The Role Of KLF2Regulated Nrf2/bach1in Mechanism Of Oxidation And Antioxidation In Rats’ Airway Epithelial Cells Induced By CSE

【Objective】To research the expression of Klf2and Nrf2/Bach1in rats’ airwayepithelial cells stimulated by CSE，and define the regulated function of target genesγ-GCS feeling of the oxidative stress. To investigate the effect of CSE on the apoptosisof airway epithelial cells and its relationship with the oxidation and antioxidationreaction.To observe the influence of Nrf2、Bach1and γ-GCS expression level bytransfected Klf2plasmid into cells.【Methods】The study was composed of two parts：(1)The part of cell without transfectedplasmid：The generation of rats’ bronchial epithelium cells used the enzyme digestedmethod，MTT assay was used to detect cell viability after interference with10%cigarette smoke extract (CSE) at different time points，The morphological changes ofcells treated with CSE for6hours were observed by Hoechst staining; And detected theconcentration of active oxygen species (ROS) and γ-GCS activity in cells at this timepoint. To observed nuclear transfer of Nrf2/Bach1in cells by immunohistochemistry.Western blotting and Reverse transcription-polymerase chain reaction（RT-PCR）techniques were used for research protein and its mRNA expressions of Klf2，Nrf2，Bach1，γ-GCS in cells．(2) The part of cell transfected plasmid：To transfect Klf2plasmidinto cells in order to its gene overexpression，Western blotting and RT-PCR techniqueswere used for research protein and its mRNA expressions of Klf2，Nrf2，Bach1，γ-GCSin these cells．【Results】1. The part of cell without transfected plasmid：(1)It appeared of time dependence on the inhibition of cell proliferation by CSE（P＜0.05）.Hoechst dyeing and electron microscopic observation results showed thatnuclear chromatin condensed and edge gathered even nuclear chipped.(2)The ROS content and γ-GCS activity were all rose in CSE group compared withnormal group（P＜0.01）.(3)Immunohistochemical results showed that Nrf2was translocated from cytoplasm to nucleus in response to stimulation by CSE. To the contrary，Bach1appears a transferfrom nucleus to cytoplasm in the same conditions（P＜0.05）.(4)Western blotting results showed that protein levels of Klf2、Nrf2、Bach1andγ-GCS were higher in the CSE group than that were in the control group（P<0.05）(5)RT-PCR results were the same as Western blotting results（P<0.05）.2. The part of cell transfected plasmid：(1)After Klf2overexpression plasmid successed transfecting cells，the mRNA andprotein levels of Nrf2and γ-GCS were observably increased compared with blankplasmid group in rats’ airway epithelial cells(P＜0.01)，while Bach1mRNA expressionlevel was lower than blank plasmid group’s(P＜0.05)，it’s protein level was nosignificant difference between the two groups(P＞0.05).(2)In blank plasmid group，mRNA and protein levels of Nrf2and γ-GCS treated6hours with10%CSE were all increased compared with blank plasmid group withouttreatment(P＜0.05).Bach1mRNA expression level was lower than that in blankplasmid group without treatment(P＜0.05)， its protein level was no significantdifference compared with control group(P＞0.05).(3)In CSE-treated Klf2plasmid group，mRNA and protein levels of Nrf2、γ-GCSwere all markedly elevated compared with untreated group(P＜0.05).Bach1mRNA andprotein expression levels were all lower than those in untreated group(P＜0.05).(4)mRNA and protein levels of Nrf2、γ-GCS in CSE-treated Klf2plasmid group were allhigher than those in blank plasmid group with the same treatment(P＜0.05).Bach1mRNAand protein expression levels were all lower than blank plasmid group with the sametreatment(P＜0.05).【Conclusions】1. CSE induced cells to occur oxidative stress reaction，it increased intracellular ROScontent，the finally effect was induced cells proliferation inhibition and apoptosisincreased， however antioxidant enzymes γ-GCS compensated rose to resistoxidative stress in the early days，along with the severity of oxidative damagedeepening γ-GCS gradually decompensated declined. 2. Nrf2palys a crucial role in up-regulating γ-GCS，which may benefit fromcytoplasm translocation of Bach1.3. When cells assumed oxidative stress，the expression of Nrf2inside cells increased，Klf2strengthen Nrf2’s endonuclear localization and stability，thus competitivelyinhibited Bach1，made Bach1transferring form nucleus to cytoplasm，both theinteraction raised antioxidative gene γ-GCS expression.