Effects Of NRF2 On NRF2 Inhibitory Factor And Its Relationship With MAPK Gene

Objective:To inhibit the effect of NRF2 on NRF2 inhibitory factor and its relationship with MAPK gene,and to provide the theoretical basis for the mechanism of arsenic toxicity induced by NRF2 gene in human keratinocyte cell line HaCaT.Methods:1.Construction of NRF2 shRNA: The human NRF2 gene was used as target gene to design small interfering RNA with NRF2 gene,and its shRNA and recombinant lentivirus expression vector were constructed and sequenced.2,lentivirus packaging: recombinant vector expression after the successful transfection and packaging cells 293 T,collection,concentration of the virus supernatant,the determination of its titer.3,inhibition of NRF2 gene Hacat cell model construction,the construction of three kinds of lentivirus were infected HaCaT cells(MOI=5),48 hours after infection,with 1μg / ml of puromycin screening stable cell lines.4.Identification of stable cell lines: After screening for stable cell lines,real-time quantitative PCR(Real-Time Quantitative PCR)was used to detect the interference efficiency.5,cell culture and arsenic treatment: the cells used in the conventional culture method of cell culture.The cell growth status was determined by MTT assay.The concentration of arsenic was determined.The concentration of arsenic was determined by 1/100,1/50,1/10.7 LC50 of toxicology standard.Cell viable oxygen was detected by flow cytometry.The expression levels of Nrf2,KEAP1,N6AMT1,Bach1,MAPK / ERK mRNAin the cells were detected by real-time fluorescent quantitative PCR.The expression levels of GSH,N6AMT1 and COX-2 in the cells were detected by ELISA.Results: 1.Sequencing of lentivirus interference vector construction showed that the recombinant vector was successfully constructed.2,lentivirus titer test results: according to the titer(TU/ml)= cell number×percentage×MOI(1)×virus dilution fold×103 select the best titer of the virus.3,NRF2 shRNA3 Hacat cells were selected,and the inhibition efficiency of the gene was 87%.4,arsenic treatment of low,medium and high dose groupswere 2.10μmol/L,4.20μmol/L,21.00μmol/L,inhibition of Nrf2,inhibition of Keap1,blank control group arsenic treatment concentration of 0μmol/L.5,cell proliferation:2.10μmol/L arsenic treated cells,cells proliferated;4.20μmol/L arsenic treated cells began to suppress at 72h;21.00μmol/L arsenic treated cells 48 h began to significantly inhibit cells.The cell rate of the blank group was slightly lower than that of the control group,which was higher than that of the Nrf2 control group.6,reactive oxygen species(ROS)in the blank group showed a stable trend with time,stable inhibition of Keap1 gene ROS expression,inhibition of Nrf2 gene ROS decreased,arsenic treatment ROS down.The expression of Bach1,N6AMT1,MAPK/ERK mRNA was promoted by blocking the Nrf2 gene for 8h and 24 h after treatment with 21μmol/L arsenic.The expression of Bach1,N6AMT1 and MAPK/ERK mRNA was inhibited by 21.00μmol/L arsenic for 72 h,,The expression of N6AMT1 and MAPK/ERK mRNA was down-regulated,the difference was statistically significant(P<0.001).The expression of N6AMT1,COX-2 and GSH protein was up-regulated in HaCaT cells treated with 24 h,and the expression of N6AMT1 and COX-2 in the HaCaT cells inhibited by NRF2 gene at 72 h and 29.00μmol/L 2,GSH protein expression was down-regulated,the difference was statistically significant(P<0.05);Conclusion: 1,arsenic with low effect.2,N6AMT1 can regulate oxidation and oxidation levels.3,GSH regulate the level of cell oxidation.4,ROS in the body to maintain a dynamic balance.5,Bach1,COX-2 can compensate for the antioxidant reaction.6,MAPK / ERK activates COX-2 expression.