Control Of ANGⅡ-P22PHOX-ROS Signaling Pathway In Lung Injury In Rats And Correlation With Active Repair Gene HOGG₁

Part one Control of Angâ…¡-p22phox-ROS signaling pathway on acute lung injury in rat induced by hyperoxiaBackgroundAs the direct result of receiving oxygen therapy and mechanical ventilation, more and more newborn especially the premature children are suffering from bronch-opulmonary dysplasia (BPD) and chronic lung disease (CLD). That caused serious impact on children's lung function and lung development. Replacement of mechanical ventilation mode, using of hormones, surfactant replacement therapy and immuno- therapy had little effect. In recent years, AngII-p22phox-ROS signaling pathway involved in vascular remodeling and organ fibrosis caused for more attention. In this study, choosing SD rats at different developmental stages as the research object, animal model of lung injury were established to exploring the changes of AngII- p22phox-ROS signaling pathway in pulmonary under the high-oxygen conditions, as well as the relationship between lung injury and PPARγfactor and active repair gene hOGG1 in the repair of lung injury.ObjectUsing the similar characteristics between rat and alveolar in premature children, animal model of lung injury were established successfully. Trying to explore the following questions by morphology and molecular biology methods:â‘ whether space-time dependencies between levels of index of Ang II- p22 phox-ROS signaling pathway and degree of lung injury exist or not.â‘¡Whether lung injury could be reduced by activating intracellular PPARγthrough antagonizing Ang II.â‘¢whether the level of gene expression of hOGG1 and repair capacity of damaged cells is closely related initiative.Method90 newborn rats (full-term rats born for 3 days) were divided randomly into the neonatal air group(A group), neonatal high-oxygen group (B group), the neonatal intervention group(C group), adult high-oxygen group (D group) included 30 adult rats (age 90 days). B,C,D groups (neonatal groups with maternal) were placed in the high concentration of oxygen of 90%±2% continuously to establish the high-oxygen animal models. The air group inhaled the air, and the other experimental factors were same with the model groups. Chymotrypsin inhibitor, NK3201 was injected by intravenous continuously for 7d into the intervention group after exposing to high oxygen; 5mg/kg/d. Six rats were selected randomly in each group at 1,3,7,14,21d after beginning of experiment. Executing the rats, the left lungs were fixed respectively in 4% parafor- maldehyde and 2.5% Glutaraldehyde after full irrigation. Part sliced and stained with hematoxylin-Iraq Red for morphology changes, and part for TEM. The right lung tissue was cryopreservated in - 80℃for next tests.The indicators responding to the effect of hyperoxia and survival of rats were recorded daily. Changes of general morphology and ultra-structure were observed by HE and electron microscopy. AngII (angiotensin II) content in lung tissue was measured by radioimmunoassay; AT1R (angiotensin type 1 receptor) and p22phox mRNA expressions of lung tissue were measured by RT-PCR; Method of determination of ROS levels in lung tissue was DCFH-DA; Apoptosis in lung tissue was estimated by TUNEL and SABC; hOGG1, PPARγprotein levels were tested by Western Blot.ResultA series of changes were observed in high-oxygen group including from the activity decreased to dependency on high oxygen concentration. As time going on, mortality rate increased. Tachypnea and decreased activity were the only performances in adult group, and no anomalies in air group. Light and electron microscopy showed morphological changes following the order: pulmonary congestion, neutrophil infiltration, alveolar interstitial edema and alveolar type II epithelial cells (AT-II) apoptosis, fibrosis tendency at t last.High oxygen can cause AngII, AT1R, p22phox mRNA increased. AngII and p22phox mRNA gradually decreased after hitting the peak at 14d, but AT1R remained slightly increased. Chymotrypsin inhibitors significantly reduced their expression (P <0.05).Compared to adult group at the corresponding time points, AT1R and p22phox mRNA levels of high-oxygen group were higher, while AngII levels showing the contrary trend. It speculated that p22phox mRNA in the lungs may be controlled by AngII and AT1R at the same time.Univariate