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Study of the Roles and Mechanisms of Vitamin D3 Up-regulated Protein 1(VDUP1) in Oxidative Stress in Asthma

Author: ZhaoHaiJin
Tutor: CaiShaoZuo
School: Southern Medical University,
Course: Pulmonology
Keywords: Asthma Oxidative Stress Vitamin D3 increases protein 1 (VDUP1) Thioredoxin (TRX) Normal human bronchial epithelial cells (NHBE)
CLC: R562.25
Type: PhD thesis
Year: 2008
Downloads: 303
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Abstract


Background and Significance: bronchial asthma (the asthma) is a serious impact on human health of chronic respiratory diseases to nonspecific airway inflammation, airway hyperresponsiveness and airway reconstruction as the main feature. However, chronic asthma, variability, paroxysmal features show the complexity of disease mechanisms and refractory. Asthma occurrence and development of gene regulation by many to determine various clinical phenotypes of asthma. Recent evidence supports the genetic and environmental factors coordinating role in the development of asthma plays a vital role. Oxidative stress is closely related with the development of asthma, involving natural history of asthma, airway inflammation, airway hyperresponsiveness, airway increased vascular permeability, tissue damage and airway reconstruction. Nevertheless, the role of oxidative stress in asthma, and the mechanism of slow progress. With N-acetylcysteine ??(NAC), represented antioxidant therapy is widely used in treatment of lung disease, but their clinical practice is still relatively limited. This may be associated with oxidative stress molecules such as reactive oxygen species (ROS) has variability, and can activate a variety of intracellular signal transduction pathways and ROS induced current on the exact physiological and pathological effects is not clear about. Therefore, oxidative stress mechanisms of asthma depth research to find disease-specific oxidative stress factors, prevention and treatment of asthma may provide a new theoretical basis and the role of targets. Airway epithelial cells (AEC) has a complex barrier function, as a defensive inhalation of harmful substances and pathogenic organisms first line of defense. Over the past decade, \AEC with a variety of environmental factors (including the environment) interact through AEC and epithelial - mesenchymal nutrition unit (EMTU) at start-up and maintenance of airway inflammation and airway remodeling play a major regulatory role. Now that, AEC structural integrity defects or functional disorder is asthma start link. AEC often in people with asthma exogenous and endogenous oxidative stress environment, anti-oxidation occurs when the AEC functional defects or excessive oxidative stress, this balance is broken, causing the innate immune system and the acquired immune system activation. Studies have shown that asthma patients are more susceptible to oxidative stress AEC damage occurs, it is easier to apoptosis, increased permeability AEC. This fully Tip AEC oxidative imbalance in antioxidant important role in the pathogenesis of asthma. The study focused primarily targets: Vitamin D3 increases protein 1 (VDUP1) is 2005 and confirmed the initial screening laboratory asthma eosinophils (EOS) survived, release of inflammatory mediators and changes in lung function was significantly related genes. Previous studies support: VDUP1 as an early response gene, with a variety of biological functions involved in oxidative stress, stress signal transduction, cell growth and differentiation, lipid metabolism and mucosal immunity process, confirmed with diabetes, cancer, cardiovascular and other complex diseases are closely related. VDUP1 major redox regulatory protein thioredoxin (TRX) key inhibitory factor, the TRX, and glutathione (GSH) systems are important antioxidant parallel control system. Therefore, this study was based on preliminary studies to further explore VDUP1 oxidative stress in asthma and the possible role of oxidative stress in AEC molecular mechanism of regulation for the future against oxidative stress in asthma treatment and intervention to provide new theoretical basis. Method: 1. Establish a mouse model of asthma in asthmatic lung tissue VDUP1 explore the expression and distribution of antioxidant effect of NAC on VDUP1 intervention. SPF level female BALB / c mice were randomly divided into control group, asthma group and asthma NAC treatment groups of five. Ovalbumin (OVA)-induced asthma model established by reverse transcription - polymerase chain reaction (RT-PCR) assay VDUP1 mRNA expression levels in lung tissue by immunohistochemistry assay VDUP1 in the lung tissue distribution and protein expression. (2) establish