The Role Of Long-isoform Transcription Factor Nrf1 In Inorganic Arsenic Toxicity In Mouse Bone Marrow Mesenchymal Stem Cells And Its Underlying Mechanisms

Object: Arsenic is a common toxic environmental metal pollutant,which is widely distributed in nature.Long-term exposure to inorganic arsenic could cause a variety of health effects such as malignant tumor,skin damage and metabolic diseases.Adipose tissue and bone tissue are important targets of inorganic arsenic.Even at low dose,longterm exposure of arsenic will cause damage to these tissues,leading to the occurrence of diseases,such as type 2 diabetes and osteoporosis.Mesenchymal stem cells(MSCs)are pluripotent adult stem cells with the potential of self-renewal and pluripotent differentiation,which can differentiate into adipocytes and osteoblasts.Studies have shown that mesenchymal stem cells are important target cells for arsenic toxicity.Arsenic can affect the cell viability of mesenchymal stem cells through oxidative stress and other ways,thereby inhibiting the formation of fat and osteoblasts and affecting the health of fat and bone tissues.Nuclear factor E2-related factor 1(Nrf1),as an important transcription factor in the CNC-basic leucine zipper family,not only regulates redox homeostasis,but also plays an important regulatory role in cell proliferation and aging,glucose metabolism,mitochondrial respiration,embryonic development and cell differentiation.This studies have found that Nrf1 plays an important role in the cytotoxicity induced by inorganic arsenic.This study intends to use mouse Bone Marrow Mesenchymal Stem Cells(mBMMSCs)as a cell model to investigate the role of long silencing of Nrf1 in the injury of mesenchymal stem cells induced by acute inorganic arsenic and explore its potential mechanism It will provide new ideas for future research on the mechanism of stem cell dysfunction caused by Nrf1 exposure to inorganic arsenic.Methods: Isolate the primary mBM-MSCs by adherent culture,and identify the cells by the identification method recommended by the International Cell Therapy Association.This separation technique has been explored in the early stage of the study group.The previous work of our study group has completed the exploration of this separation technology condition.After exposure to different concentrations of inorganic arsenic,the cell viability was measured by the CCK-8 method,and the reactivity of different molecular weight proteins of NRF1 to inorganic arsenic was detected by immunoblot experiments.Lentivirus transfection with shRNA was used to establish Scramble and L-Nrf1-KD stably transfected cells.After exposure to inorganic arsenic,the differences in arsenic sensitivity of Scramble and L-Nrf1-KD cells were determined by observing cell morphological integrity,detecting cell viability,apoptosis rate,expression of apoptosis-related proteins.After 24 hours of 5 μM inorganic arsenic exposure,the cells were collected;after 24 hours of exposure to the same concentration of inorganic arsenic,cell culture medium were replaced with inorganic arsenic-free medium and cultured for another 24 hours,and then the cell culture medium was collected.Atomic fluorescence method was used to detect the accumulation and efflux of arsenic in cells;mRNA-seq data was used to analyze the abundance of Aqp and Abcc family mRNAs in mBM-MSCs;The effects of basal and inorganic arsenic exposure state on mRNA and protein expression of key molecules were examined.Both Nrf2 mRNA,protein,and downstream gene expression levels were detected,and MitoSox Red probes were used to detect mitochondrial-derived ROS levels to evaluate the basic and inorganic arsenic exposure state on Nrf2-antioxidant defense effects.After mtROSspecific scavenger Mito-Q intervention,observed the cell morphological integrity,apoptosis rate,and expression of apoptotic proteins of the cells.Result: Compared with control cells,the level of long isoform NRF1 protein in arsenicexposed cells was significantly increased(p < 0.05).Compared with Scramble cells,cell morphology and mesenchymal stem cell surface marker expression of L-Nrf1-KD cell did not change.After exposure to arsenic(> 10 μM)for 24 hours,the apoptosis rate and apoptosis-related proteins cleaved-caspase 3 and cleaved-PARP expression was significantly higher than Scramble cells(p < 0.05).Compared with Scramble cells,there was no significant difference in arsenic accumulation in L-Nrf1-KD cells,but the efflux was reduced 15%(p < 0.05).L-Nrf1 gene silencing leads to decrease the of ABCC4,which is most likely the main reason for the reduction of arsenic efflux.When treated with 15 μM arsenic for different periods,the protein expression of NRF2 was significantly lower than Scramble cells after 12 hours(p < 0.05),and the mRNA levels of its downstream antioxidant genes Gclc,Gclm,Nqo1,and Ho-1 were also significantly lower than Scramble cells(p < 0.05).After exposure to 15～20 μM arsenic for 24 hours,the mitochondrial reactive oxygen species level of L-Nrf1-KD cells was significantly higher than Scramble cells(p < 0.05).Mito-quinone,a mitochondrial reactive oxygen species-specific scavenger,significantly reduced the apoptotic rate of L-Nrf1-KD cells induced by arsenic exposure(p < 0.05).Conclusion:1.Inorganic arsenic exposure induced NRF1 protein expression in mBM-MSCs,and mainly L-NRF1 protein expression.2.L-Nrf1 deficiency can not only inhibit the expression of ABCC4 that can promote inorganic arsenic efflux,but also increase the mtROS level,thereby enhancing the inorganic arsenic toxicity in mBM-MSCs.