correlation analysis showed that the level of ROS positively correlated with AgII, p22phox and AT1R (r = 0.775, P <0.01; r = 0.838, P <0.01; r = 0.712, P <0.05). TUNEL and caspase-3 showed the same trend with the increasing of ROS. All of three indicators of high oxygen were higher than those of adult group, and significantly different can be observed (P <0.05).HOGG1 and PPARγprotein in air group maintained a relatively stable level, while rapid increase can be caused by high-oxygen condition in high oxygen group, with the characters of early peak time,short persistent,low level and rapid decreasing. Compared with high oxygen rats, hOGG1 and PPARγprotein expression in adult group were significantly increased.Intervention of Chymotrypsin inhibitor could not completely prevent lung injury, but compared with high oxygen group, pulmonary ROS levels, apoptotic index, caspase-3 levels decreased significantly (P <0.05). IOD of hOGG1 and PPARγwere 549.47±1.31 and 604.49±1.77, increased respectively by 23.01% and 28.90% (P <0.05).ConclusionsGeneral performance combined mortality and lung morphology indicators that animal model of lung injury established successfully. Continuous exposure to high oxygen levels can be significantly increased AngII level in lung of rats and cause AngII-p22phox-ROS signaling pathway corresponding changes. Degree and time sequence were consistent with the morphological changes. It suggested that RAS involved in lung injury by upgrading TUNEL, caspase-3 and promoting the development of pulmonary fibrosis.AngII and AT1R levels were significantly higher in high oxygen group than that in adult group probably due to the worse injury of lungs.The limited ability of upgrading hOGG1 and PPARγlevels may be another reason for hyperoxia-induced lung injury and the worse changes in lung in high oxygen group.Chymotrypsin inhibitor can inhibit AngII production, reduce ROS production, and enhance PPAR-γand hOGG1 expression, enhanced repair capacity of the lung function of active cells, so that lung stem cells may play a role in active rehabilitation. Part two Construction and identification for biological characteristics of A549 cells with over expression of hOGG1 geneBackgroundAlveolar epithelial injury could be repaired only by the AT-â…¡cells, so apoptosis of AT-â…¡caused by DNA damage because of imbalance between oxidation and antioxidant status leads to pulmonary fibrosis. Therefore, only increasing ability to resist oxidative damage, enhancing the capacity of active repair of damaged DNA, AT-â…¡can play a lung stem cells to repair the injury. For this purpose, this study attempts to establish new cell line with high expression of active repair gene, and further applied to lung injury experiment.ObjectThrough preparation of eukaryotic plasmids of pcDNA3.1(+)/Myc His A by plasmid extraction and purification, and amplification of full hOGG1 gene by RT-PCR, the eukaryotic expression vector pcDNA3.1(+) / MycHis A-hOGG1 was successfully constructed. Combined with luciferase reporter plasmid PGL3 promoter, pcDNA3.1(+)/MycHis A-hOGG1 was stably cotransfected into A549 cells to get the cell line with high expression of hOGG1. The biological characteristics of new cell line were identified for further study. MethodFull-length cDNA sequence of hOGG1 gene was amplified referring to Gen Bank (NM₀16821.2). Plasmid was extracted from Ecoli DH5a cells after conventional preparation, transformation and amplified. Solution concentration of extracted plasmid was determined by spectrophotometer, and the integrity of the plasmid was determined by UVI gel electrophoresis imaging system.HOGG1 gene and pcDNA3.1(+)/Myc-His A were digested with restriction endonuclease EcoRI and XbaI reaction system, and determined with the application of 0.8% agarose gel electrophoresis products. Appropriate pcDN3.1(+)/Myc-His A and hOGG1 gene were taken to link with adding T4 DNA ligase into. Competent bacteria freshly prepared were conversed by the linking products, and then planted in LB flat containing AMP (final concentration 10μl/ml) to screen the positive colonies. Observation of morphological changes of recombinant finished with human eyes. Four positive colonies were picked randomly, and plasmid was extracted using mini prep two-enzyme kit. Digestion products were determined by electrophoresis 2.5% agarose gel electrophoresis, and PCR amplification DNA sequence