The Effect Of Acute Arsenite Exposure On The Expression Of Short Isoforms Of NRF1 In Pancreatic ? Cells And The Underlying Mechanisms

Objective:Long-term exposure of arsenic can lead to skin lesions,substantial organ damage,and the induction of various cancers.More and more epidemiological studies have revealed environmental arsenic exposure is closely related to Type 2 diabetes.Our previous study found the long-isoforms knockdown of the transcription factor NRF1?Nuclear factor E2-related factor 1,NFE2L1?in MIN6 pancreatic?cells was more sensitive to cell damage caused by arsenic exposure,suggesting that the long isoforms NRF1?L-Nrf1s?play an important role to protect pancreatic?cells from apoptosis.Mouse Nrf1 gene have four isoforms transcripts.The dynamics of these various isoforms of Nrf1 in response to iAs-induced pancreatic?cells cytotoxicity are unknown.In the present study,we firstly found short isoforms of NRF1 are responsive to acute arsenite exposure and they play significant roles in iAs-induced pancreatic?cells cytotoxicity and apoptosis.The results provide important insights into the initial molecular response to iAs in pancreatic?cells of arsenic diabetogenic effects.Methods:1.Nrf1 in MIN6 cells and mouse islets with arsenic exposure were detected by Real-time PCR and Western Blot.2.OE-Cont and OE-583,OE-453 MIN6 cells were stably constructed,and NRF1 protein was verified by Western Blot.3.With acute arsenic treatment in OE-Cont and OE-583,OE-453 MIN6 cells,cell viability were detected by trypan blue staining and CCK kit,apoptosis protein Cleaved-Caspase-3 was detected by Western Blot,and cell cycle changes were measured by flow cytometry.4.Anti-oxidant enzyme genes,apoptosis process-related genes,proteasome subunit gene,phase II enzyme genes and arsenic absorption and efflux genes in OE-Cont,OE-583 and OE-453 MIN6 cells were detected by Real-time qPCR analysis.5.Measurement of influx and efflux of arsenic were detected by atomic fluorescence.6.Serial 5'-deleted S-Nrf1s promoter-driven luciferase reporters were designed and luciferase activity were analyzed using a Luciferase Reporter Assay System.7.mRNA stability of Nrf1 in normal MIN6 cells were measured by Real-time qPCR.Results:1.Acute iAs³⁺exposure induces nuclear S-Nrf1s accumulation in pancreatic?cells.2.Stable OE Nrf1-583 and OE Nrf1-453 MIN6 cells were constructed successfully by transducing with lentivirus.3.CCK testing results,trypan blue staining count cells,the apoptotic protein level of Cleaved-Caspase-3,and the number of dead cells with flow cytometry in MIN6 cells all indicate that the OE S-Nrf1s MIN6 cells are more tolerant to cytotoxicity caused by acute arsenic exposure.4.With arsenic exposure,the mRNA levels of antioxidant genes Gclc,Gclm and Ho-1did not increase in the OE S-Nrf1s MIN6 cells,so it cannot explain the protection of S-NRF1s;the genes involved in the apoptosis process IRF1,MAPT,RASSF5 and BNIP3,the proteasome subunit gene PsmB6,and the enzyme genes Nqo1,Gsto1 and Gstm1 mRNA levels in OE-Cont and OE-583,OE-453 cells no statistically significant;The mRNA level of Abcc family related genes involved in arsenic efflux in OE S-Nrf1s MIN6 cells were significantly higher than the control cells.5.The luciferase activity of different length responsive region in the S-Nrf1s gene promoter did not be induced under the exposure of iAs,and the increased expression of Nrf1 induced by iAs³⁺is partially related to its mRNA degradation.Conclusion:1.Acute iAs³⁺exposure can induce short isoforms Nrf1?S-NRF1s?accumulation in the pancreatic?cells.2.Over-expression of S-NRF1s in MIN6 cells is more tolerant to cytotoxicity caused by acute arsenic exposure.Preliminary results indicate that the OE S-Nrf1s MIN6 cells decreases intracellular arsenic accumulation and reduces intracellular toxicity.