亚硝酸钠增强自噬减轻肝脂肪变性的细胞脂肪蓄积  

Sodium nitrite reduces lipid accumulation in steatotic cells by enhancing autophagy

作　　者：张优敬 郑乃汭 刘彬[2] 吉爱玲[1] 李彦章[1] 皇甫超申[1] 

机构地区：[1]河南大学医学院环境医学研究所,河南开封475004 [2]河南大学护理学院,河南开封475004

出　　处：《药学学报》2015年第8期1000-1007,共8页Acta Pharmaceutica Sinica

基　　金：国家环保公益项目专项资助项目(200809115);省部共建河南大学科研项目(SBGJ090702)

摘　　要：最近发现抑制自噬会加重肝细胞内脂肪蓄积,亚硝酸盐可以降低高脂饲养小鼠体内甘油三酯含量。因此,本实验目的在于观察亚硝酸盐对单纯性肝细胞脂肪变性的影响和自噬在其中的可能作用。首先用1.2 mmol·L-1油酸诱导人肝细胞系L-02细胞24 h建立肝细胞脂肪变性模型,再用0.2 mmol·L-1亚硝酸钠加或不加三甲基腺嘌呤(3-MA)或氯喹处理24 h。肝细胞脂质蓄积评价采用油红O染色和细胞内甘油三酯含量测定。1.2 mmol·L-1油酸诱导人肝细胞系L-02细胞24 h可以导致肝细胞明显的脂肪变性。用0.2 mmol·L-1亚硝酸钠处理L-02肝脂肪变性的细胞,可以明显减少肝细胞脂质蓄积,而3-MA可以减弱亚硝酸钠的这种效应;LC3-II免疫荧光、Western blot结果显示,亚硝酸钠促进L-02脂肪变性细胞LC3-II荧光颗粒和蛋白表达增加,氯喹同样可以增加这种现象;相差显微镜下显示细胞内空泡增多,溶酶体示踪剂荧光显微镜下发现,细胞溶酶体数量增多;电镜显示自噬溶酶体数量增加,氯喹可以减弱这种现象。这些结果表明,亚硝酸钠能够促进肝脂肪变性的L-02细胞自噬,促进脂质分解,减轻肝脂肪变性细胞脂质蓄积。Recent data have revealed that inhibiting autophagy exacerbates lipid accumulation in hepatocytes and nitrite treatment reduces total triglyceride levels in the high-fat diet mice. Therefore, the present study aimed to determine the effects of nitrite on simple hepatic steatosis and the possible role of autophagy. Firstly, steatotic L-02 cells were induced by incubating L-02 cells with 1.2 mmol·L^-1 oleic acid （OA） for 24 h. Secondly, steatotic L-02 cells were treated with 0.2 mmol·L^-1 sodium nitrite （SN） plus 3-methyladenine （3-MA）, or chloroquine （CQ） for 24 h, and then lipid accumulation was measured with oil red O staining and triglyceride quantification. The notable steatosis could be observed in L-02 cells following exposure to 1.2 mmol·L^-1 OA for 24 h. Treatment with 0.2 mmol·L^-1 sodium nitrite reduced lipid accumulation in steatotic L-02 cells. 3-MA weakened the ability of sodium nitrite to ameliorate hepatic steatosis. Additionally, the sodium nitrite increased number of LC3-II immunostaining puncta and LC3-II protein expression was confirmed by immunofluorescence or Western blot analysis, and the effects were enhanced by CQ treatment. The number of increased cytoplasm vacuoles and lysosomes increased was confirmed by phase contrast and fluorescence microscope respectively. The increased autolysosome was detected by electron microscopy, this phenomenon could be reversed by CQ treatment. These data demonstrated that sodium nitrite enhanced the autophagic flux and decomposition of triglycerides in steatotic L-02 cells.

关 键 词：亚硝酸盐 L-02细胞 油酸 肝细胞脂肪变性 自噬 

分 类 号：R963[医药卫生—微生物与生化药学]