循环外泌体通过AMPKα-SIRT1通路调控酮病奶牛肝脂代谢紊乱的机制研究

Ketotic dairy cows display high blood concentration of NEFA and hepatic lipid metabolism disorderd. SIRT1 is closely associated with hepatic lipid metabolism disorder. Exosomes are membrane vesicles in exchanging signals between cells. Proteins and nucleic acids carried by exosomes can regulate hepatocytes function. Studies have reported that plasma circulating exosomes could promote the proliferation of kidney cells in dairy cows. However, the effects of circulating exosomes on dairy cows hepatocytes are still poorly reported. Accordingly, we hypothesized that plasma exosomes would integrate into dairy cows hepatocytes and deliver endogenous protective signals to liver via AMPKα-SIRT1 pathway to alleviate hepatic lipid metabolism disorder. In the present study, plasma exosomes miRNAs in dairy cows will be analyzed by high-throughput sequencing techniques (HTS). In vitro, dairy cows hepatocytes co-cultured with high concentration of NEFA are treated with plasma circulating exosomes. The activity of hepatocytes, indicators of lipid metabolism, indicators of liver function and AMPKα-SIRT1 signaling pathway are detected. SIRT1 overexpression adenovirus were transfected to hepatocytes to further study the effect of circlulating exosomes on hepatic lipid metabolism disorder. This study will clarify the repair effect and mechanism of plasma exosecreting on hepatic lipid metabolism disorder in ketotic cows and provide a theoretical and experimental foundation for finding suitable targeted therapy of ketosis.

酮病奶牛存在高NEFA血症和肝脂代谢紊乱，SIRT1与肝脂代谢紊乱密切相关。外泌体是细胞间信号交流的膜泡，其携带的蛋白质和核酸可作为信号分子调节肝细胞功能。已有文献证实奶牛血浆外泌体可促进肾细胞增殖，但未见其对奶牛肝细胞功能影响的相关报道。因此，我们提出奶牛血浆外泌体可以进入奶牛肝细胞通过AMPKα-SIRT1途径向肝脏传递内源性保护信号，减轻肝脂代谢紊乱的科学假设。本项目利用高通量测序技术筛选奶牛血浆外泌体中的miRNA；采用血浆外泌体处理体外高NEFA共培养的肝细胞，研究其对肝细胞活性、脂代谢指标、肝功能指标和AMPKα-SIRT1信号通路的影响，再将过表达SIRT1的腺病毒转染到肝细胞中，进一步证实外泌体对肝脂代谢紊乱的影响，以明确血浆外泌体对酮病奶牛肝脂代谢紊乱的修复效果和作用机制，为寻找合适的酮病靶向治疗药物奠定理论和实验基础。

本试验探究循环外泌体对奶牛肝细胞AMPKα-SIRT1通路关键蛋白和mRNA相对表达水平的影响。筛选15头围产期非酮病、亚临床酮病和临床酮病奶牛，采集血液样本检测肝功能、肾功能和脂代谢等指标，提纯外泌体用于miRNA组学分析，筛选差异miRNA。同时，体外分离奶牛原代肝细胞，用外泌体刺激1.2 mM和2.4 mM NEFA诱导的肝细胞，分别采用实时荧光定量PCR法（qRT-PCR）和蛋白质印迹法（western blotting）检测肝细胞pAMPKα、AMPKα、SIRT1、PGC-1α和SREBP-1C的mRNA相对表达水平和蛋白表达量。再将过表达SIRT1腺病毒转染到NEFA诱导后的肝细胞中，与最佳浓度的循环外泌体相比较，采用上述方法检测AMPKα-SIRT1通路关键基因和酶的表达量的变化。结果表明，外泌体在TEM下为茶托状结构，western blotting检测到表面标记蛋白Alix、CD9、CD63和HSP70的表达，NTA追踪到粒径分布在30-150 nm。miRNA组学分析结果表明，差异miRNA主要参与代谢通路，且共筛选出差异miRNA上调top10和下调top10。40 μg/mL循环外泌体刺激12 h是提高NEFA诱导的奶牛原代肝细胞活性的最佳浓度和时间。采用1.2 mM和2.4 mM NEFA处理肝细胞后，与对照组相比，添加40 μg/mL非酮病奶牛和亚临床酮病奶牛循环外泌体，AMPKα、PGC-1α和SIRT1的mRNA相对表达量和蛋白表达水平增加，SREBP-1C的mRNA相对表达量和蛋白表达水平降低，生化指标AST、ALT、ALP、LDH、TC、TG的含量显著降低，VLDL的分泌量显著升高。采用1.2 mM NEFA诱导肝细胞后，与空白对照组相比，SIRT1过表达组中SIRT1和PGC-1α的mRNA相对表达量和蛋白表达水平增加，SREBP-1C的mRNA相对表达量和蛋白表达水平降低。总之，酮病奶牛存在肝脂代谢紊乱，且miRNA测序结果表明围产期奶牛循环外泌体与肝脏脂代谢紊乱有内在联系。体外试验结果表明，添加的外泌体通过介导AMPKα-SIRT1通路调控NEFA诱导的肝脏脂代谢紊乱，修复肝细胞损伤，与转染过表达SIRT1腺病毒效果一致，且亚临床酮病奶牛循环外泌体的效果优于非酮病奶牛循环外泌体。