Sodium Arsenite Induces Apoptosis Of BRL-3A Cells Through DNMT1 Regulation Of SOCS1/NF-κB Pathway

Objective:Arsenic is a common toxic metalloid in nature.The human body can be exposed to arsenic through various ways.When excessive arsenic enters the human body,it will cause harm to health,especially the liver is the most important metabolic organ of the human body.Arsenic is likely to cause more serious damage to the liver.However,the specific mechanism of its damage to the liver is not fully clear.Previous studies have shown that the activation of NF-κB pathway by sodium arsenite can cause inflammatory damage and apoptosis in BRL-3A cells,and Suppressor of cytokine signaling 1（SOCS1）can regulate NF-κB signal pathway.It has been reported that the high expression of DNMT1 can inhibit the expression level of SOCS1.Therefore,our purpose of this study is to explore whether sodium arsenite can regulate SOCS1/NF-κB pathway through DNMT1 and finally induce apoptosis of BRL-3A cells.Methods:In this study,CCK-8 method was used to detect the cell activity of BRL-3A cells treated with 0,5,10,20 μmol/L sodium arsenite.Western Blot method was used to detect p65,p-p65,κBα,p-IκBα,apoptosis-related proteins Bax,Bcl-2,inflammatory factors IL-1β,IL-6,TNF-α and DNMT1,SOCS1 under the intervention of different concentrations of sodium arsenite and DNMT1 inhibitor DC 517,and DNMT1 mRNA expression was detected by qRT-PCR method.Determination of protein expression of DNMT1 and SOCS1 by immunofluorescence.Apoptosis was detected by Annexin-FITC/PI method.Analysis of variance was used for comparison between groups,Dunnett method was used for comparison with control group,and Tukey’s method was used for multiple comparison between groups,α=0.05.Results:1.Effect of NaAsO2 on BRL-3A cells:After BRL-3A cells were exposed to different concentrations of 0,5,10,20 μmol/L NaAsO2 for 24 hours,with the increase of NaAsO2 treatment concentration,the number of dead cells gradually increased,and the cell viability decreased with the increase of the dose and time of NaAsO2 intervention.2.Effect of NaAsO2 on BRL-3A cells apoptosis and inflammatory factors:10 and 20 μmol/L NaAsO2 can increase the number of apoptotic cells in BRL-3 A cells and promote the protein expression level of the inflammatory cytokine IL-1β,TNF-α and IL-6（P<0.05）,the expression level of antiapoptotic protein Bcl-2 decreased（P<0.05）,the expression level of proapoptotic protein Bax increased（P<0.05）.3.Effect of NaAsO2 on SOCS1/NF-κB pathway in BRL-3A cells:Compared with the control group,the protein expression of p-p65 and p-IκBα in BRL-3A was higher,and the protein expression of IκBα was decreased.After BRL-3A cells were treated with different concentrations of NaAsO2 for 24 hours,the expression of SOCS1 was detected.The results showed that compared with the control group,the expression of SOCS1 decreased gradually with the increase of NaAsO2 concentration,and the green fluorescence intensity was weaker.4.Effect of NaAsO2 on DNMT1 in BRL-3A cells:When measuring the expression of DNMT1 protein,we found that with the increase of NaAsO2 concentration,the expression of DNMT1 increased（P<0.05）,and in the immunofluorescence experiment,it was also found that the green fluorescence in the nucleus of the 10 and 20 μmol/L NaAsO2 groups was more than that of the control group.5.The role of DNMT1 in NaAsO2 induced BRL-3A cell apoptosis:After the combination of DNMT1 inhibitor DC 517 and NaAsO2,the number of apoptotic cells detected by Annexin-FITC/PI method was lower than that of the arsenic alone group,while the expression of Bcl-2 increased（P<0.05）,the level of Bax protein decreased（P<0.05）,and the level of proinflammatory cytokines protein expression also decreased（P<0.05）.6.DNMT1 affects the regulatory effect of NaAsO2 on SOCS1/NF-κB signal pathway:After DC₅17 and NaAsO2 co-interfere with BRL-3A cells,the protein level of p-p65 and p-IκBα arsenic was lower than that of arsenic alone intervention group（P<0.05）,however,the level of IκBα was up（P<0.05）;after DNMT1 was inhibited,NaAsO2 was added.At this time,the expression of SOCS1 protein was higher than that of arsenic alone（P<0.05）.In the cell immunofluorescence test,the fluorescence intensity of SOCS1 protein also had the same trend.Conclusion:NaAsO2 promotes DNMT1 expression,down-regulates SOCS1,and activates NF-κB.Inhibit the expression level of Bcl-2,promote Bax and promote inflammatory factor IL-1β,TNF-α and IL-6 expression,make BRL-3A cell apoptosis and induce inflammatory injury,which provides a theoretical basis for elucidating the molecular mechanism of arsenic-induced liver injury.