ERO1α介导同型半胱氨酸诱导的肝细胞内质网应激  

Endoplasmic reticulum oxidoreductin 1α mediates homocysteine-induced hepatocyte endoplasmic reticulum stress

作　　者：周龙霞[1] 杨安宁[1] 陈久凯 赵丽[2] 王艳华[2] 刘现梅 蔡欣[2] 张鸣号[1] 姜怡邓[1] 曹军[1] 

机构地区：[1]宁夏医科大学基础医学院病理学与病理生理学系,宁夏回族自治区银川市750004 [2]宁夏医科大学检验学院临床检验诊断学教研室,宁夏回族自治区银川市750004

出　　处：《世界华人消化杂志》2014年第34期5228-5234,共7页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;Nos.81260063;81360073~~

摘　　要：目的:探讨内质网氧化还原酶-1α(endoplasmic reticulum oxidoreductin 1α,ERO1α)在同型半胱氨酸(homocysteine,Hcy)诱导的肝细胞内质网应激(endoplasmic reticulum stress,ERS)中的作用及机制.方法:培养人肝细胞,用Hcy(100μmol/L)干预,同时设对照组,使用酶联免疫法(ELISA)检测ERS相关蛋白葡萄糖调节蛋白78(glucoseregulated protein 78,GRP78)、X盒结合蛋白-1(X-box binding protein-1,XBP-1)、内质网类似激酶(protein kinase RNA-like endoplasmic r e t i c u l u m k i n a s e,P E R K)及激活转录因子6(activating transcription factor 6,ATF6)的水平;用不同浓度Hcy(0、50、100、200、500μmol/L)和100μmol/L Hcy+叶酸+维生素B12(VB12)干预,运用实时定量PCR和Western blot检测ERO1αm RNA和蛋白水平;构建ERO1α基因重组质粒和RNA干扰片段并感染肝细胞,检测ERO1αm RNA和蛋白表达变化;采用ELISA法检测其对ERS相关蛋白的调控作用.结果:与对照组比较,Hcy组GRP78、ATF6、P E R K、X B P-1水平升高(P<0.01,P<0.01,P<0.05,P<0.01);不同浓度Hcy干预后,肝细胞内ERO1αm RNA及蛋白水平呈下降趋势,且随着Hcy浓度的增加而减少(P<0.01),呈剂量依赖关系;将ERO1α重组质粒转染肝细胞后,ERO1αm RNA及蛋白表达明显增加(P<0.01);将三个不同的ERO1αsi RNA片段转染肝细胞后,ERO1αm RNA及蛋白表达降低(P<0.01),其中si RNA2作用最明显(P<0.01);与Hcy组相比,Hcy+p ERO1α组GRP78、XBP-1、PERK及ATF6均明显降低(P<0.01),而Hcy+si RNA2组均明显升高(P<0.01).结论:Hcy可能通过下调ERO1α引起肝细胞GRP78-XBP-1/PERK/ATF6表达增加促进ERS发生.AIM： To assess the role of endoplasmic reticulum oxidoreductin 1α（ERO1α） in homocysteine（Hcy）-induced endoplasmic reticulum stress（ERS）.METHODS： Hepatocytes were cultured in the presence or absence of Hcy（100 μmol/L）, and ELISA was used to determine the concentrations of of glucose-regulated protein 78（GRP78）, X-box binding protein-1（XBP-1）, protein kinase RNA-like endoplasmic reticulum kinase（PERK） and activating transcription factor 6（ATF6）. Hepatocytes were then cultured with different concentrations of Hcy（0, 50, 100, 200, 500 μmol/L） and 100 μmol/L Hcy plus folic acid and vitamin B12, and the expression of ERO1α was detected by q RT-PCR and Western blot. ERO1α recombinant plasmid and ERO1α small interfering RNAs were then used to transfect hepatocytes, and the expression of ERO1α and the concentrations of GRP78, PERK, ATF6 and XBP-1 were measured. RESULTS： Compared with non-treated cells, the concentrations of GRP78, PERK, ATF6 and XBP-1 significantly increased in Hcy-treated cells（P 〈 0.01, P 〈 0.01, P 〈 0.05, P 〈 0.01）. Hcy decreased the expression of ERO1α at m RNA and protein levels（P 〈 0.01） in a dose-dependent manner. Transfection with ERO1α recombinant plasmid significantly increased the expression of ERO1α（P 〈 0.01）, while transfection with three ERO1α small interfering RNAs significantly decreased the expression of ERO1α, with si RNA2 having the most significant effect（P 〈 0.01）. Compared with the Hcy group, the concentrations of GRP78, PERK, ATF6 and XBP-1 significantly decreased in the Hcy ＋ p ERO1α recombinant plasmid group（P 〈 0.05）, but increased in the Hcy ＋ si RNA2 group（P 〈 0.01）. CONCLUSION： ERO1α may be involved in Hcy-induced hepatocyte ERS possibly by regulation of the GRP78-XBP-1/PERK/ATF6 signal pathway.

关 键 词：同型半胱氨酸 内质网应激 内质网氧化还原酶-1α 肝细胞 GRP78-XBP-1/PERK/ATF6 

分 类 号：R363[医药卫生—病理学]