| ObjectiveArsenic(Arsenic,As),as a metal-like poison,exists widely in the environment.Arsenic exposure can cause a variety of injuries and diseases,especially the most serious damage to the liver,but its toxicity mechanism has not been fully elucidated.Nuclear transcription factor kappa B(NF-κB)is an important transcription factor mediating inflammatory response,which can regulate the expression of miR-21,while miR-21 can regulate a variety of target proteins such as PDCD4.However,it has not been reported whether arsenic induces hepatocyte inflammatory injury through NF-κB/miR-21/PDCD4.In this study,BRL-3A,of normal rat hepatocytes was exposed to sodium arsenite to explore the inflammatory damage of arsenic to hepatocytes and the role of NF-κB/miR-21/PDCD4 pathway in it,in order to provide theoretical reference for the study and prevention of arsenic toxicity.MethodsIn this study,normal rat hepatocytes BRL-3A were treated with 0,5,10 and 20 μmol/L sodium arsenite for 24,48 and 72 hours.Cell viability was detected by CCK-8 kit,apoptosis was detected by Annexin V-FITC/PI apoptosis kit,oxidation products and inflammatory factors were detected by ROS kit and ELISA kits of LPO,IL-1β and IL-6.BRL-3A cells were treated with 0,5,10 and 20 μmol/L sodium arsenite for 24 hours,and BRL-3A cells were treated with 10 μmol/L sodium arsenite alone or in combination with NF-κB inhibitor BAY11-7082 and miR-21 inhibitor for 24 hours.NF-κB signal pathway and PDCD4 protein expression were detected by Western blot,and miR-21 expression was detected by RT-q PCR.Single factor analysis of variance(ANOVA)was used to compare the differences of molecules between groups.Multiple comparisons between groups were conducted with LSD,and Spearman for intermolecular correlation analysis.Results1.Toxicity of arsenic to BRL-3A cells.The cell viability of BRL-3A cells treated with 0,5,10 and 20 μmol/L arsenic for 24,48 and 72 hours was detected.The results showed that compared with the blank control group,the cell viability of 5,10 and 20 μmol/L arsenic groups decreased gradually(P<0.05),showing a dose-effect relationship,and the cell activity after 48 and 72 hours of arsenic treatment was similar to that of 24 hours.The morphology of cells was observed under microscope.the results showed that with the increase of arsenic concentration,the number of BRL-3A living cells decreased,the number of floating small round transparent dead cells gradually increased,and the cells gradually changed from fusiform to small to transparent,and became floating dead cells.The results showed that with the increase of arsenic concentration,the number of red nuclei decreased gradually,while the number of green cell membrane gradually increased,while the number of cells with green halo also increased after nucleus and membrane Merge,suggesting that the apoptotic toxicity of arsenic to BRL-3A increased with the increase of arsenic concentration,showing a dose-effect relationship.The production of ROS was detected.Compared with the blank control group,the intracellular green fluorescence increased gradually with the increase of arsenic concentration for 24 hours,which showed that arsenic could significantly induce the expression of ROS.Similarly,compared with the blank control group,arsenic could induce lipid peroxidation in BRL-3A cells,and the expression of LPO in the supernatant increased with the increase of arsenic concentration(P<0.05).The expression levels of IL-1β and IL-6 in the supernatant of BRL-3A cells were detected.The results showed that with the increase of arsenic concentration and time,the expression of IL-1β and IL-6 in the supernatant of BRL-3A cells increased gradually(all P<0.05),and showed a certain dose-and time-effect relationship.2.Effects of arsenic on the expression of NF-kappa B,miR-21 and PDCD4 in BRL-3A cells.After BRL-3A cells were treated with 0,5,10 and 20 μmol/L arsenic for 24 hours,the changes of NF-κB signal pathway were detected.The results showed that compared with the blank control group,the increase of arsenic concentration could promote the expression of IκB α kinase IKKα/β,increase the expression of p-IκBα and p-IκBα/IκBα,activate NF-κB,and up-regulate the expression of p-P65 and P65/p-P65(all P<0.05).However,there was no significant change in the expression of IκBα and NF-κB p65.Compared with the blank control group,10 and 20 μmol/L arsenic could induce the expression of miR-21 in BRL-3A cells,and the expression level of miR-21 increased with the increase of arsenic concentration(all P<0.05).Compared with the blank control group,arsenic 5,10 and 20μmol/L could decrease the expression of PDCD4 in BRL-3A cells(all P<0.05).3.Analysis of the correlation between NF-κB,miR-21 and PDCD4 in BRL-3A cells after arsenic interventionAnalysis of the correlation between NF-κB,miR-21 and PDCD4 in BRL-3A cells after arsenic intervention Spearman correlation analysis was used to analyze the correlation between NF-κB and miR-21 and PDCD4.The results showed that miR-21 was positively correlated with p-P65 and p-P65/P65,and negatively correlated with PDCD4,while PDCD4 was negatively correlated with p-P65 and p-P65/P65(all P<0.05).4.Effect of arsenic on the expression of miR-21 and PDCD4 after inhibition of NF-κB.BRL-3A cells were treated with 10 umol/L BAY11-7082 and 10 umol/L arsenic for 24 hours,and the expression of miR-21 and PDCD4 was detected.The results showed that the expression of miR-21 in BAY11-7082 and arsenic combined treatment group was higher than that in BAY11-7082 group,but lower than that in arsenic group(all P<0.05).However,the PDCD4 of BAY11-7082 combined with arsenic group was higher than that of arsenic group(P<0.05).5.Effect of arsenic on PDCD4 expression after knocking down miR-21.Four hours after transfection with miR-21 inhibitor and miRNAs Negative Control,BRL-3A cells were treated with 10 umol/L arsenic for 24 hours.The expression levels of miR-21 and PDCD4 in cells were detected.The results showed that compared with miR-21 inhibitor group,miR-21 expression increased in miR-21 inhibitor transfection combined with10umol/L arsenic treatment group,while PDCD4 expression decreased in miR-21 inhibitor transfection combined with 10 umol/L arsenic treatment group(all P<0.05).Conclusion1.Arsenic can induce oxidative damage and inflammatory damage in BRL-3A cells,and show a certain time and dose-effect relationship;medium and high concentrations of arsenic can reduce the activity of BRL-3A cells and promote apoptosis.2.Arsenic may induce inflammatory injury in BRL-3A cells by activating NF-κB to up-regulate miR-21 and inhibit the expression of PDCD4. |
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