Reduction Of Inorganic Arsenic On The Nuclear Transcription Factor Nrf2 And Heme Oxygenase 1 HO-1

ObjectiveArsenic is the earth's crust common type of metal elements,widely distributed in the environment.Human beings contact with it through food,drinking water,breathing and other means,epidemiological investigation and laboratory studies have shown acute or chronic arsenic exposure will produce injury.So far,all over the world there have been near 200 million people in the high-arsenic drinking water threats.Chronic exposure to arsenic in drinking water can cause skin hyperkeratosis,pigment loss and calm,nervous system damage,cardiovascular disease and the organ tumor.At present, the pathogenesis of arsenic poisoning is not clear,but in recent years,many mechanisms in the study,arsenic-induced reactive oxygen species and lead to oxidative damage arising from the theory by more and more attention.Data confirmed that there is crowd of chronic arsenic exposure can cause oxidative stress in vivo,reactive oxygen species(reactive oxygen species,ROS) generation increased;On the other hand,in recent years,low concentrations of reactive oxygen species ROS act as an intracellular signal reaction between transfersomes through some of the body-mediated oxidative stress-sensitive signal transduction pathways and play an important role.At present,the nuclear transcription factor(transcription factor NF-E2-related factor 2,Nrf2) in the the role of regulation of oxidative stress is being more and more and more attended. Nuclear transcription factor Nrf2,are a member of zinc finger protein(bZIP) Cap'n'Collar a family,a lot of coding in the activation of antioxidant enzymes and phaseâ…¡expression of drug metabolizing enzymes play an important role in the regulation and control,and its regulation antioxidant response element through the (antioxidant response elements,AREs) implementation. Among many of the Nrf2 activation of antioxidant enzymes,heme oxygenase 1, (HO-1) expression induced by a wide range of concerns,in enhancing the body of its antioxidant capacity plays an important role.HO-1 protein is a heat shock protein HO In normal tissues they were minimal expression,only in circumstances of oxidative stress generated,increasing the body resistance and maintain the body to survive.In summary,this study used for the exposure factors NaAsO₂ to normal human liver cell line Chang liver cells,observation of inorganic arsenic on the Cell viability,ros,GSH et al of the effects of oxidative stress.And nuclear transcription factor Nrf2 and its regulation of downstream target genes HO-1 protein expression effect of Chang liver cell;on the cells produce reactive oxygen species ROS and intracellular GSH conten,as well as the factors that add to intervene after BSO effect of these indicators, so as to the pathogenesis of arsenic poisoning to provide a basis for research.Methods一,Experimental methods(一) Cell CultureChang liver cell line were purchased from the Shanghai Cell Bank,Chinese Academy of Sciences(ZN1003).Cells were cultured in complete medium(containing 10%FBS,1×105 U/L Green,streptomycin),in a fully humidified atmosphere with 5%CO₂ at 37℃.When cells reached 80%-90%integration to 1:3 for passage.(二) Cell proliferation dynamic DetectedAlamarblue method used.Experimental group as follows:arsenite and BSO pretreatment group.Determination of cell microplate reader using the absorbance value of the hole to calculate the percentage reduction Alamarblue,to restore the higher the rate of cell proliferation express stronger vitality.(三) BSO pretreatment on intracellular GSH contentGSH kit measured intracellular GSH content,Coomassie brilliant blue method of intracellular protein content.Experimental grouping for simple dye arsenic and BSO pretreatment group.(å››) Determination of intracellular ROSUsing 2',7'-dichloro-diacetyl fluorescein(DCFH-DA) Determination of intracellular ROS content.Experimental grouping for simple dye arsenic and BSO pretreatment group.(五) western blotting experiments1,Protein extraction(Nrf2 and HO-1)Experimental grouping as follows:pure dye arsenic group,BSO pretreatment group.After the extraction of cell lysate protein,Coomassie brilliant blue method of protein quantification.2,SDS-PAGE3,Membrane switchUsing semi-dry transfer method to transfer protein to PVDF membrane(216 mA, 1h).4,HybridHybrid antibody concentration:the concentration of anti-1:Nrf2(1:1000),HO-1 (1:1000) and the internal-actin(1:5000);two anti-concentration are as follows:1:5000.5,Chemi luminescence reactionECL kit in accordance with the steps,using gel imaging analyzer exposure imaging,gel imaging analysis system for protein analysis of gray value,the results of the experimental group with the average gray value with the control group,the average gray value of the relative value express.