analysis on the machine.After that, vector pcDN3.1(+)/Myc-His A-hOGG1 and pGL3 promoter were allocated according to 9:1 ratio with adding polyfection and serum free DMEM, and cultured continuously under 37℃, 95% humidity, 5% CO2 to transfected A549 cell. Stable transfected clones were randomly selected respectively at 1,2,3,4,5d to detect luciferase activity (RLU) with the Bright-Glo TM Luciferase Assay System, while hOGG1 protein expression was tested with Western Blot to identify the cell lines with high expression of hOGG1 gene.A549, A549-P and stably transfected A549-T cells were randomly selected to test the biological characteristics, including: cells growth image observed with Olympus camera system, cell growth rate observed for 7 day with MTT then drawing the growth curve, cell cycle detected with Flow cytometry.Result1. Full coding sequence of hOGG1 about 1200 bp was successfully amplified. Electrophoresis results showed good agreement with the expected size.2. The length of digested amplified product and vector determined with gel electrophoresis (hOGG1 was 1200bp, pcDNA3.1(+)/Myc-His A to 5346bp) were consistent with the estimated size. Sequenced report showed that segment sequence: CMVF (TH1 -5) CMVR (TH2-5) and TH1-3209, hOGG1 sequence for splicing.3. Transfected clones were selected under fluorescence microscope (PGL3 without antibiotics antagonist gene expression, so fluorescent indicated successful cotransfection). With the G418, stably transfected cell clones to be selected to culture continuously.Then choosing cell clones randomly to test hOGG1 gene expression: transfected cells luciferase activity to 0.444±1.88 (RLU×105), negative control luciferase activity was only 0.021±3.37 (RLU×105). HOGG1 protein expression was detected with WB. The transfected group level was 485.37±0.21 (IOD), while the control group and negative control group were 147.35±2.04 (IOD) and 159.41±4.37 (IOD). Statistically significant differences between the two groups (P <0.05 & P <0.01) was observed that suggest transfected cells increased the expression of hOGG1 gene successfully, and A549 cell line with high expression of hOGG1 gene was successfully established.4. No morphology difference and cell cycle difference were found between the three groups of cells, suggesting that the process of gene transfer did not affect their biological characteristicsConclusions1. Eukaryotic plasmid of pcDNA3.1(+)/Myc-HisA-hOGG1 was constructed successfully With different restriction enzyme sites to ensure that the fragment was inserted one way.2. PGL3 promoter carrying luciferase reporter gene is an internationally popular reporter gene. Cotransfection with pcDNA3.1(+)/Myc-His A has the advantages of high sensitivity, strong correlation, easily screening and good visibility. 3. Fluorescence intensity indicating hOGG1 mRNA expression levels of transfected cells was significantly higher than that of (the blank cell group and the negative control group (P<0.05), and that had nothing to do with the plasmid pcDNA3.1(+)/Myc-His A (the blank cell group and the negative control group, P> 0.05) itself. It suggested high expression of hOGG1 gene in A549 cells was successfully constructed.4. By growth rate, morphology detection, cell cycle and other identification means, no significant changes in biological characteristics were found between three group cells. Successful construction of A549 cell line with high expression of hOGG1 gene provides a very effective biological tool for further in-depth study about oxidative DNA damage and repair.Part three Effect of A549 cells with hOGG1 gene overexpression on oxygen-induced DNA damage and repairBackgroundThe first part of the study, we observed the lung injury in rats with rapid emergence of ROS. In the initial phase, expression level of the active DNA repair gene hOGG1 increased. But with the high oxygen stimulus time going no, hOGG1 expression rapidly decreased when a variety of substances increasing that evoking apoptosis. This phenomenon indicates that the limited ability of upgrading active DNA repair gene may be one of reasons due to high oxygen lung injury. The last stage of the experiment we successfully established A549 cell line with overexpression of hOGG1. And in this portion, capability of transfected A549 cells against oxidative damage would be further studyMethodThree experimental groups were designed: the