糖基因GLT25D1和GLT25D2缺失对APAP诱导的急性药物性肝损伤的影响

Effects of Glucose Gene GLT25D1 and GLT25D2 Deletions on Acute Liver Injury Induced by APAP

目的 研究糖基因Glt25D1和Glt25D2在对乙酰氨基酚(acetaminophen,APAP)诱导的急性药物性肝损伤中的可能作用及机制.方法 抽取6~8周雄性糖基因Glt25D1和Glt25D2敲除小鼠(Glt25D1^(Δhep) Glt25D2^(-/-))及野生型小鼠(Wild type,WT),构建药物性肝损伤模型.实验组腹腔内注射500 mg/kg APAP,对照组给予相同剂量生理盐水.造模6 h后处死小鼠.检测小鼠血浆中丙氨酸氨基转移酶(alanine aminotransferase,ALT)和天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)水平,观察肝组织病理.分离提纯小鼠肝原代细胞,分别加APAP和衣霉素(Tunicamycin,Tm)刺激6 h后收取细胞,提取细胞蛋白和RNA.q-PCR、Western印迹检测小鼠肝脏组织及原代肝细胞Glt25D1和Glt25D2、内质网应激相关蛋白、凋亡相关蛋白、炎性因子的表达变化趋势.结果 Glt25D1^(Δhep) Glt25D2^(-/-)造模组小鼠ALT和AST水平均显著高于WT造模组.小鼠肝脏HE染色可见,Glt25D1^(Δhep) Glt25D2^(-/-)造模组小鼠肝损伤程度比WT造模组更严重.Glt25D1^(Δhep) Glt25D2^(-/-) APAP造模组GRP78、chop以及cleaved caspase-12、cleaved caspase-3等内质网应激相关蛋白的表达量高于WT造模组,同时伴有线粒体细胞色素C的释放.结论 Glt25D1和Glt25D2基因敲低加重小鼠APAP诱导的急性肝损伤,促进内质网应激,促进肝细胞凋亡.

Objective To investigate the role and mechanism of sugar genes Glt25D1 and Glt25D2 in acute liver injury induced by acetaminophen(APAP).Methods Six to eight weeks old male Glt25D1^(Δhep) Glt25D2^(-/-) mice and WT mice were used to establish drug-induced liver injury model.Mice in administered group were given APAP via intraperitoneal injection with a dose of 500 mg/kg body mass,while mice in control group were injected with the same dose of saline.Mice were sacrificed after being treated by APAP for 6 h.The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in plasma of mice were detected,and the pathology of liver tissue was observed.Primary hepatocytes were isolated and purified from male Glt25D1^(Δhep) Glt25D2^(-/-)mice and WT mice after stimulation with APAP and Tunicamycin(Tm)respectively for 6 h,and the cellular proteins and RNA were extracted.The expression levels of Glt25D1 and Glt25D2,the changing trends of ER stress-related proteins,apoptotic proteins and imflammatory factors were detected by Western blotting.Results The ALT and AST levels of Glt25D1^(Δhep) Glt25D2^(-/-)APAP administration group were significantly higher than those in the WT administration group.HE staining showed that,compared with mice of WT administration group,wider range of necrosis and more significant infiltration of inflammatory cells in the portal area were found in the liver of Glt25D1^(Δhep) Glt25D2^(-/-)APAP administration group.Western blotting demonstrated that administration of APAP to mice and primary hepatocytes caused significant elevated expression of endoplasmic reticulum stress(ERS)-related proteins and apoptotic proteins,such as GRP78,chop,ATF6,p-eIF2α and cleaved caspase-12,and cleaved caspase-3,accompanied with the release of mitochondrial cytochrome C.Conclusion Glt25D1 and Glt25D2 gene knockdown could aggravate APAP-induced acute liver injury in mice,promote endoplasmic reticulum stress and hepatocyte apoptosis.