围产期能量负平衡导致的高NEFA血症是诱发奶牛脂肪肝的重要因素。NEFA导致肝细胞内的氧化应激、内质网应激以及NFκB的激活在介导肝脂沉积中均发挥重要作用，且有研究表明细胞内的高钙也可引起肝脂沉积，而申请者前期研究发现NFκB能通过激活Orai1调控细胞内钙池引发的Ca2+的进入。据此，本项目提出高NEFA可能通过激活NFκB-Orai1通路促进钙离子内流导致内质网应激，从而导致脂肪酸的从头合成增加，进而引起脂质沉积的科学假设。本项目拟通过体外培养犊牛肝细胞，经高NEFA处理模拟能量负平衡状态，采用基因沉默技术以及NFκB/Orai1抑制剂处理方法，监测细胞内钙离子的释放与进入过程，评价肝细胞氧化应激状态，内质网应激水平及脂肪酸从头合成因子的调控作用，从而明确NFκB-Orai1通路及细胞内钙对高NEFA介导奶牛肝细胞脂沉积的调控机制，为阐明奶牛脂肪肝的发病机制提供理论依据。

Fatty liver disease of dairy cows during the transition period, which caused by the negative energy balance, are closely associated with high blood concentration of nonesterified fatty acid (NEFA). NEFA can active the oxidative stress and endoplasmic reticulum (ER) stress and NF-κB signaling pathway to induce hepatic lipidosis. Moreover, high concentration of intracellular Ca2+ can also induce hepatic lipidosis in human NAFLD. NFκB is known to upregulate Orai1, the Ca2+ channel accomplishing store operated Ca2+ entry (SOCE). We hypothesize that high blood concentration of NEFA would activate NFκB-sensitive Orai1 expression signaling pathway to increase ER stress, thus induce hepatic lipidosis in dairy cows. In this program, the calf hepatocytes in vitro are cultured and treated with NEFA, while the hepatocytes are silencing p65,Orai1 and treated NFκB or Orai1 inhibitors, respectively. SOCE, p65, Orai1, H2O2/NADPH enzyme, GRP78, SREBP-1c, ACC1, FAS expressions levels and activities are measured. The effects of NEFA on the NFκB- sensitive Orai1 expression signaling pathway are evaluated to clarify the regulation role of NFκB-Orai1 expression on hepatic lipid metabolism in dairy cows. This study will further provide valuable information to reveal the pathogenesis of fatty liver in dairy cows.