摘要目的 观察燃煤污染型砷中毒大鼠肝组织中蛋白激酶Cδ(PKCδ)、核因子E2相关因子2(Nrf2)的表达,并探讨其作用.方法 采用成组设计,将30只Wistar大鼠(雌雄各半)按体质量(80 ～ 100 g)采用随机数字表法分为对照组、饮水型砷中毒组(水砷组)和低、中、高砷粮食污染组(低、中、高砷组),每组6只.对照组常规喂养,水砷组自由饮用100 mg/L三氧化二砷(As2O3)水溶液,低、中、高砷组分别喂饲病区高砷煤烘烤玉米粉所配制的饲料(砷含量分别为25、50、100mg/kg),均喂养3个月.大鼠处死前,抽取外周静脉血2 ml,检测血清丙二醛(MDA)含量,铜锌超氧化物歧化酶(SOD1)、谷胱甘肽过氧化物酶(GPx)活力;处死后,分离肝脏,检测肝脏二酯酰甘油(DAG)含量、PKCδ和Nrf2 mRNA和蛋白表达;采用Pearson相关分析方法,对蛋白表达进行相关性分析.结果 大鼠血清MDA含量,SOD1、GPx活力组间比较差异有统计学意义(F=26.441、3.327、120.645,P均＜ 0.05),其中各染砷组血清MDA含量均高于对照组(P均＜0.05);SOD1、GPx活力均低于对照组(P均＜0.05).大鼠肝组织DAG含量,Nrf2 mRNA表达组间比较差异有统计学意义(F=8.237、8.656,P均＜0.05),其中各染砷组肝组织DAG含量分别为(2.67±0.25)、(2.36±0.19)、(2.54±0.22)、(2.69±0.32)μg/L,均高于对照组[(2.05±0.24)μg/L,P均＜0.05];各染砷组肝组织Nrf2 mRNA表达分别为1.16±0.09、1.09±0.12、1.14±0.15、1.27±0.16,均明显高于对照组(0.94±0.08,P均＜0.05).大鼠肝组织细胞膜和细胞质中磷酸化PKCδ(pPKCδ)蛋白表达组间比较差异有统计学意义(F=15.925、6.699,P均＜0.05),各染砷组肝组织细胞膜pPKCδ蛋白表达分别为0.49±0.06、0.33±0.05、0.37±0.06、0.50±0.08,除低砷组外均高于对照组(0.28±0.04,P均＜0.05);各染砷组肝组织细胞质中pPKCδ蛋白表达分别为0.38±0.06、0.31±0.05、0.35±0.05、0.36±0.05,均高于对照组(0.24±0.05,P均＜0.05).大鼠肝组织细胞质及细胞核Nrf2、磷酸化Nrf2(pNrf2)蛋白表达组间比较差异有统计学意义(F=9.024、9.709、10.396、25.532,P均＜0.05),其中各染砷组肝组织细胞质Nrf2蛋白表达分别为0.76±0.09、0.58±0.07、0.64±0.09、0.73±0.07,除低砷组外均高于对照组(0.52±0.08,P均＜0.05);pNrf2蛋白表达分别为0.50±0.07、0.43±0.06、0.48±0.06、0.54±0.07,均高于对照组(0.32±0.06,P均＜0.05).各染砷组肝组织细胞核中Nrf2蛋白表达分别为0.44±0.07、0.41±0.06、0.47±0.06、0.54±0.09,均高于对照组(0.30±0.05,P均＜0.05);pNrf2蛋白表达分别为0.35±0.04、0.29±0.04、0.41±0.05、0.43±0.06,均高于对照组(0.20±0.03,P均＜0.05).相关性分析显示,DAG含量与肝细胞膜和细胞质pPKCδ蛋白表达均呈正相关(r=0.663、0.604,P均＜0.05);细胞膜pPKCδ蛋白表达与细胞质和细胞核pNrf2蛋白表达均呈正相关(r=0.642、0.670,P均＜0.05).结论 砷可诱导氧化应激性肝损伤;同时,砷可上调Nrf2 mRNA表达和蛋白表达、增加pPKCδ蛋白表达和DAG含量,促进pPKCδ发生膜转位后活化,进而磷酸化Nrf2使其发生核转位激活,从而调节机体的氧化应激反应.

