Deletion Of Nrf2 Increase Endoplasmic Reticulum Stress Sensitivity Through Disturb The Unfolded Protein Response Of Pancreatic β Cell

Objective:The prevalence of diabetes is increasing and diabetes have become a global public health problem.The core of most diabetes pathogenesis is the islet of pancreaticβ-cell dysfunction and death.Understanding the molecular mechanisms behind cell failure are important for developing methods to prevent and reverse disease.Proinsulin production constitutes a major load on pancreaticβcell endoplasmic reticulum（ER）,and chronic serious ER stress is a cause of pancreaticβcell failure and loss in diabetes.Enhance the degradation of endoplasmic reticulum related misfolded protein and restore pancreaticβcell endoplasmic reticulum homeostasis are important treatment for diabetes.Nuclear factor E2-related factor 2（NRF2）is a CNC-b ZIP transcriptional factor that regulates a wide range of cell protection genes to respond to environmental pressure.The role of NRF2 in preventing pancreaticβcell damage is still unclear.Our previous study found that genetic ablation of Nrf2 gene in Akita diabetes mice aggravated pancreaticβcell apoptosis,indicating that NRF2 plays an important role in endoplasmic reticulum stress mediated apoptosis in pancreaticβcell.This study aimed to elucidate the role of the transcription factor NRF2 in pancreaticβcell under ER stress and specific mechanism in cell dysfunction,in order to provide new ideas for the prevention and treatment of diabetes by regulating NRF2 activity.Methods:1.Gene ablation was performed on Nrf2 mice in Akita diabetes model,which contained heterozygous mutation of mouse insulin 2 gene（Ins2WT/C96Y）leading to the formation of insulin-like disulfide bonds and the accumulation of a large number of misfolded proteins in the endoplasmic reticulum of pancreaticβcell,as well as the disorder of endoplasmic reticulum function,and ultimately leading to pancreaticβcell apoptosis.Mice of different genotypes were randomly divided into four groups:Nrf2-WT;Ins2^+/+、Nrf2-KO;Ins2^+/+non diabetes group,Nrf2-WT;Ins2^+/Akitaand Nrf2-KO;Ins2^+/Akitadiabetes group.Insulin and apoptotic index Cleaved Caspase-3 were detected by immune-histochemical staining.The ultrastructure of mouse pancreas was observed by electron microscopy.2.The Scramble and Nrf2-KD MIN6 cells established by lentivirus transduction were treated with endoplasmic reticulum stress inducers（thapsigargin（TG）and tunicamycin（TUN））in vitro.The transcriptome was sequenced by KC digital RNA seq absolute quantitative UMI technique,and the differentially expressed genes were analyzed with the difference criteria of log2（Fold Change）>1 and p<0.05.The biological function analysis of differentially expressed genes is enriched through GO pathways.After treatment with TUN and TG,the cell activity of Scramble and Nrf2-KD of MIN6cell was detected by trypan blue staining,and the expression of unfolded protein response related gene was quantitatively analyzed by Western blot and RT-q PCR.The cytoplasmic and nucleoprotein extraction kit was used to separate cell components and the protein levels of ATF6α、el F2α、ATF4 and BIP in different cell components were detected.Results:1.The Nrf2 gene ablation model of Akita diabetes model was successfully stablished.Immunohistochemical staining analysis of Insulin and Cleaved Caspase-3showed that the isletsβcell damage was aggravated of Nrf2-KO;Ins2^+/Akitamore than Nrf2-WT;Ins2^+/Akita,and the difference was statistically significant（P<0.05）.2.The ultrastructure of pancreatic islets observed by transmission electron microscope showed that there were a large number of secretory granules in the cytoplasm of non diabetes mice,which were in a large round shape and were surrounded by boundary membrane.The mitochondria,endoplasmic reticulum and Golgi complex in the cytoplasm were rich and developed.The structure of pancreatic tissue in the diabetes group changed significantly,the number of secretory granules decreased,there was degranulation,and the rough endoplasmic reticulum expanded to varying degrees.The shape and quantity of secretory granules in Nrf2 gene knockout mice changed abnormally,the degranulation phenomenon increased,and the endoplasmic reticulum expanded more.3.The Nrf2 gene and protein of Scramble and Nrf2-KD cells were verified.Compared with the control group,the expression of Nrf2 and downstream genes in Nrf2-KD cells decreased significantly after their knockout,and the difference was statistically significant（P<0.05）.It was confirmed that Nrf2 was effectively silenced in MIN6 cells.4.After TUN treatment of Scramble and Nrf2-KD cells at different times,the KC-digital RNA-seq absolute quantitative UMI technology was used for transcriptome sequencing,the m RNA of the protein-coding genes annotated by the genome was analyzed,and the gene expression quantity was counted,and the correlation between the gene expression characteristics of intra-group and inter-group samples and the differentially expressed genes were evaluated.Gene Ontology comprehensive database is used to define and describe gene and protein functions.The database standardizes the biological terms of genes and gene products in different databases and is applicable to all species.After screening the genes that respond to endoplasmic reticulum stress,study the distribution of Scramble and Nrf2-KD cell differential genes in Gene Ontology,and clarify the embodiment of sample differences in gene function in the experiment.5.TUN（2μg/ml）and TG（150n M）treatment Scramble and Nrf2-KD cells for 0,6,18,and 24 hours,it was observed that the cell morphology changed significantly with the prolongation of treatment time,and obvious cell death occurred at 18 hours,and the number of Nrf2-KD cell death was significantly greater than that of Scramble cells.TUN（2μg/ml）treatment for 24 hours showed that the number of living cells was lower than that of control cells,which proved that Nrf2-KD group was more sensitive to ER stress than Scramble group.This was consistent with the results of in vivo experiments,and the difference was statistically significant.6.TUN（2μg/ml）/TG（150n M）,the m RNA expression levels of UPR molecules in Scramble and Nrf2-KD cells were significantly different after different time treatment,which was consistent with the results of cell transcriptome sequencing.The expression levels of ATF6,ATF4,GADD34,ATF5 and CHOP were significantly decreased after Nrf2 silencing.7.Western blot results showed that the sensitivity of Nrf2-KD to endoplasmic reticulum stressors compared with Scramble could be at least partially through BIP,ATF6αand P-PERK protein level.ATF6αdifferently expressed under the influence of different endoplasmic reticulum stressors,when Ca²⁺-ATPase inhibition leads to pancreaticβcells.Nrf2 silencing significantly reduces ATF6αduring endoplasmic reticulum stress but has little effect on its nuclear translocation.Conclusions:Nrf2 silencing blocks the induction of molecular chaperone glucose regulatory protein BIP by reducing the main branches of UPR P-PERK and ATF6α.The effect of the expression of ATF4,GADD34 and CHOP contributes to the vulnerability of pancreaticβcell to endoplasmic reticulum stress,and then aggravates the severity of diabetes.