BRL-3A Cell Injury By NaAsO₂ Via The SOCS1-TLR4/MyD88/NF-κB Pathway And The Intervention Of Proanthocyanidins

ObjectiveArsenic poisoning is a serious health risk for humans,and long-term chronic arsenic exposure can cause arsenic to accumulate in the liver,leading to liver lesions,yet the molecular mechanisms of arsenic-induced liver damage are not fully understood.Proanthocyanidins（PC）,a natural polyphenolic compound,have been shown to reduce arsenic poisoning,however,the potential mechanism for alleviating arsenic-induced liver damage still needs to be investigated.Therefore,the aim of this study was to investigate whether sodium arsenite（NaAsO₂）could induce apoptosis and inflammatory in BRL-3A cells via the SOCS1/TLR4/My D88/NF-κB signalling pathway,and whether PC could antagonise arsenic-induced BRL-3A cells injury.To provide a theoretical basis for the possible mechanism of hepatotoxicity of arsenic and a reference for the prevention and treatment of arsenic poisoning by proanthocyanidins.MethodsIn this study,BRL-3A cells were selected as the object of study.Cells were treated with different concentrations（0,5,10,20μmol/L）of NaAsO₂,and intervened by SOCS1 plasmid transfection,TLR4inhibitor TAK-242,PC in combination with NaAsO₂.Subsequently,cell activity was detected using CCK-8assay;Annexin V-FITC/PI to detect apoptosis in BRL-3A cells;and cellular immunofluorescence to detect the intracellular localization and expression of SOCS1,TLR4,and My D88.The expression of pathway-related proteins:SOCS1,TLR4,My D88,IκBα,p-IκBα,p65,p-p65,BAX,Bcl-2,caspase-3,IL-1β,IL-6,and TNF-αin BRL-3A cells was detected by Western Blot.The assay indicators were described in the form of mean±standard deviation,and the statistical methods such as ANOVA and Bonferroni were used to statistically analyse the differences in the results,with the test levelα=0.05.Results1.Effect of NaAsO₂ and PC on BRL-3A cell activity Compared with the control group,cell activity decreased when BRL-3A cells were intervened with 5,10 and 20μmol/L NaAsO₂ for 24 h（P<0.05）;and cell activity gradually decreased when BRL-3A cells were intervened with 10μmol/L NaAsO₂ for 24 h,48 h and72 h（P<0.05）.In addition,there was no significant change in cell activity after 25 and 50 mg/L PC intervention in BRL-3A cells for 24 h.The activity of BRL-3A cells decreased remarkably after 75 and 100mg/L PC intervention（P<0.05）.When 25 and 50 mg/L PC were combined with 10μmol/L NaAsO₂intervention,BRL-3A cell activity was substantially higher compared with the NaAsO₂ group（P<0.05）;after75 mg/L PC combined with 10μmol/L NaAsO₂ intervention,BRL-3A cell activity did not change appreciably compared with the NaAsO₂ group.After the combined intervention of 100 mg/L PC and 10μmol/L NaAsO₂,BRL-3A cell activity was dramatically reduced compared with the NaAsO₂ group（P<0.05）.2.Effects of NaAsO₂ intervention on apoptosis and inflammatory injury in BRL-3A cells Compared with the control group,the apoptosis was obviously increased（P<0.05）,and the expression of BAX/Bcl-2ratio and caspase-3 was increased after 24 h of 10 and 20μmol/L NaAsO₂ intervention in BRL-3A cells.In addition,compared with the control group,the expression of inflammatory factors IL-1βand TNF-αwas significantly increased after 24 h of intervention with 5,10,and 20μmol/L NaAsO₂ in BRL-3A cells（P<0.05）.The expression of inflammatory factor IL-6 was markedly increased after 24 h of intervention with 10 and20μmol/L NaAsO₂ in BRL-3A cells（P<0.05）.3.Role of SOCS1/TLR4/My D88/NF-κB signaling pathway in NaAsO₂-induced apoptosis and inflammatory injury in BRL-3A cells Compared with the control group,NaAsO₂ induced a dramatically lower expression level of SOCS1 and Bcl-2（P<0.05）and a markedly higher expression level of TLR4,My D88,p-IκBα,p-p65,BAX/Bcl-2 ratio,caspase-3,IL-1β,IL-6,and TNF-α（P<0.05）.Compared with the NaAsO₂ alone group,the expression of apoptotic and inflammatory factors（IL-1β,IL-6,TNF-α）was reduced after the combined intervention of SOCS1 overexpression transfection and NaAsO₂,and the expression of SOCS1,Bcl-2 was restored,and TLR4,My D88,p-IκBα,p-p65,BAX/Bcl-2,and caspase-3 expression levels were obviously reduced（P<0.05）.The combined intervention of TLR4 inhibitor TAK-242 and NaAsO₂resulted in attenuated apoptosis and inflammatory injury in BRL-3A cells,increased expression of Bcl-2,and the expression of TLR4,My D88,p-IκBα,p-p65,BAX/Bcl-2 ratio,caspase-3,IL-1β,IL-6,and TNF-αcompared to the NaAsO₂ group was noticeably lower（P<0.05）.4.PC antagonized NaAsO₂ to induce apoptosis and inflammatory injury in BRL-3A cells Compared with the control group,the apoptosis rate and inflammatory factor expression were not dramatically altered after PC intervention,and there were no significant changes in SOCS1/TLR4/My D88/NF-κB pathway indicators.Compared with the NaAsO₂ group,apoptosis was attenuated and inflammatory factor expression was reduced after combined NaAsO₂ and PC intervention,and SOCS1,TLR4,My D88,p-IκBαand p-p65were significantly reduced（P<0.05）.ConclusionNaAsO₂ lead to apoptosis and inflammation in hepatic BRL-3A cells through inhibition of SOCS1,upregulation of TLR4/My D88,and activation of NF-κB signaling pathway.PC may alleviate NaAsO₂-induced apoptosis and inflammatory injury in BRL-3A cells through SOCS1/TLR4/My D88/NF-κB signaling pathway.