KLF2Regulate Nrf2/Bach1in The Bronchical Epithelial Cells Sensitized By Derp1Allergen Against Oxidative Stress

Objective: The possible underlining mechanisms of asthma have not been welldefined. At present, There are an increasing awareness of the airway “free radicaldamage” and “oxidation/antioxidant imbalance” theory, which need immediateattention. The respiratory tract epithelium is a critical mechanical barrier to preventthe lung from the entrance of stimulation, such as allergens. Airway epithelial cellscan release chemotactic factors after allergen exposure and induce inflammatory cellsrecruitment which produce a number of ROS（reactive oxygen species) and leads tooxidative damage in the lung. Derp1（Dermatophagoides pteronyssinus1,Derp1)allergen is an ubiquitous and important indoor allergen，and it is also believed to takepart in the processes of asthma. To study the influence of Derp1allergen may activateoxidative stress induced the damage to the rat bronchial epithelial cells and caused theexpression of antioxidant genes in damaged cells. To detect the effect of Derp1allergen on the regulating expression of Krüppel-like factor2（KLF2)、NF-E2relatedfactor2（Nrf2）、BTB and CNC homology1（Bach1) andγ-glutamylcysteinesynthetase(γ-GCS) in rat bronchial epithelial cells. In addition, To explore themechanisms of overexpressing KLF2influence the expressson levels of Nrf2、Bach1and γ-GCS in cells and the effect of that in the pathogenesis of bronchial asthma. Itwill be a new theoretical basis for the preventment and treatment of asthma in future.Methods:（1）Tracheas were removed from120-to140-weight SD male rats sacrificed byPentobarbital Sodium. The rat bronchia epithelial cells were isolated by4℃digestionwith0.5%pronase on the epithelial side of trachea and bronchus before brushing withcell brush and cultured in serum-free hormone supplemented K-SFM. The cells wereidentified with immunofluorescence and electron microscopy, both the cell viability and the membrane integrity of cells were evaluated.（2）Using different intervention and treatment, The rat bronchia epithelial cellswere divided into three groups: control group (group A), Derp1allergen (5μg/ml)group (group B) and DEX (5μg/ml Derp1allergen+1×10-6mol/l dexamethasone)group (group C), cultured for8h, thenγ-GCS activity was measured by two enzymesmethod；To detect the levels of KLF2、Nrf2、Bach1andγ-GCS mRNA in each groupby reverse transcription-polymerase chain reaction (RT-PCR)；The protein expressionof KLF2、Nrf2、Bach1andγ-GCS in each group were observed by western blot(WB).（3）The rat bronchia epithelial cells were divided into blank plasmid group (groupA) and KLF2recombinant plasmid group (group B) by using different plasmidtransfected into cells. Furthermore,To observe the transfection effect between A and Bgroup by using immunofluorescence, and test the transfection efficiency in each groupby using RT-PCR and WB. However, Group A and B were cultured and divided into3groups according to dissimilar intervention and treatment like before. We nextevaluated the KLF2overexpressing by plasmid trnsfected into bronchial epithelialcells, then detected the influence of KLF2overexpressing on the mRNA and proteinexpressions of Nrf2、Bach1andγ-GCS in each group by using RT-PCR and WB.Results:（1）The method for Low concentration of Pronase digestion and mechanicaldissociation were adopted to obtain rat bronchia epithelial cells in high purity andhigh viability. The cells grow well in entire K-SFM media with no serum.Immunofluorescence analysis of cytokeratin19expressed in cytoplasma of cells,accorded the characteristic of bronchial epithelium cells. It is clear that the typical ratstrachea and bronchial epithelium of ciliated columnar cells in the electron microscopy,cellular membrane is intact, intracellular organelles are healthy，cells have a largenumber of cilia on one side.（2）The activity ofγ-GCS in the group B was higher than that in group A（P<0.05）;In the group C, γ-GCS activity was elevated compared to controls (P<0.05), but not reach statistical difference compared with group B（P>0.05).