内质网应激诱导的自噬对肝细胞凋亡的影响
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摘要
目的:观察二硫苏糖醇(DTT)诱导人正常肝细胞LO2发生内质网应激的过程中自噬相关指标表达的变化及其对细胞凋亡的影响。方法:采用2.0 mmol/L DTT处理LO2细胞0、6、12和24 h,诱导细胞发生内质网应激;real-time PCR及Western blot检测葡萄糖调节蛋白78(GRP78)、蛋白激酶R样内质网激酶(PERK)、活化转录因子4(ATF4)、C/EBP同源蛋白(CHOP)、自噬相关基因12(Atg12)、自噬相关基因5(Atg5)和微管相关蛋白1轻链3(LC3) mRNA及蛋白水平的变化;流式细胞术检测细胞凋亡情况;透射电镜观察自噬小体的产生情况。采用400 nmol/L的雷帕霉素预处理LO2细胞1 h,再给予2.0 mmol/L的DTT处理24 h,观察雷帕霉素预处理对细胞凋亡的影响。结果:2.0 mmol/L DTT处理LO2细胞6、12和24 h后,细胞中GRP78、PERK、ATF4、CHOP、Atg12、Atg5和LC3的mRNA及蛋白水平较0 h组显著上调(P <0.05);同时LC3Ⅱ/LC3Ⅰ也较0 h组显著增高(P <0.05);透射电镜检测发现,DTT处理6、12和24 h后,细胞中均可见自噬小体的产生;流式细胞术检测发现,DTT处理细胞6、12和24 h后,细胞凋亡较0 h组显著增加;而经自噬诱导剂雷帕霉素预处理后,DTT诱导的细胞凋亡显著下降(P<0.05)。结论:内质网应激可诱导自噬的发生,而雷帕霉素预处理可在一定程度上减轻内质网应激诱导的LO2细胞凋亡。
AIM:To observe the changes of autophagy-related indexes during endoplasmic reticulum stress(ERS)induced by dithiothreitol(DTT)and its effect on apoptosis in human normal hepatocytes.METHODS:LO2 cells were treated with DTT at 2.0 mmol/L for 0,6,12 and 24 h to induce ERS.The expression of glucose-regulated protein 78(GRP78),protein kinase R-like endoplasmic reticulum kinase(PERK),activating transcription factor 4(ATF4),C/EBP homologous protein(CHOP),autophagy-related gene 12(Atg12),autophagy-related gene 5(Atg5)and microtubule-associated protein 1 light chain 3(LC3)at mRNA and protein levels was determined by real-time PCR and Western blot.The apoptosis was analyzed by flow cytometry.The formation of autophagosomes was observed under transmission electron microscope.After the LO2 cells were pretreated with rapamycin at 400 nmol/L for 1 h and treated with DTT at 2.0mmol/L for 24 h,the effect of rapamycin pretreatment on the apoptosis was analyzed by flow cytometry.RESULTS:After treatment with DTT at 2.0 mmol/L for 6,12 and 24 h,the mRNA and protein levels of GRP78,PERK,ATF4,CHOP,Atg12,Atg5 and LC3 in the LO2 cells were significantly higher than those in 0 h group(P<0.05).At the same time,the ratio of LC3Ⅱ/LC3Ⅰwas also increased after DTT treatment(P<0.05).Observation under transmission electron microscope showed that autophagosomes were found in the LO2 cells treated with DTT for 6,12 and 24 h.After DTT treatment for 6,12 and 24 h,the apoptosis rate of LO2 cells was significantly higher than that in DTT 0 h group,while the apoptosis induced by DTT was significantly decreased after rapamycin pretreatment(P<0.05).CONCLUSION:ERS induces autophagy and rapamycin pretreatment alleviates the apoptosis of LO2 cells to some extent.
