Regulation Of The Nrf2/Mafk Pathway On GPx In Cristaria Plicata

Keap1/Nrf2-ARE signaling pathway is an endogenous antioxidant pathway that protects cells from oxidative damage.When aquatic organisms exposed to environmental toxicant stress,the Keap1/Nrf2-ARE signaling pathway is triggered to produce responses.In this study,We studied the relationship between the core transcription factor Nrf2 and Mafk in the Keap1/Nrf2-ARE signaling pathway,and their regulation of downstream glutathione peroxidase(GPx).The expression of CpMafk in hepatopancreas,gills and kidneys was up-regulated under microcystins stimulation.In hepatopancreas,CpMafk mRNA expression was significantly up-regulated at 12 and 72 h.In gills,the expression of CpMafk was significantly up-regulated at 12 and 48 h.In the kidney,the expression of CpMafk was significantly upregulated at 72 and 96 h after silencing CpNrf2 and microcystin(MC)treatment.Comparison with MC group,the expression of CpMafk in hepatopancreas,gills and kidneys showed a downward trend.The hepatopancreas showed the most obvious changes,and the mRNA expression of CpMafk was significantly down-regulated at 12,24,48,96 h.In gills,the expression of CpMafk was significantly down-regulated at 48,72 and 96 h.In the kidney,CpMafk expression was extremely significantly up-regulated at 24 h and extremely significantly down-regulated at 96 h.Comparison with the NS group,CpMafk mRNA expression level decreased by 45%at 48 h after CpMafk injection,respectively.CpMafk mRNA expression in ODNI group decreased by 46%at 24 h.In ODN2 group,CpMafk mRNA expression level decreased by 32%and 49%at 24 and 48 h,respectively.However,comparison with ODNyx group,CpMafk mRNA expression level only showed interference effect at 48 h.In ODN3 group,CpMafk mRNA expression was decreased by 41%and 46.8%at 24 and 48 h,respectively.Comparison with ODNyx group,CpMafk mRNA expression was significantly decreased.In ODN4 group,CpMafk mRNA expression decreased by 60%and 32%at 24 and 24 h,respectively.Comparison with ODNyx group,CpMafk mRNA expression decreased significantly at 24 h.In ODN5 group,CpMafk mRNA expression level decreased by 45%and 44%at 24 and 48 h,respectively.Comparison with ODNyx group,CpMafk mRNA expression level decreased significantly.Under different interference effect,comparison with NS group,CpMafk disturbance effects were 41%and 46.8%,CpNrf2 mRNA expression significantly decreased,CpMafk interference effect was 60%,CpNrf2 mRNA expression decreased significantly.Comparison with ODNyx group,CpNrf2 mRNA expression decreased significantly,while CpMafk interference effect was 46.8%.Comparison with NS group,CpGPx mRNA expression was significantly down-regulated.Comparison with the ODNyx group,CpGPx mRNA expression was significantly down-regulated and extremely significantly down-regulated,while CpMafk interference effect was 41%and 60%,respectively.The lack of Nrf2 in Cristaria plicata would affect the transcription level of Mafk,and the lack of Mafk in the Cristaria plicata would also cause the decline of Nrf2 transcription level.These results were suggested that a direct or indirect regulatory relationship was between transcription factors of CpNrf2 and CpMafk.The prokaryotic expression of CpNrf2 with GST label showed that GST-CpNrf2 protein could bind to Mafk protein in the Cristaria plicata tissue protein.The yeast double-hybrid test further verified that a direct interaction was between CpNrf2 and CpMafk.The total length of the CpGPx promoter sequence was 1974 bp,and the prediction showed that CpGPx promoter sequence had three TATA-boxes,three CAAT-boxes,and a variety of transcription factor binding sites,including Nrf2:Mafk,AP-1,c-myc,NF-kappaB,Smad3,Smad4,ARE elements.GST-CpNrf2 protein was unable to bind to the promoter of CpGPx in vitro through EMSA.The dual luciferase reporter assay indicated that CpNrf2 and CpMafK negatively regulated the promoter of CpGPx.