（3）RT-PCR results showed that the expression of KLF2mRNA in the group Bwas obviously higher than group A（P<0.01）and group C (P<0.05), The levels ofKLF2mRNA in the group C is better than group A, the difference wassignificant(P<0.05); The expression of Nrf2mRNA in the group B was evidentlyhigher than group A（P<0.01）and group C (P<0.05), The levels of Nrf2mRNA in thegroup C is better than group A, the difference was significant(P<0.01); The expressionof Bach1mRNA in the group B was lower than that in group B and group C（P<0.05）,while there were no statistical difference between group C and group A(P>0.05); Theexpression ofγ-GCS mRNA in the group B was distinctly higher than group A andgroup C (P<0.05), but there were no statistical difference between group C and groupA (P>0.05).（4）Western blot results showed that the expression of KLF2protein in the groupB was obviously higher than group A（P<0.01）and group C (P<0.05), The levels ofKLF2Protein in the group C is better than group A, the difference wassignificant(P<0.05); The expression of Nrf2Protein in the group B was evidentlyhigher than group A（P<0.01）and group C (P<0.05), The levels of Nrf2Protein in thegroup C is better than group A, the difference was significant(P<0.05); Theexpression of Bach1Protein had no discernible difference among the three groups（P>0.05）; The expression ofγ-GCS Protein in the group B was higher than groupA(P<0.05), The levels of γ-GCS Protein in the group C is better than group A, thedifference was significant(P<0.05).（5）Immunofluorescence results showed that lipo2000vector can effectivelytransfected contrast blank plasmid and KLF2recombinant plasmid into the ratsbronchial epithelial cells，analysis of transfection effect revealed group A can reach to60.4%and group B can reach to41.6%. Based on the results of RT-PCR and Westernblot，the expression of KLF2mRNA and KLF2Protein in the group B was higherthan group A，the difference was significant(P<0.05).（6）RT-PCR results showed that the expression of Nrf2mRNAin the group B was evidently increased than group A（P<0.01），especially in the group A3，the differenceof the expression of Nrf2mRNA was higher than group A1and A2have statisticallysignificant（P<0.05）；The expression of Bach1mRNA were negative or low positivein the group A and B，but there are no statistical difference between two groups(P>0.05). The expression of r-GCS mRNA in the group B was increased than groupA（P<0.05），especially in the group A2and group B2，the difference of the expressionof r-GCS mRNA were respectively higher than group A of others and group B ofothers，the difference was significant（P<0.05）.（7）Western blot results showed that the expression of Nrf2protein in the group Bwas increased than group A（P<0.05），especially in the group A2and group B2，thedifference of the expression of nrf2protein were respectively higher than group A ofothers and group B of others have statistically significan（tP<0.05）；The expression ofBach1protein were no statistical difference between group A and group B(P>0.05)；The expression of r-GCS protein in the group B was markly increased than group A（P<0.01），especially in the groupA2and group B2，the difference of the expressionof r-GCS protein were respectively higher than group A of others and group B ofothers，the difference was significant（P<0.05）.Conclusions:（1）Derp1allergen had direct effect on the oxidative Stress of the rat bronchialepithelial cells，it could result in the dynamic expression of Antioxidant genes fromthe rat bronchia epithelial cells via sensitizing them. Bronchia epithelial cells mayplay the most important effects on the allergens sensitization and may take part in thepathogenesis of asthma.（2）Transcription factors KLF2、Nrf2and Bach1may play important roles inoxidative stress of the rat bronchial epithelial cells via regulatingγ-GCS，then thiseffect was an important protection measure in order to maintain homeostatic functionsof cells. There were positive correlation between the expression of KLF2、Nrf2andthe expression ofγ-GCS in the cells，but a negtive correlation between the expressionof Bach1and the expression ofγ-GCS in the cells. （3）Bronchial epithelial cells with the over-expressed KLF2gene revealed thatKLF2may enhance antioxidant activity of Nrf2by increasing its nuclear localizationand expression，which would further affect the expression of the downstream targetgene such as γ-GCS was increased. We also found that the expression of KLF2andBach1had no direct correlation in this research.（4）The dexamethasone would have protective effect to the bronchial epithelialcells by influencing the expression of KLF2and Nrf2which may be key inanti-oxidation mechanism of asthma，then this effect would reduce the risk ofoxidative damage attributed to allergen to cells，and supplied a new theoretical basisto treat asthma.