Inorganic Arsenic Induces Pyroptosis And β Cells Dysfunction Through Stimulating The IRE1α/TNF-α Pathway And Protective Effect Of Taurine

Objective:Chronic exposure to inorganic arsenic（iAs）is a risk factor forβcells dysfunction.Our previous studies demonstrated that iAs induced endoplasmic reticulum stress and pyroptosis in C57/BL6J mice,and taurine（Tau）decreased the activation of pyroptosis.Inositol-requiring enzyme 1 alpha（IRE1α）,one of the three classic ER stress signaling pathways,is a key driver of inflammation.However,the mechanism of IRE1αregulates pyroptosis is still unclear.Therefore,in this study,we investigated the mechanism of As₂O₃-inducedβcells dysfunction and the protection of Tau in pancreas of rat offspring.Methods:In vivo,the Wistar mice were used as experimental model.All the pregnant rats and offspring were exposed to As₂O₃（2⁸ mg/kg）and Tau（150 mg/kg）once a day.The pregnant rats were given As₂O₃ and Tau by gavage from GD 6 until pup postnatal day 28（PND 28）.The offspring were given As₂O₃ and Tau by gavage from PND 28 until PND 42.At last,the offspring were euthanized,and the pancreas were collected for the following experiments.The morphology of pancreas was observed by hematoxylin-eosin（HE）staining.The expression of inositol-requiring enzyme 1 alpha（IRE1α）,phosphorylated protein p-IRE1α,tumor necrosis factor-α（TNF-α）,NOD-like receptor family pyrin domain-containing 3（NLRP3）,pro-caspase-1,caspase-1,apoptosis-associated speck-like protein（ASC）,pro-interleukin（IL）-1β,IL-1βand MPO in pancreas were detected by Western blot.In vitro,we used INS-1 cells for the study.The cytotoxicity of As₂O₃ was detected by MTT assay.The expression of p-IRE1αin INS-1 cells treated with 1⁴μM As₂O₃for 24 h was detected by immunofluorescence staining;The expression of TNF-α,NLRP3,pro-caspase-1,caspase-1,ASC,pro-IL-1β,IL-1βwas detected by Western blot.INS-1 cells were pretreated with NLRP3 inhibitor（MCC950）,TNF-αinhibitor（Lenalidomide）,IRE1αinhibitor（Irestatin 9389）and Tau before treatment with 4μM As₂O₃ for 24 h.Immunofluorescence staining was used to detect the expression of p-IRE1α;Western blot was used to detect the expression of TNF-α,NLRP3,caspase-1,IL-1βin INS-1 cells.Flow cytometry was used to examine the pyroptotic INS-1 cells.Lactate dehydrogenase（LDH）assay kit was used to measure the LDH release.The Rat INS（Insulin）ELISA Kit was used to measure the insulin release.Results:In vivo,the structures of pancreas in As₂O₃-treated groups were irregularly arranged and not clear,with glands swelling.After Tau treatment,improvements on the morphology of pancreas were observed;The p-IRE1α,TNF-α,NLRP3,caspase-1,the level of IL-1βand MPO were significantly increased in the As₂O₃ treated group at 8 mg/kg and all were significantly decreased in Tau treated group compared with only As₂O₃-treated group.In vitro,INS-1 cells were treated with different concentrations of As₂O₃ for 24 h,and INS-1 cells viability decreased with the increase of As₂O₃ concentration.After treated with 1⁴μM As₂O₃ for 24 h,the expression of p-IRE1α,TNF-α,NLRP3,caspase-1 and IL-1βwere increased.After treated with NLRP3 inhibitor（MCC950）,TNF-αinhibitor（Lenalidomide）,IRE1αinhibitor（Irestatin 9389）and Tau the expression of NLRP3 inflammasome related proteins were decreased compared with the As₂O₃-treated group;The results of flow cytometry showed that the percentages of pyroptotic cells was significantly decreased compared with the As₂O₃-treated group;The level of LDH release rate in INS-1 cells were decreased compared with the As₂O₃-treated group;Importantly,the insulin release suppressed by As₂O₃ were promoted.Conclusion:In conclusion,our study demonstrated that As₂O₃ upregulated the level of IRE1α,TNF-αand triggered NLRP3 inflammasome activation,leading to INS-1 cells pyroptosis and dysfunction;however,these effects could be improved by Tau.Tau markedly protected As₂O₃-inducedβcells dysfunction by reduced the phosphorylation of IRE1α,secretion of TNF-αand activation of pyroptosis.