摘要
目的探讨GLT25D2基因在对乙酰氨基酚（APAP）诱导的肝毒性损伤中是否对自噬有调控作用。方法选择GLT25D2/野生型C57B/J小鼠和GLT25D2/基因敲除C57B/J小鼠为研究对象。①体内实验：将20只野生型小鼠和20只GLT25D2/小鼠分别按随机数字表法分为磷酸盐缓冲液（PBS）对照组和APAP干预组，每组10只。腹腔注射25 /的APAP溶液500 m/g建立小鼠肝毒性损伤模型；PBS对照组腹腔注射等量PBS。制模成功后立即处死小鼠取肝组织，采用蛋白质免疫印迹试验（Western Blot）检测肝组织自噬相关蛋白ATG5、ATG7、微管相关蛋白1轻链3（LC3）、P62的表达；电镜下观察肝组织超微结构改变，评估自噬水平。②体外实验：另取野生型小鼠和GLT25D2/小鼠各1只，采用两步胶原灌注法提取原代肝细胞并分为两部分：一部分以5 mmo/ APAP溶液干预0、8、12 h后收集细胞，采用Western Blot试验检测肝细胞自噬相关蛋白ATG5、ATG7、LC3、P62的表达；另一部分用绿色荧光蛋白-LC3质粒（GFP-LC3）转染24 h，分别给予PBS（PBS对照组）和5 mmo/ APAP（APAP干预组）温育12 h，荧光显微镜下观察GFP-LC3阳性表达以反映自噬体的形成情况。结果①体内实验结果显示：与相应PBS对照组相比，经APAP干预后，野生型及GLT25D2/小鼠肝组织中自噬正性相关蛋白ATG5、ATG7、LC3 -Ⅱ表达下调，而负性相关蛋白P62表达上调，说明APAP处理后自噬整体水平降低。与野生型小鼠相比，GLT25D2/小鼠肝组织自噬正性相关蛋白ATG7、ATG5表达上调（ATG/-actin：1.21±0.29比0.84±0.19，ATG/-actin：1.29±0.14比1.54±0.40，均P〉0.05），LC3 -Ⅱ表达略下调（LC3 -/-actin：0.52±0.06比0.58±0.06，P〉0.05），而负性相关蛋白P62表达下调（P6/-actin：1.13±0.94比1.54±0.40，P〉0.05），说明基因敲除后小鼠自噬表达水平高于野生型小鼠。电镜下观察超微结构显示，与相应PBS对照组相比，经APAP干预后，野生型小鼠肝组织自噬体数量无明显变化，但GLT25D2/小鼠自噬体数量增多。②体外实验结果显示：随APAP干预时间延长，野生型及GLT25D2/小鼠原代肝细胞自噬正性相关蛋白ATG5、ATG7表达逐渐上调，LC3略有波动，而负性相关蛋白P62表达逐渐下调，12 h分别达峰值或谷值，说明APAP刺激细胞自噬表达增强，并呈一定时间依赖性；与野生型小鼠相比，GLT25D2/小鼠12 h原代肝细胞自噬正性相关蛋白ATG5、ATG7、LC3 -Ⅱ及负性相关蛋白P62表达上调（ATG/-actin：0.93±0.09比0.74±0.06，ATG/-actin：0.80±0.09比0.65±0.10，LC3 -/-actin：1.35±0.30比1.15±0.20，P6/-actin：0.36±0.02比0.31±0.03，均P〉0.05），说明基因敲除后自噬水平增强。荧光显微镜下观察显示，经APAP干预后，野生型及GLT25D2/小鼠原代肝细胞GFP-LC3阳性细胞均较PBS对照组明显增加；GLT25D2/小鼠GFP-LC3阳性细胞比例明显高于野生型小鼠（0.64±0.08比0.36±0.05，P〈0.05）。
结论GLT25D2是自噬负性调控因子，敲除GLT25D2基因能够增强APAP诱导肝毒性损伤小鼠的自噬水平。
ObjectiveTo investigate whether GLT25D2 gene regulates autophagy in acetaminophen （APAP）-induced hepatotoxicity injury.
