Nrf2-Keap1-ARE信号通路在燃煤型砷中毒大鼠肝损伤中的作用

Role of Nrf2-Keap1-ARE signaling pathway in liver injury in rats with coal-burning-induced arsenism

目的探讨核因子E2相关因子2-Kelch样ECH联合蛋白1-抗氧化反应元件(Nrf2-Keap1-ARE)信号通路在燃煤型砷中毒大鼠肝损伤中的作用。方法将30只健康清洁级Wistar大鼠按体重随机分为5组,分别为对照(去离子水,无砷标准饲料)组、饮水型砷中毒(100 mg/L)组和25、50、100 mg/kg砷粮食污染组,每组6只,雌雄各半。采用自由摄食和饮水方式进行染毒,连续染毒90 d,采用实时荧光定量PCR法检测肝组织中铜锌超氧化物歧化酶(SOD1)、谷胱甘肽过氧化物酶1(GPx1)m RNA的表达水平,采用免疫组化法检测肝组织中Nrf2、pNrf2、Keap1、SOD1和GPx1蛋白表达,采用Western blot法检测肝细胞浆和细胞核中Nrf2和pNrf2的蛋白表达水平;采用化学法检测SOD1和GPx的活力。结果与对照组比较,各染砷组肝组织SOD1、GPx1 mRNA表达量及Nrf2、pNrf2蛋白表达阳性率均增加(P<0.05);Keap1蛋白表达阳性率无明显改变;50、100 mg/kg砷粮食污染组肝组织SOD1蛋白表达阳性率降低(P<0.05);各砷粮食污染组GPx1蛋白表达阳性率均降低(P<0.05)。与饮水型砷中毒组比较,100 mg/kg砷粮食污染组GPx1蛋白表达阳性率降低(P<0.05)。与对照组比较,各染砷组肝组织细胞浆、细胞核中Nrf2和pNrf2蛋白表达量增加(P<0.05)。与饮水型砷中毒组比较,100 mg/kg砷粮食污染组肝细胞核Nrf2和50、100 mg/kg砷粮食污染组肝细胞核pNrf2蛋白表达量均增加(P<0.05)。与对照组比较,各砷染毒组大鼠肝组织SOD1和GPx活力均较低,除25 mg/kg砷粮食污染组SOD1活力外,差异均有统计学意义(P<0.05)。与饮水型砷中毒组比较,25 mg/kg砷粮食污染组大鼠肝组织GPx的活力较高,而50、100 mg/kg砷粮食污染组大鼠肝组织GPx的活力较低,差异均有统计学意义(P<0.05);而各剂量砷粮食污染组大鼠肝组织SOD1的活力均无明显改变。相关性分析显示,大鼠肝组织细胞核Nrf2、pNrf2蛋白的表达水平与SOD1、GPx1 mRNA的表达水平均呈正相关(P<0.05);大鼠肝组织SOD1、GPx1蛋白的表达水平与其酶活力也呈正相关(P<0.05)。结论砷可增加Nrf2蛋白表达和磷酸化,促进Nrf2核转位,从而诱导SOD1和GPx1 mRNA表达上调;同时,砷可能通过影响基因的转录后调控机制,使SOD1和GPx1蛋白表达及其酶活力降低,导致砷中毒肝损伤的发生发展。

Objective To understand the role of Nrf2-Keap1-ARE signaling pathway in liver injury in rats with coal-burninginduced arsenism.Methods Thirty Wistar rats(half male and half female),80-100 g,were randomly divided into five groups,six in each,the control group(no arsenic exposure),drinking water arsenic exposure group(100 mg/L)and food arsenic exposure groups(the content of arsenic in corn flour:25,50 and 100 mg/kg),and the treatments were conducted for 90 consecutive days.The mRNA expression of SOD1 and GPx1 in the liver was detected by RT-PCR,the protein expression of Nrf2,pNrf2,Keap1,SOD1 and GPx1 in the liver was detected by immunohistochemistry.The protein expression of Nrf2 and pnrf2 in the cytoplasm and nucleus in the liver was detected by Western blot.The activity of SOD1 and GPx was detected by chemical method.Results Compared with the control group,the mRNA expression levels of SOD1,GPx1,and the protein expression rates of Nrf2 and pNrf2 in arsenic exposed groups increased(P<0.05);No significant difference was seen in the protein expression rate of Keap1 among groups;The protein expression rates of SOD1 decreased in the 50 and 100 mg/kg arsenic-contaminated grain groups(P<0.05),the protein expression rate of GPx1 decreased in the arsenic-contaminated grain groups(P<0.05).Moreover,the protein expression rate of GPx1 decreased also in the 100 mg/kg arsenic-contaminated grain group compared with that in drinking water arsenic exposure group(P<0.05).The protein expression levels of Nrf2 and pNrf2 in the cytoplasm and nucleus in liver were higher than that in the control group(P<0.05).The protein expression levels of Nrf2 in the nucleus in the 100 mg/kg arsenic-contaminated grain group,and the protein expression levels of pNrf2 in the nucleus in the 50,100 mg/kg arsenic-contaminated grain group were higher than those in the drinking arsenic group(P<0.05).Compared with the control group,the activity of SOD1 and GPx in the liver in each arsenic-exposed group was significantly lower,except for the activity of SOD1 in the 25 mg/kg arsenic-contaminated grain group(P<0.05).Compared with the drinking water arsenic exposure group,the activity of GPx in the liver in the 25 mg/kg arsenic contaminated grain group was higher,while that of 50 and 100 mg/kg arsenic contaminated grain group was lower,the difference was statistically significant(P<0.05);The activity of SOD1 in the liver in all food arsenic exposure groups had no significant change.The protein expression levels of Nrf2 and pNrf2 in the nucleus in the liver were positively correlated with the transcription expression levels of SOD1 and GPx1(P<0.05).Furthermore,the protein expression levels of SOD1,GPx1 in the liver were positively correlated with the enzyme activity of SOD1 and GPx1(P<0.05).Conclusion Coal-burning arsenic exposure may increase Nrf2 protein expression and phosphorylation,and promote nuclear translocations of Nrf2,which may induce the up-regulation of the transcription expression of SOD1 and GPx1.In addition,the arsenic may affect the post-transcriptional mechanisms of SOD1 and GPx1,which may decrease their protein expression and enzyme activity and lead to liver injury.