摘要
目的研究在饱和脂肪酸软脂酸钠诱导肝细胞脂毒性凋亡过程中,蛋白激酶R样内质网激酶(PKR-like ER kinase,PERK)/活化转录因子4(activating transcription factor 4,ATF4)/C/EBP同源蛋白(C/EBP-homologous protein,CHOP)信号通路的作用,对参与饱和脂肪酸诱导肝细胞脂毒性凋亡的主要UPR信号通路进行探讨。方法MTT法筛选不同浓度(0、36、72、108、144、180μmol/L)软脂酸钠作用L02和HepG2细胞株的最佳浓度后,将细胞分为正常对照组和模型组(软脂酸钠108μmol/L),甘油三酯试剂盒检测细胞内甘油三酯含量,流式细胞术Annexin V-PE/7-AAD双染检测细胞凋亡率,Western blot法检测Bip、t-PERK、磷酸化PERK(p-PERK)、ATF4、CHOP蛋白的表达。将PERK shRNA转染L02和HepG2细胞沉默PERK基因,以Control shRNA作为对照,将细胞分为Control shRNA、Control shRNA+软脂酸钠、PERK shRNA、PERK shRNA+软脂酸钠组,Western blot法检测t-PERK、ATF4、CHOP蛋白的表达,流式细胞术检测细胞凋亡率。结果软脂酸钠成功诱导建立肝细胞脂变模型。在体外实验中,软脂酸钠能够诱导肝细胞发生脂肪变性以及脂变肝细胞凋亡,细胞凋亡率随软脂酸钠诱导时间延长而增加,与对照组相比,模型组48 h细胞凋亡率明显增加,差异有统计学意义(P<0.05)。软脂酸钠诱导L02和HepG2细胞0、12、24、48 h,各时间点Bip、p-PERK、ATF4和CHOP蛋白的表达呈时间依赖性增加(P<0.05)。在使用和不使用软脂酸钠诱导下,PERK shRNA组细胞t-PERK蛋白表达水平显著低于Control shRNA组(P<0.05);软脂酸钠诱导下,与Control shRNA组相比,PERK shRNA组细胞ATF4和CHOP蛋白表达水平显著降低,细胞凋亡率也明显降低(P均<0.05)。结论饱和脂肪酸能够诱导脂变肝细胞凋亡,并且启动过度内质网应激(endoplasmic reticulum stress,ER stress),激活PERK/ATF4/CHOP信号通路。阻断PERK通路能够在一定程度上抑制脂变肝细胞凋亡,提示ER stress UPR信号途径PERK/ATF4/CHOP参�
Objective To investigate the role of PKR-like ER kinase（PERK）/activating transcription factor 4（ATF4）/C/EBP-homologous protein（CHOP） signaling pathway in the lipoapoptosis induced by saturated fatty acid in human liver cells, and to search the primary unfolded protein response（UPR） pathway in this process. Methods The optimal sodium palmitate concentration for treatment of L02 and HepG2 cells was screened by MTT assay of the cells treated with sodium palmitate at various concentrations（0, 36, 72, 108, 144, 180 μmol/L）. The cells were divided into normal control and model groups. The cells in normal control group were untreated, while those in model group were treated with180 μmol/L, and determined for triglyceride content by triglyceride kit, for apoptosis rate by flow cytometry with Annexin V-PE/7-AAD staining, and for expressions of Bip, t-PERK, p-PERK, ATF4 and CHOP by Western blot. PERK gene was silenced by transfection of L02 and HepG2 cells with PERK shRNA, using Control shRNA as control. The cells were divided into Control shRNA, Control shRNA ＋ sodium palmitate, PERK shRNA and PERK shRNA ＋ sodium palmitate groups, and determined for expressions of t-PERK, ATF4 and CHOP by Western blot, and for apoptosis rate by flow cytometry. Results The model of hepatic steatosis was established successfully by induction with sodium palmitate. In vitro test showed that sodium palmitate induced the hepatic steatosis and apoptosis, and the apoptosis rate increased with the increasing time for induction. The apoptosis rate of cells 48 h after incubation in model group was significantly higher than that in control group（P〈0. 05）. L02 and HepG2 cells were induced with sodium palmitate for 0, 12, 24 and 48 h,in which the expressions of Bip, p-PERk, ATF4 and CHOP increased with the time for induction（P〈0. 05）. The expression levels of t-PERK in PERK shRNA group with and without induction of sodium palmitate were significantly lower than those in Control shRNA group（P〈0. 05）. However,
出处
《中国生物制品学杂志》
CAS
CSCD
2017年第8期811-818,共8页
Chinese Journal of Biologicals
基金
重庆市卫生局重点课题(2012-1-033)