abstractsObjective To observe the expression of Protein Kinase C Delta (PKCδ) and NF-E2-related factor 2 (Nrf2) in the liver of arsenic poisoning rats induced by coal-burning,and explore their roles.Methods According to body weight (80-100 g),thirty Wistar rats (half male half female) were divided into five groups of 6 each using random number table method,the control group,and drinking arsenic,low,medium and high arsenic contaminated grain groups.The control group was fed normally for 3 months;drinking arsenic,low,medium and high arsenic contaminated grain groups were fed respectively with 100 mg/L As2O3 solution and different concentrations of arsenic-containing feed (25,50 and 100 mg/kg).At the end of the experiment period,non-anticoagulant whole blood 2 ml from peripheral vein was collected.Malondialdehyde (MDA) contents,activities of superoxide dismutase 1 (SOD1) and glutathione peroxidase (GPx) were detected.After sacrificing the animals,the liver was separated and then diacylglycerol (DAG) contents,mRNA and protein expressions of PKCδ and Nrf2 were determined,and the correlation was analyzed by Pearson.Results There were significant differences in serum MDA contents,SOD1 and GPx activities among groups (F =26.441,3.327,120.645,P ＜ 0.05).The serum MDA contents in arsenic-exposed groups were higher than that of the control group (P ＜ 0.05).However,activities of SOD1 and GPx1 were lower than those in the control group (P ＜ 0.05).There were significant differences in liver DAG contents,Nrf2 mRNA expression levels among groups (F =8.237,8.656,P ＜ 0.05).DAG contents in the liver tissues of the drinking arsenic,low,medium and high arsenic contaminated grain groups were respectively (2.67 ± 0.25),(2.36 ± 0.19),(2.54 ± 0.22) and (2.69 ± 0.32) μg/L,which were significantly higher than that in the control group [(2.05 ± 0.24) μg/L,P ＜ 0.05].The expression levels of Nrf2 mRNA in liver tissue were respectively 1.16 ± 0.09,1.09 ± 1.20,1.14 ± 0.15 and 1.27 ± 0.16,which were higher than that in the control group (0.94 ± 0.08,P ＜ 0.05).There were significant differences in the expression of pPKCδ protein in the cell membrane and cytoplasm of liver tissue between groups (F =15.925,6.699,P ＜ 0.05).The protein expression levels of pPKCδ in the cell membrane of liver tissue were 0.49 ± 0.06,0.33 ± 0.05,0.37 ± 0.06 and 0.50 ± 0.08,respectively,which were significantly higher than that in the control group (0.28 ± 0.04,P ＜ 0.05).The protein expression levels of pPKCδ in the cytoplasm were 0.38 ± 0.06,0.31 ± 0.05,0.35 ± 0.05 and 0.36 ± 0.05,respectively,which were higher than that in the control group (0.24 ± 0.05,P ＜ 0.05).There were significant differences in the expression of Nrf2 and pNrf2 in cytoplasm and nucleus of liver tissues among groups (F =9.024,9.709,10.396,25.532,P ＜ 0.05).The protein expression levels of Nrf2 in the cytoplasm were respectively 0.76 ± 0.09,0.58 ± 0.07,0.64 ± 0.09 and 0.73 ± 0.07,which were higher than that of the control group (0.52 ± 0.08,P ＜ 0.05),except the low arsenic contaminated grain group.The protein expression levels of pNrf2 in the cytoplasm were respectively 0.50 ± 0.07,0.43 ± 0.06,0.48 ± 0.06 and 0.54 ± 0.07,which were higher than that in the control group (0.32 ± 0.06,P ＜ 0.05).The expression levels of Nrf2 protein in the nucleus were respectively 0.44 ± 0.07,0.41 ± 0.06,0.47 ± 0.06 and 0.54 ± 0.09,which were higher than that in the control group (0.30 ± 0.05,P ＜ 0.05).The protein expression levels of pNrf2 in the nucleus were respectively 0.35 ± 0.04,0.29 ± 0.04,0.41 ± 0.05 and 0.43 ± 0.06,which were higher than that in the control group (0.20 ± 0.03,P ＜ 0.05).The correlation analysis showed that DAG contents and the protein expression of pPKCδ in the cell membrane and the cytoplasm were positively correlated (r =0.663,0.604,P ＜ 0.05).Furthermore,the protein expression of pPKCδ in the cell membrane and pNrf2 in the cytoplasm and nucleus were also positively correlated (r =0.642,0.670,P＜ 0.05).Conclusions Arsenic could induce oxidative stress liver injury,and upregulate the mRNA and protein expression of Nrf2.Moreover,arsenic could also increase the protein expression of pPKCδ and DAG content,and then promote pPKCδ membrane transposition,phosphorylate Nrf2,and induce its nuclear transposition,which could regulate oxidative stress reaction.