipid metabolism and antioxidant defense in mice and human.However,the role of SIRT3 in lipid metabolism and antioxidant defense in liver of dairy cows during transition period is not known so far.In order to test the hypothesis that SIRT3 regulates hepatic lipid metabolism and antioxidant defense during transition period via deacetylation of key enzymes,hepatic SIRT3 expression and acetylation of mitochondrial proteins during transition period are measured,and primary cultured cow hepatocytes modified by SIRT3 gene RNAi and overexpression are also tested for acetylation and activity of the key enzymes involved in lipid metabolism and antioxidant defense.The present project will make clear the effect of SIRT3 on hepatic lipid metabolism and antioxidant defense,which will further reveal new mechanism of energy regulation during transition period and offer new insight into the pathogenesis of common metabolic disorders like ketosis and fatty liver ect. in dairy cows so as to search new target or strategy for prevention and treatment for those diseases.

围产期是奶牛感染性和代谢性疾病高发期，能量负平衡是该类疾病的病理学基础，肝脂代谢紊乱和氧化应激是其病理学特征。沉默信息调控因子2样蛋白3（SIRT3）是线粒体主要去乙酰化酶，在小鼠及人类已证实，SIRT3在能量负平衡条件下活化，是肝脂代谢及抗氧化防御的重要调节者，但SIRT3是否介入围产期奶牛肝脂代谢和抗氧化防御调节暂不明确。本项目以“SIRT3可通过调控关键酶活性从而发挥调节围产期奶牛肝脏能量代谢及抗氧化防御作用”为假设，在体测定围产期不同阶段奶牛肝脏SIRT3表达特征及线粒体蛋白乙酰化，调控SIRT3表达测定其对培养肝细胞脂代谢和抗氧化相关酶乙酰化水平及活性的影响，明确SIRT3对脂质代谢与抗氧化防御系统的调节作用，以期拓宽对围产期能量代谢调节的理解，并为揭示能量代谢障碍性疾病（如酮病、脂肪肝等）发病的分子机制提供启示，进而为探寻防治新靶标奠定理论基础。

奶牛营养代谢性疾病多发生在围产期，能量负平衡是该类疾病的病理学基础，肝脂代谢紊乱和氧化应激是其病理学特征。沉默信息调控因子2样蛋白3（SIRT3）是线粒体主要去乙酰化酶，在小鼠及人类已证实是肝脂代谢及抗氧化防御的重要调节者，但SIRT3是否介入围产期奶牛肝脂代谢和抗氧化防御调节暂不明确。以山羊作为模式动物，本项目采集了围产期不同阶段肝脏样本及血液样本，检测肝SIRT3表达水平及其与血液相关指标相关性。同时，采集了健康奶牛及严重能量负平衡奶牛肝脏样本，检测其肝脏SIRT3表达水平。在犊牛原代肝细胞中沉默/过表达SIRT3，观测脂代谢关键酶的表达及胞内甘油三酯含量，检测抗氧化防御关键酶的表达、活性和总抗氧化能力。结果显示，与产前3周以上相比，产后1周和产后2周肝脏SIRT3蛋白水平显著降低，且产犊当天肝SIRT3蛋白水平显著增高。另发现，血清Mn-SOD活性与肝SIRT3 mRNA水平呈正相关（r=0.46）。血清NEFA水平与SIRT3 mRNA水平呈负相关（r=-0.41）。而且脂肪肝奶牛肝脏SIRT3表达水平相比健康奶牛较低。过表达SIRT3抑制脂合成关键酶ACC-1和FAS的表达，促进脂分解关键酶CPT-1、CPT-2和ACO的表达，显著降低甘油三酯在奶牛肝细胞的沉积。si-SIRT3处理显著降低CPT-1、CPT-2和ACO的表达。SIRT3沉默显著降低抗氧化防御关键酶Mn-SOD和IDH2 mRNA水平，显著降低该两酶的活性；而SIRT3过表达提高了肝细胞总抗氧化能力，提高Mn-SOD和IDH2活性。本项目明确了SIRT3通过调控某些关键酶的表达及活性从而发挥调节肝细胞脂质代谢与抗氧化防御的作用，拓宽了对围产期能量代谢调节的理解，并为揭示能量代谢障碍性疾病（如酮病、脂肪肝等）发病的分子机制提供启示，为探寻防治新靶标奠定理论基础。