MethodsGLT25D2/ wild-type C57B/J mice and GLT25D2/ C57B/J mice were selected as subjects. ① In vivo experiment： 20 for wild-type mice and 20 for GLT25D2/ mice were respectively divided into phosphate buffer （PBS） control group and APAP intervention group according to random number table, with 10 mice in each group. The hepatotoxicity injury model of mice was reproduced by intraperitoneal injection of 25 / APAP solution 500 m/g. The PBS control group was intraperitoneally injected with the same amount of PBS. The mice were sacrificed immediately after model reproduction, and the liver tissues were harvested. Western Blot was used to detect the expressions of autophagy-related proteins ATG5, ATG7, microtubule-associated protein 1 light chain 3 （LC3） and P62. The ultrastructural changes in liver tissue were observed under electron microscope to observe the level of autophagy. ② In vitro experiment： primary hepatocytes extracted by two-step collagen perfusion from one GLT25D2/ wild-type mouse and one GLT25D2/ mouse were divided into two parts respectively. One part was treated with 5 mmo/ APAP solution. The cells were harvested at 0, 8, and 12 hours, and the expressions of autophagy-related proteins ATG5, ATG7, LC3, and P62 were determined by Western Blot. The other part was transfected with the green fluorescent protein-LC3 plasmid （GFP-LC3） for 24 hours. The cells were cultured with PBS （PBS control group） or 5 mmo/ APAP （APAP intervention group） for 12 hours, and the positive expression of GFP-LC3 was observed under the fluorescence microscope, thereby reflecting the expression of autophagosomes.
Results① In vivo experiment： compared with the corresponding PBS control group, the expressions of the positive-associated proteins ATG5, ATG7 and LC3-Ⅱ in liver tissue of the APAP intervention group were down-regulated in the wild-type and GLT25D2/ mice, while the expression of the negative correlation protein P62 was up-regulated, indicating that the overall level of autophagy decreased after treatment with APAP. Compared with wild-type mice, the expressions of autophagy positive correlation proteins ATG5 and ATG7 were up-regulated in GLT25D2/ mice （ATG/-actin： 1.21±0.29 vs. 0.84±0.19, ATG/-actin： 1.29±0.14 vs. 1.54±0.40, both P 〉 0.05）, LC3-Ⅱ expression was slightly down-regulated （LC3-/-actin： 0.52±0.06 vs. 0.58±0.06, P 〉 0.05）, while negative correlation protein P62 was down-regulated （P6/-actin： 1.13±0.94 vs. 1.54±0.40, P 〉 0.05）, indicating that the expression of autophagy in GLT25D2/ mice was higher than that in wild-type mice. Ultrastructural observation under electron microscope showed that the number of autophagosomes in the liver tissue of wild-type mice did not change significantly after APAP intervention as compared with that in PBS control group, but the number of autophagosomes in GLT25D2/ mice was increased. ② In vitro experiment： with the prolongation of APAP intervention, the expressions of ATG5 and ATG7 in the primary hepatocytes of wild-type and GLT25D2/ mice were up-regulated, LC3 was slightly fluctuated, and the expression of negative-related protein P62 was gradually down-regulated. The peak value or the trough value reached at 12 hours. It was indicated that the expression of autophagy in APAP-stimulated cells was enhanced with a time-dependent manner. Compared with wild-type mice, the expressions of autophagy correlation proteins ATG5, ATG7, LC3-Ⅱ and P62 were up-regulated in GLT25D2/ mice at 12 hours （ATG/-actin： 0.93±0.09 vs. 0.74±0.06, ATG/-actin： 0.80±0.09 vs. 0.65±0.10, LC3-/-actin： 1.35±0.30 vs. 1.15±0.20, P6/-actin： 0.36±0.02 vs. 0.31±0.03, all P 〉 0.05）, indicating that the expression of autophagy was enhanced after gene knockout. Fluorescence microscopy showed that GFP-LC3 positive cells in both wild-type and GLT25D2/ mice hepatocytes were significantly increased after APAP intervention as compared with those of PBS control group, and the proportion of GFP-LC3 positive cells in GLT25D2/ mice was significantly higher than that in wild-type mice （0.64±0.08 vs. 0.36±0.05, P 〈 0.05）.
ConclusionsGLT25D2 is a negative regulator of autophagy. Knockout of GLT25D2 gene can enhance the autophagy level of APAP-induced hepatotoxicity injury in mice.
作者
郭乐乐
刘贞利
张晶
任锋
Zhang Xiaohui;Guo Lele;Liu Zhenli;Zhang Jing;Ren Feng(Department of Hepatitis C and Toxic Liver Disease,Beijing Youan Hospital,Capital Medical University,Beijing 100069,China;Beijing Institute of Hepatology,Beijing Youan Hospital,Capital Medical University,Beijing 100069,China)
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2018年第9期882-887,共6页
Chinese Critical Care Medicine
基金
国家自然科学基金（81770611,81270532）
北京市自然科学基金（7162085）
北京市科技计划项目（Z161100000516113）
北京市卫生系统高层次卫生技术人才培养计划项目（2013-3-075）
