Study On Regulation Of Antioxidant Genes By JNK Mediated Nrf2-ARE Pathway From Cristaria Plicata

JNK are members of the mitogen-activated kinase(mitogen-activated protein kinase,MAPK)family.It can regulate physiological and pathological processes such as cell growth and differentiation,environmental stress and inflammatory response.Nrf2-ARE pathway is an important pathway to resist endogenous or exogenous electrophilic attacks and protect cells from oxidative damage.NQO1,as Nrf2 classical downstream target gene,plays a crucial role in quinone metabolism,scavenging peroxides and resisting oxidative damage.This paper studies the regulation function of MAPK family members JNK on Nrf2.Moreover,Nrf2 transcriptional regulation of downstream target gene NQO1 was detected.In this study,the cDNA sequences of CpJNK and CpNQO1 were cloned.The size of CpJNK's 5'-untranslated region is 240bp,the 3' untranslated region is 2372bp,the open reading frame is 1209bp,a total of 402 amino acids are encoded,including an S TKc domain(24-320aa).JNK conserved motifs T-P-Y motifs exist within the activation ring in this domain and are highly conserved at Gly-225 sites.The size of CpNQO1's 5'-untranslated region is 47bp,the 3' untranslated region is 718bp,an the open reading frame is 888bp,codes for 295 amino acids,and contains a Flavodoxin 2 domain(15-224aa).Real-time PCR was used to analyze the distribution of CpJNK and CpNQO1 in the tissues of Cristaria plicata.CpJNK and CpNQO1 were expressed in all the detected tissues.CpJNK was the highest expressed in hepatopancreas,CpNQO1 was the highest expressed in kidney,and both were the lowest expressed in blood cells.The expression of CpJNK mRNA in the hepatopancreas,gills and kidneys overall showed a down-regulation trend after MC stimulation.The expression of CpJNK mRNA was extremely significantly down-regulated at 12 and 24 h in hepatopancreas;at 12,24 and 72 h in gills;at 12 and 96 h in kidneys.After knock-down CpNrf2,the expression of CpJNK mRNA in hepatopancreas,gills and kidneys was up-regulated compared with MC group.The expression of CpJNK mRNA was extremely significantly upregulated at 12 and 24 h in hepatopancreas;at 72 h in gills;at 12 and 24 h in kidney.After MC stimulation,the CpNQO1 showed an up-regulation trend compared with NS group.The expression of CpNQO1 mRNA was significantly upregulated at 48 and 72 h in hepatopancreas;at 24,48 and 72 h in gills,and the highest expression was found at 24 h.at 48 and 72 h in kidney.The expression of CpNQO1mRNA in hepatopancreas,gills and kidneys showed a down-regulation trend compared with MC group after knock-down CpNrf2.The expression of CpNQO1mRNA was significantly downregulated at 12,24,48 and 72 h in hepatopancreas;at 24,72 and 96 h in gills;at 12,24,48 and 96 h in kidney.When stimulated by MC,the enzyme activity of Cu/Zn-SOD was significantly up-regulated at 96 h,GPx activity increased significantly at 24 and 48 h,MDA content was significantly up-regulated at 72 and 96 h.Cu/Zn-SOD activity was significantly down-regulated at 24 and 72 h,GPx activity at 24,48,96 h,while MDA content was significantly up-regulated at 12 h after knock-down CpNrf2.The expression of CpNrf2 was significantly higher than that of negative control group after inhibition of JNK by SP600125.and the expression of downstream target genes CpNQO1?CpMn-SOD?CpCu/Zn-SOD?CpPRX was up-regulated to some extent.the expression of CpNrf2 and its downstream target genes was up-regulated after MC stimulation.the expression of CpNrf2 and its downstream target genes was upregulated compared with MC group after addition of inhibitors and MC treatment.Westrem-blot experimental results showed that the expression of P-JNK was down-regulated after MC treatment,while the expression of P-JNK was up-regulated after knock-down CpNrf2;The expression of CpNrf2 in SP group was up-regulated;compared with MC group,the expression of CpNrf2 in SP+MC group was up-regulated.Gel affinity analysis showed that CpNrf2 could bind to CpNQO1 promoter in vitro.The double luciferase report assay showed that CpNrf2 could up-regulate CpNQO1 transcription level in a dose-dependent manner;The individual CpMafK can not regulate the transcription level of CpNQO1,and it must ombined with CpNrf2 to play its transcriptional regulatory role.