内质网应激/蛋白激酶样内质网激酶/DNA损伤诱导因子153通路在七氟烷致大鼠海马神经元细胞凋亡中的作用

Role of endoplasmic reticulum stress/protein kinase R-like endoplasmic reticulum kinase/DNA damage inducing factor 153 pathway in sevoflurane-induced neuronal apoptosis

目的观察内质网应激/蛋白激酶样内质网激酶(PERK)/DNA损伤诱导因子153(CHOP)通路在七氟烷致新生大鼠海马神经细胞凋亡中的作用.方法(1)按照海马神经元暴露在3%七氟烷不同时间长度分为4组:对照组(Control组)、七氟烷暴露1h组(Sev1组)、七氟烷暴露3h组(Sev2组)、七氟烷暴露6h组(Sev3组),用蛋白质印迹法(Western blot)检测各组海马神经元凋亡蛋白半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-12、B细胞淋巴瘤/白血病-2相关X蛋白(bax)以及内质网应激蛋白PERK、CHOP的蛋白表达;(2)将海马神经元分为4组:对照组(Control组)、七氟烷暴露6h组(Sev组)、加入内质网应激特异性抑制剂牛磺熊脱氧胆酸(TUDCA)100μmol/L并暴露于3%七氟烷6h组(Sev+T组)以及加入TUDCA 500μmol/L暴露于3%七氟烷6h组(Sev+HT组).采用Western blot的方法评估各组细胞凋亡蛋白Caspase-12、bax的表达.(3)将海马神经元分为5组:对照组(Control组)、暴露于3%七氟烷6h组(Sev组),经PERK小干扰RNA(siRNA)转染并暴露于3%七氟烷6h组(PERK siRNA),经CHOP siRNA转染并暴露于3%七氟烷6h组(CHOPsiRNA),以及加入TUDCA 100μmol/L并暴露于3%七氟烷6h组(TUDCA组).利用Western blot的方法对比分别加入内质网应激特异性抑制剂TUDCA、PERK siRNA、CHOP siRNA后凋亡蛋白Caspase-12、bax的表达.应用SPSS 20.0统计软件分析,组间比较采用单因素方差分析.结果(1)海马神经元暴露在3%七氟烷3h及6h后均出现亡蛋白Caspase-12、bax以及内质网应激蛋白PERK、CHOP的蛋白表达的上调(3组bax表达分别是1.00±0.00、3.20±0.42、4.10±0.51,F=76.450,Caspase-12表达分别是1.00±0.00、3.30±0.34、5.20±0.41,F=214.500;PERK表达分别是1.00±0.00、3.20±0.31、5.10±0.29,F=307.800;CHOP表达分别是1.00±0.00、2.80±0.31、4.90±0.33,F=247.800,P<0.05);(2)七氟烷引起的细胞凋亡加入不同浓度内质网应激特异性抑制剂TUDCA后,细胞凋亡蛋白Caspase-12、bax较之前下调(Caspase-12:5.20±0.41、3.90±0.35、2.30±0.13,F=315.500;bax:4.10±0.51、3.70±0.29、2.20±0.21,F=38.760;P<0.05),且高浓度TUDCA下调细胞凋亡表现更明显(Caspase-12:5.20±0.41比2.30±0.13,bax:4.10±0.51比2.20±0.21,P<0.05);(3)PERK抑制剂PERK siRNA、CHOP抑制剂CHOPsiRNA均可不同程度地下调Caspase-12、bax凋亡蛋白的表达.Sev、PERK siRNA、CHOPsiRNA3组bax分别是4.10±0.51、2.90±0.32、3.50±0.23,F=12.470,Caspase-12分别是:5.20±0.41、3.60±0.11、2.90±0.36,F=61.030,P<0.05.结论内质网应激在七氟烷致海马神经细胞凋亡中起着重要的作用,抑制内质网应激可以抑制七氟烷引起的海马神经元凋亡.

Objective To investigate the role of endoplasmic reticulum stress in sevoflurane-induced neuronal apoptosis.Methods(1)The hippocampal neurons were divided into four groups according to the exposure time of 3%sevoflurane:control group,sevoflurane inhalation 1 h group(Sev 1),sevoflurane inhalation 3 h group(Sev 2),sevoflurane inhalation 6 h group(Sev 3).Western blotting was used to detect the expression of apoptotic proteins:cysteinyl aspartate-specific protease(Caspase)-12,B cell lymphoma/leukemia-2 associated X protein(bax)and endoplasmic reticulum stress proteins[protein kinase R-like endoplasmic reticulum kinase(PERK),DNA damage inducing factor 153(CHOP)];(2)The hippocampal neurons were divided into four groups:control group,sevoflurane inhalation 6 h group(Sev),endoplasmic reticulum stress-specific inhibitor taurobear deoxycholic acid(TUDCA)(100μmol/L)and sevoflurane inhalation 6 h group(Sev+T),and endoplasmic reticulum stress-specific inhibitor TUDCA(500μmol/L)and sevoflurane inhalation 6 h group(Sev+HT).Western blotting was used to evaluate the expression of Caspase-12 and bax after adding different concentrations of TUDCA;(3)The hippocampal neurons were divided into five groups:control group,sevoflurane inhalation 6 h group(Sev),PERK small interfering RNA(siRNA)and sevoflurane inhalation 6 h group(PERK siRNA),CHOP siRNA and sevoflurane inhalation 6 h group(CHOP siRNA),and TUDCA(100μmol/L)and sevoflurane inhalation 6 h group(TUDCA).Western blotting was used to detect the expression of apoptotic proteins Caspase-12 and bax.Results(1)After 3%sevoflurane exposure for 3 h and 6 h,hippocampal neurons showed up-regulation of Caspase-12,bax,PERK and CHOP proteins(bax:1.00±0.00,3.20±0.42,4.10±0.51,F=76.450;Caspase-12:1.00±0.00,3.30±0.34,5.20±0.41,F=214.500;PERK:1.00±0.00,3.20±0.31,5.10±0.29,F=307.800;CHOP:1.00±0.00,2.80±0.31,4.90±0.33,F=247.800,P<0.05).(2)When TUDCA was added,the apoptotic proteins Caspase-12 and bax were down-regulated(Caspase-12:5.20±0.41,3.90±0.35,2.30±0.13.F=315.500;bax:4.10±0.51 and 3.70±0.29,2.20±0.21,F=38.760,P<0.05),especially in high concentration of TUDCA(Caspase-12:5.20±0.41 and 2.30±0.13;bax:4.10±0.51 and 2.20±0.21,2.20±0.21,F=38.760,P<0.05).(3)TUDCA,PERK siRNA and CHOP siRNA could down-regulate the expression of Caspase-12 and bax to varying degrees(bax:4.10±0.51,2.90±0.32,3.50±0.23,F=12.470,P<0.05;Caspase-12:5.20±0.41,3.60±0.11,2.90±0.36,F=61.030,P<0.05).Conclusion Endoplasmic reticulum stress plays an important role in sevoflurane-induced apoptosis of hippocampal neurons.Inhibiting endoplasmic reticulum stress can inhibit sevoflurane-induced apoptosis of hippocampal neurons.