人ether-α-go-go钾通道减少肝脏的内质网应激和凋亡

Effect of human ether-α-go-go related gene channels on hepatic endoplasmic reticulum stress and apoptosis

目的研究人ether-α-go-go(human ether-α-go-go related gene,h ERG)钾通道在肝细胞中与内质网应激和凋亡的关系。方法选取雄性20周龄h ERG基因敲除(knockout,KO)及同窝野生(wild type,WT)小鼠,测量体质量、摄食量;肝脏石蜡切片HE染色; Western blotting法检测糖异生关键酶葡萄糖-6-磷酸酶(glucose-6-phosphatase,G6pase)和磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate carboxykinase,PEPCK)在肝脏的表达水平; Western blotting和荧光定量PCR方法检测肝脏凋亡相关基因活化的半胱天冬酶半胱天冬酶3(cleaved-cysteine aspartyl protease 3,cleaved-caspase 3),B细胞淋巴瘤因子2(B-cell lymphoma 2,Bcl2)和Bcl2相关X蛋白(Bcl2 associated X protein,Bax)表达水平;同时检测内质网应激相关基因磷酸化真核细胞翻译起始因子2(phosphorylated-eukaryotic translation initiation factor 2α,p-eIF2α)、激活转录因子4(activating transcription factor 4,ATF4)以及C/EBP同源蛋白(C/EBP-homologous protein,CHOP)等基因的表达。经尾静脉给KO小鼠注射hERG慢病毒(lentivirus,LV)后,Western blotting法检测胰岛及肝脏内质网应激和凋亡相关指标。结果与WT小鼠相比,KO小鼠体质量及摄食量差异无统计学意义,肝细胞轻度肿胀,肝脏糖代谢G6Pase表达水平无明显变化,而PEPCK表达上调提示hERG敲除引起肝脏糖代谢异常。KO小鼠肝脏的内质网应激及凋亡相关指标均上调,提示KO小鼠体内存在内质网应激和凋亡。KO小鼠体内过表达h ERG后,改善了小鼠的肝脏内质网应激及凋亡。结论 hERG钾通道可以通过减少肝脏内质网应激和凋亡改善肝细胞内糖代谢。

Objective To study the relationship between human ether-α-go-go related gene( h ERG) potassium channel and endoplasmic reticulum stress and apoptosis in hepatocytes. Methods The body weight and food intake of male 20-week-old h ERG knockout( KO) and wild type( WT) mice,and HE staining of liver paraffin sections was performed. Western blotting was used to detect key enzymes of gluconeogenesis including glucose-6-phosphatase( G6 pase) and phosphoenolpyruvate carboxykinase( PEPCK). Western blotting and realtime qPCR methods were used to detect liver apoptosis related genes including cleaved-cysteine aspartyl protease 3( cleaved-caspase 3),Bcell lymphoma 2( Bcl2) and Bcl2 associated X protein( Bax). Liver endoplasmic reticulum stress related genes including,phosphorylatedeukaryotic translation initiation factor 2α( p-eIF2α),activating transcription factor 4( ATF4) and C/EBP-homologous protein( CHOP) were also detected. hERG lentivirus was injected via tail vein of KO mice,and Western blotting was used to detect the protein levels of islet and liver endoplasmic reticulum stress and apoptosis related indicators. Results Compared with WT mice,no statistically significant differences was observed in body weight and food intake in KO mice. G6 Pase expression level has no significant change,while the up-regulation of PEPCK expression suggested that hERG knockout caused abnormal liver glucose metabolism. The endoplasmic reticulum stress and apoptosis related genes were up-regulated suggesting the presence of endoplasmic reticulum stress and apoptosis in KO mice. After the overexpression of hERG in KO mice,the liver endoplasmic reticulum stress and apoptosis were improved. Conclusion hERG potassium channel canimprove glucose metabolism in liver cells with reducing liver endoplasmic reticulum stress and apoptosis.