blank control group (A549), and transfected group (PGL3 + pcDNA3.1(+)/Myc-HisA-hOGG1 A549-T). Each group further divided into time groups and concentration group.Time group cells exposed to environment with 90% oxygen concentration were collected specimens respectively at 0h (blank control), 12h, 24h, 48h. Concentration group cells were placed in incubator with 50%, 60%, 80%, 90% oxygen concentration, cultured for 24h.A portion of cells exposed to 90% of oxygen concentration cultured for 24h and 48h, set into containing bovine serum to culture after removing the stimulation, maintaining medium 37℃for 180 min., the cells respectively collected at the time of 0,60,120min to assess DNA damage repair capacity.Subjects were observed including general morphology changes and ultra structure changes, apoptosis with Annexin V/PI apoptosis assay and cell cycle with FL2-H. Apoptosis-related indicators, PPAR-γ, Bcl-2 and Caspase3 were detected with Western Blot. Capacity against high oxygen-induced DNA damage and active repair on three groups of cells were evaluated by Modified comet assay.Conclusions1. Morphological changes: the high oxygen exposure can result in general morphology and ultra structure changes in three group cells and degree gradually increase with the level of oxygen concentration and exposure time. Changes in transfected group were slighter significantly compared to the blank group.2. Apoptosis and cell cycle comparison: High oxygen can induce cell apoptosis, the proportion of apoptotic cells was positively correlated with oxygen concentration and exposure time. Apoptosis ratio of A549-T cells decreased compared to the blank group (P <0.05, part of the index P <0.01). Cell cycle arrest could be observed in all of groups, showing increased G0G1 phase cell ratio and decreased S phase cells ratio. The cell block phenomenon showed that there was limited cellular DNA replication and blocked cell division. Compared with blank control group, there were less G0G1 phase cells and more S phase cells in A549-T (P<0.05).3. Apoptosis index: Expression of PPARγ, Caspase3 and apoptosis-related Bcl-2 protein showed the different trends. The expression of PPARγprotein gradually decreased while Bcl-2 protein expression increased with the exposure time and oxygen concentration. Caspase3 expression first increased and then decreased, and the peak appeared respectively at 24h and 90% oxygen concentration.Indicators above-mentioned showed he same trends in A549-T cells, but statistically significant differences can be observed compared with control group. Among Which, PPARγprotein levels were significantly higher while the Caspase3 and the Bcl-2 protein levels significantly lower than that in control group (P<0.05 & P <0.01).4. DNA damage and repairHigh oxygen exposure resulted in DNA damage in varying degrees. Both of trailing tail rate and Olive Tail Moment(TM) were increased with high oxygen exposure time increasing. Tail moment of the blank control group at 24h came up to 19.3±0.95, significantly increased compared to 8.90±1.88 in A549-T group (P <0.05). Comet head became from round strong fluorescence intensity to point-like weaker fluorescence intensity with the incremental large tails. In contrast, A549-T cells slightly changed significantly.Statistically significant beginning time of repair was 60 min in A549-T cells, while that was delayed to 120 min in control groups. Furthermore, hyperoxia-induced DNA damage in A549-T cells could be fully restored at different times, while 11% of damaged cells in the control groups remained not to be repaired completely (P<0.05). When the exposure time extended to 48h, cells rate failed to be complete repaired was 40% and beginning time of repair was delayed to 180 min.Conclusions1. Except inducing direct apoptosis, high oxygen could affect the alveolar epithelium to repair by blocking the cell cycle.2. PPARγ, Caspase3 and Bcl-2 expression is closely related with apoptosis levels. But affection mechanism and regularity were very differen.3. Capability against hyperoxia in A549-T cells was markedly increased. A549-T cells could repair the high oxygen-induced DNA damage much better stronger, faster and more complete than A549 cells.4. In this experiment, hOGG1 gene that over expressed in transfected cells could enhance the anti-damage repair capacity to prevent apoptosis and improve the pathological changes.