二,statistical treatment Using SPSS 11.5 statistical software for statistical analysis,group between groups using two independent samples t-test;groups compared using single factor analysis of variance(ANOVA),2-2 compared the use of LSD-t test.Results(一) Changes in cell morphologyExposure to As for 24 h,Chang liver cell of the control group is in good condition, the cells showed typical epithelial cell morphology,for the flat triangular or polygonal cytoplasm near the Central Office,there is one or two round nuclei,cell morphology consistent boundary clear;NaAsO₂ of 10μmol/L group of some cell contraction deformation,the volume of smaller,normal cells lose their close connection,a small number of cells from the matrix and suspended cultivate;50μmol/L group round cell contraction,deformation,become smaller,cell decrease in the number of gaps increases,the majority of stromal cells from the culture,suspended in culture medium; 100μmol/L group of cells from the wall even more serious situation,the majority of round cells,compared with the control group,only a small number of cells can be seen adherent.(二) Dynamic changes in cell proliferation1,NaAsO₂ separate roles:kinds of NaAsO₂ solution treatment for 24 h, Alamarblue reduction rate,compared with the control group were statistically significant differences(P＜0.01),and with the increasing dose of arsenic pollution, reduction rate is gradually reduced,100μmol/L group,the lowest rate of reduction for the control group 0.35±0.06-fold.See Table 1,Figure 1 and Figure 2.2,BSO pretreatment on the Chang liver cell activity:3 mmol/L BSO pretreatment 12h after transfection of arsenic 24 h group,the cell to restore the rate of dyeing with arsenic alone group compared to reduction rate were significantly decreased(P＜0.01),Table 2 and Figure 3. (三) BSO pretreatment on Chang liver cells of GSH contentArsenic alone stained 24 h,1,2.5 and 5μmol/L group of intracellular GSH content was gradually increased,10μmol/L group GSH content is lower than the control group,respectively,compared with the control group the differences were statistically significant(p＜0.01);BSO pretreatment of 12 h and then add NaAsO₂ after 24 h treatment,intracellular GSH and stained with a separate group arsenic group differences were statistically significant(p＜0.01).Table 3 and Figure 4.(å››) BSO pretreatment on Chang liver cells the effects of ROSBSO pretreatment of 12 h,add the concentrations of NaAsO₂ solution 24h, intracellular ROS were significantly elevated.With the same group NaAsO₂ separate solution treatment group,the difference has statistical significance(p＜0.01),Table 4 and Figure 5.(五) NaAsO₂ of cell nuclear transcription factor Nrf2 protein expression of activation0-24 h after exposure,NaAsO₂ s can ustained induce cell core transcription factor Nrf2 protein expression,5μmol/L NaAsO₂ solution exposed to 12 h,10μmol/L NaAsO₂ solution 2,6,12 h exposure of the nuclear transcription factor Nrf2 protein expression are stronger than the control group,the difference has statistical significance (P＜0.01).And nuclear transcription factor Nrf2 protein expression in a time-related activation that NaAsO₂ solution at 12h when exposed to nuclear transcription factor Nrf2 protein expression in the activation peak,then gradually reduced to normal levels. Table 5 and Figure 6.(å…­) NaAsO₂ of cell heme oxygenase -1 protein expression of activationExpoured to NaAsO₂ 10μmol/L,time was 6 h and 12 h group,the HO-1 protein content gradually increased,with the control group(0 h) as compared(p＜0.05 or p ＜0.01);5 and 10μmol/L group,6 h,12 h and 24 h between the groups,with the exposure time increased,cell HO-1 protein expression gradually increased,the difference has statistical significance(p＜0.01).Table 6 and Figure 7.Conclusions1,NaAsO2 patterns can change,the survival rate of decline in its toxic effect.2,At BSO on Chang liver cells,the role of non-toxic dose range simultaneously with the NaAsO2 role in Chang liver cells,may exacerbate the cytotoxicity NaAsO2.