摘要目的 观察贵州省燃煤污染型砷中毒病区砷暴露者外周血核因子E2相关因子2(Nrf2)与下游抗氧化反应元件(ARE)序列的结合能力(Nrf2-ARE结合能力)以及抗氧化酶基因的表达,为砷致机体肝氧化损伤机制的研究提供依据.方法 以贵州省兴仁县燃煤型砷中毒病区交乐村和长庆村为调查点,通过健康体检,选择161例砷暴露者为砷暴露组,按照《地方性砷中毒诊断标准》(WS/T 211-2001)分为病区非病例组(21例)和病例组(140例),病例组参照《职业性中毒性肝病诊断标准》(GBZ 59-2010)进一步分为轻度肝病组(52例)、中重度肝病组(36例),其余病例为无明显肝病组(52例);同时选择相邻的非病区上坝田村45例居民作为对照组.采集观察对象外周血提取血细胞核蛋白:凝胶电泳迁移试验(EMSA)检测其外周血细胞核内Nrf2-ARE结合能力,实时荧光定量PCR检测铜锌超氧化物歧化酶(Cu/Zn-SOD)和谷胱甘肽过氧化物酶l(GSH-Px1) mRNA相对表达量.结果 Nrf2-ARE结合能力检测结果:各组人群外周血Nrf2-ARE结合能力组间比较差异有统计学意义(F=116.033,P＜0.05).其中与对照组(3.14±1.34)比较,病区无明显肝病组(5.17±2.06)、轻度肝病组(13.13±4.84)及中重度肝病组(32.35±14.76) Nrf2-ARE结合能力均升高(P均＜0.05);与病区非病例组(5.15±3.23)及无明显肝病组比较,轻度肝病组及中重度肝病组Nrf2-ARE结合能力升高(P均＜0.05);与轻度肝病组比较,中重度肝病组Nrf2-ARE结合能力升高(P＜0.05).Cu/Zn-SOD和GSH-Px1 mRNA表达结果:病区无明显肝病组、轻度肝病组及中重度肝病组Cu/Zn-SOD mRNA相对表达量[中位数(M):1.127 8、1.257 8、1.6320]和GSH-Px1 mRNA相对表达量(M:1.334 5、1.940 9、2.062 6)均高于对照组(M:0.961 8、0.884 3),组间比较差异均有统计学意义(x2=13.065、19.934,P均＜0.05);轻度肝病组及中重度肝病组GSH-Px1 mRNA相对表达量均高于病区非病例组(M:1.248 4),组间比较差异均有统计学意义(P均＜0.05).相关性分析:砷暴露人群外周血Nrf2-ARE结合能力与Cu/Zn-SOD和GSH-Px1 mRNA表达量呈正相关(r=0.271、0.292,P均＜0.01).结论 砷所致Nrf2-ARE结合能力升高参与了砷对Nrf2下游抗氧化酶基因表达的调控,该过程可能参与了燃煤型砷中毒肝损伤的发生发展.

abstractsObjective To detect the combining capacity of peripheral blood NF-E2-related factor 2 (Nrf2)of arsenic-exposed residents in the coal-contaminated arsenism area in Guizhou with the sequence of downstream antioxidant response element (ARE) as well as antioxidase gene expression,and to provide a basis for in-depth revelation of arsenic oxidative damage mechanism to human body.Methods Jiaole and Changqing villages in coal-burning-borne arsenism areas in Xingren County of Guizhou were selected as the survey spots,and 161 cases of arsenic-exposed residents were selected as the arsenic exposed group on the basis of physical examination.They were divided into non-patient group (21 cases) and patient group (140 cases) according to the "Diagnostic Criteria of Endemic Arsenism" (WS/T 211-2001),and the patient group was further divided into mild hepatosis group (52 cases),moderately severe hepatosis group (36 cases) and non-apparent hepatosis group (52 cases) according to the "Diagnostic Criteria of Occupational Chronic" (GBZ 59-2010).Moreover,45 residents from one village neighboring to non-epidemic area were selected as controls.The hemocyte nucleoprotein was extracted from peripheral blood in the sampling subjects.The combining capacity of peripheral blood Nrf2-ARE was tested by electrophoretic mobility shift assay (EMSA),and the relative expression quantity of Cu/Zn superoxide dismutase (Cu/Zn-SOD) and glutathione peroxidase 1 (GSH-Pxl) mRNA was tested with real-time fluorescence quantification PCR (qPCR).Results The testing results of Nrf2-ARE combining capacity showed that the difference of Nrf2-ARE combining capacity between groups was statistically significant (F =116.033,P ＜ 0.05).Compared with the control group (3.14 ± 1.34),the Nrf2-ARE combining capacity was higher in the non-apparent hepatosis group (5.17 ± 2.06),mild hepatosis group (13.13 ± 4.84) and moderately severe hepatosis group (32.35 ± 14.76,all P ＜ 0.05);compared with the non-patient group (5.15 ± 3.23) and non-apparent hepatosis group,the Nrf2-ARE combining capacity of mild hepatosis group and moderately severe hepatosis group was higher (all P ＜ 0.05);compared with mild hepatosis group,the Nrf2-ARE combining capacity of moderately severe hepatosis group was higher (P ＜ 0.05).The results of Cu/Zn-SOD and GSH-Pxl mRNA expression showed the relative expression quantities of Cu/Zn-SOD [Median (M):1.127 8,1.257 8,1.632 0] and GSH-Pxl (M:1.334 5,1.940 9,2.062 6) mRNA of non-apparent hepatosis,mild hepatosis,moderately severe hepatosis groups were higher than those of the control groups (M:0.961 8,0.884 3),respectively,and their differences were statistically significant (x2 =13.065,19.934,all P ＜ 0.05).The relative expression quantities of GSH-Pxl mRNA of mild hepatosis group and moderately severe hepatosis group were higher than that of the non-patient group (M:1.248 4),and their differences were statistically significant (all P ＜ 0.05).The correlation analysis indicated the Nrf2-ARE combining capacity was positively related to the Cu/Zn-SOD and GSH-Px1 mRNA expression (r =0.271,0.292,all P ＜ 0.01).Conclusion The increase of Nrtf2-ARE combining capacity by arsenic participates is involved in the regulation of Nrf2 downstream antioxidase gene expression,and this process possibly participates in occurrence and development of coal-burning-borne arsenism and hepatic injury.