AIM:To observe the changes of autophagy-related indexes during endoplasmic reticulum stress(ERS)induced by dithiothreitol(DTT)and its effect on apoptosis in human normal hepatocytes.METHODS:LO2 cells were treated with DTT at 2.0 mmol/L for 0,6,12 and 24 h to induce ERS.The expression of glucose-regulated protein 78(GRP78),protein kinase R-like endoplasmic reticulum kinase(PERK),activating transcription factor 4(ATF4),C/EBP homologous protein(CHOP),autophagy-related gene 12(Atg12),autophagy-related gene 5(Atg5)and microtubule-associated protein 1 light chain 3(LC3)at mRNA and protein levels was determined by real-time PCR and Western blot.The apoptosis was analyzed by flow cytometry.The formation of autophagosomes was observed under transmission electron microscope.After the LO2 cells were pretreated with rapamycin at 400 nmol/L for 1 h and treated with DTT at 2.0mmol/L for 24 h,the effect of rapamycin pretreatment on the apoptosis was analyzed by flow cytometry.RESULTS:After treatment with DTT at 2.0 mmol/L for 6,12 and 24 h,the mRNA and protein levels of GRP78,PERK,ATF4,CHOP,Atg12,Atg5 and LC3 in the LO2 cells were significantly higher than those in 0 h group(P<0.05).At the same time,the ratio of LC3Ⅱ/LC3Ⅰwas also increased after DTT treatment(P<0.05).Observation under transmission electron microscope showed that autophagosomes were found in the LO2 cells treated with DTT for 6,12 and 24 h.After DTT treatment for 6,12 and 24 h,the apoptosis rate of LO2 cells was significantly higher than that in DTT 0 h group,while the apoptosis induced by DTT was significantly decreased after rapamycin pretreatment(P<0.05).CONCLUSION:ERS induces autophagy and rapamycin pretreatment alleviates the apoptosis of LO2 cells to some extent.
引文
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[14]陈晨,朱艳,李霞,等.过氧化氢对人小梁细胞内质网应激ATF4-CHOP通路的影响[J].潍坊医学院学报,2016,38(5):379-381.
[15]Guan Y,Li Y,Zhao G,et al.HMGB1 promotes the starvation-induced autophagic degradation ofα-synuclein in SH-SY5Y cells Atg 5-dependently[J].Life Sci,2018,202:1-10.
[16]Tang Q,Zheng G,Feng Z,et al.Trehalose ameliorates oxidative stress-mediated mitochondrial dysfunction and ER stress via selective autophagy stimulation and autophagic flux restoration in osteoarthritis development[J].Cell Death Dis,2017,8(10):e3081.
[2]刘宝琴,王华芹.内质网应激与未折叠蛋白反应的研究进展[J].中华肿瘤防治杂志,2010,17(11):869-872.
[3]全文淑,金英顺,金吉哲,等.过度内质网应激在慢性环孢素A肾毒性细胞凋亡中的作用机制[J].中国病理生理杂志,2014,30(6):1047-1051.
[4]陈娜子,姜潮,李校堃.内质网应激与疾病[J].中国生物工程杂志,2016,36(1):76-85.
[5]张旭,王煜,马娟,等.内质网应激介导的细胞凋亡在肝纤维化发生发展中的作用[J].临床肝胆病杂志,2015,31(9):1537-1539.
[6]谢汝佳.Calpain2及其抑制蛋白calpastatin在内质网应激介导的肝细胞凋亡中的作用[D].贵阳:贵阳医学院,2014.
[7]Gifford JB,Huang W,Zeleniak AE,et al.Expression of GRP78,master regulator of the unfolded protein response,increases chemoresistance in pancreatic ductal adenocarcinoma[J].Mol Cancer Ther,2016,15(5):1043-1052.
[8]Gong J,Wang XZ,Wang T,et al.Molecular signal networks and regulating mechanisms of the unfolded protein response[J].J Zhejiang Univ Sci B,2017,18(1):1-14.
[9]戴婷婷,程卉,苏婧婧.内质网应激调节的凋亡信号通路研究进展[J].中医临床研究,2017,9(5):147-148.
[10]孙小霞,姜昕.泛素-蛋白酶体系统和自噬之间的关系[J].中国医药科学,2015,5(9):42-44.
[11]刘关羽,何卫阳,朱鑫,等.氧化应激诱导自噬对骨髓间充质干细胞增殖与凋亡的影响[J].中国病理生理杂志,2015,31(12):2176-2182.
[12]B’chir W,Maurin AC,Carraro V,et al.The e IF2α/ATF4 pathway is essential for stress-induced autophagy gene expression[J].Nucleic Acids Res,2013,41(16):7683-7699.
[13]倪志华,张玉明,邓传怀,等.LC3基因在细胞自噬过程中的表达研究[J].湖北农业科学,2015,54(20):4932-4936.
[14]陈晨,朱艳,李霞,等.过氧化氢对人小梁细胞内质网应激ATF4-CHOP通路的影响[J].潍坊医学院学报,2016,38(5):379-381.
[15]Guan Y,Li Y,Zhao G,et al.HMGB1 promotes the starvation-induced autophagic degradation ofα-synuclein in SH-SY5Y cells Atg 5-dependently[J].Life Sci,2018,202:1-10.
[16]Tang Q,Zheng G,Feng Z,et al.Trehalose ameliorates oxidative stress-mediated mitochondrial dysfunction and ER stress via selective autophagy stimulation and autophagic flux restoration in osteoarthritis development[J].Cell Death Dis,2017,8(10):e3081.