hydrogen peroxide (H 2 O 2 ) induced normal human bronchial epithelial cells (NHBE) nonfatal and fatal oxidative stress model to study VDUP1, TRX Expression and localization in HBE. Tetrazolium salt (MTT) colorimetric detection of different H 2 O 2 concentration on NHBE strains HBE135-E6E7 (HBE) Vigor; choose 50μM, 200μM and 600μM treatment HBE 24h, RT-PCR detection VDUP1/TRX and β-actin gene expression level, immunofluorescence VDUP1/TRX protein expression levels and intracellular localization. Hoechests33342 detect cell apoptosis. Analysis of apoptosis and VDUP1, TRX expression correlation. Different concentrations H 2 O 2 right VDUP1, TRX expression. Compare VDUP1 and TRX in HBE to oxidative stress reactivity, and initially clarify their synergy. 3 Construct VDUP1 RNA interference (siRNA VDUP1) plasmid was observed after siRNA VDUP1 on cell growth, apoptosis and oxidative stress tolerance in rats. siRNA VDUP1 plasmid construction, transfection, the transfection efficiency by immunofluorescence identified by RT-PCR and Western blot were used to detect the interference efficiency. MTT assay after siRNA VDUP1 HBE growth, using different H 2 O 2 concentrations (50μM, 200μM, 400μM, 600μM and 10000μM) stimulation disturbance HBE, Hoechest detect different concentrations H 2 O 2 concentration on cell apoptosis. 4 Research VDUP1 in H 2 O 2 HBE VEGF expression induced effects and possible mechanisms. First with different concentrations of H 2 O 2 (50μM, 200μM, 600μM) in serum-free culture conditions treated HBE 24 hours. RT-PCR assay HBE VEGF mRNA expression, double antibody sandwich ELISA assay H 2 O 2 -induced HBE VEGF expression and PI 3 K antagonist agent Ly294002 (10μM) on VEGF expression. After confirming H 2 O 2 can PI 3 K-dependent expression of VEGF to promote on the basis of immunofluorescence Ly294002 (10μM) for 50μM H < sub> 2 O 2 -induced HBE VDUP1/TRX and VDUP1/Jab1 expression and localization effects. ELISA assay normal group, empty plasmid group and siRNA VDUP1 group H 2 O 2 -induced HBE VEGF expression, and explore VDUP1 in H 2 O 2 HBE VEGF expression induced effects and possible mechanisms. Results: 1.VDUP1 in asthmatic lung tissue was significantly increased, while NAC further increase VDUP1 expression. VDUP1 mainly located in the AEC and a small amount of mesenchymal cells. Successfully established mouse model of asthma, lung tissue VDUP1 mRNA relative expression in the control group, asthma group and NAC treatment group were 0.633 ± 0.055,0.922 ± 0.052,1.07 ± 0.076; relative to the control group, asthma group and NAC treatment groups VDUP1 expression was significantly increased (n = 5, P <0.001); NAC group relative to the asthma group, the expression also significantly increased (n = 5, P <0.001). Immunohistochemical staining VDUP1 mainly in bronchial and alveolar epithelial cells, can also be found in the nucleus and membrane. Normal group, asthma group and NAC treated gray values ??were: 66.74 ± 2.98,77.94 ± 3.23,98.38 ± 7.31. Relative to the normal group, asthma group VDUP1 expression was significantly enhanced (n = 5, P = 0.004), compared to the normal group and asthma group was significantly increased NAC group (n = 5, P <0.001). 2.H 2 O 2 can increase HBE VDUP1/TRX expressed in lower concentrations can promote VDUP1 expression, while promoting both translocation to the nucleus and cell membrane. VDUP1 and TRX fluorescence intensity of expression was positively correlated with apoptosis. Establish H 2 O 2 -induced fatal and non-fatal HBE research model induced by oxidative stress. In 200μM less H 2 O 2 for 24h on no significant effect on cell viability. Normal, 50μM H 2 O 2 , 200μM H 2 O 2 , 600μMH 2 O 2 , respectively, for 24h, VDUP1 mRNA relative expression in the four groups were 0.355 ± 0.031,0.453 ± 0.047,0.414 ± 0.047,0.507 ± 0.067; TRX mRNA expression in the four groups were : 0.366 ± 0.076,0.386 ± 0.045,0.446 ± 0.083,0.677 ± 0.025. Relative to the control group, VDUP1 at 50μM H 2 O 2 (n = 6, P = 0.008), 200μM H 2 O 2 (n = 6, P = 0.012), 600μM H 2 O 2 (n = 6, P <0.001) each treatment group were significantly increased, and with increasing concentration. TRX expression only at 600μM group was significantly increased (n = 6, P = 0.04); immunofluorescence VDUP1/TRX distributed mainly in the cytoplasm, VDUP1 colocalization with TRX at 50μM H 2 O 2 Handling, both fluorescence intensity increases, VDUP1, TRX apparent translocation to the plasma membrane, some cells translocation to the nucleus. When cells in apoptosis were significantly increased both the fluorescence intensity. 3.siRNA VDUP1 after transfection on the growth of normal HBE no significant effect, but in the role of oxidative stress can reduce the higher concentration H 2 O 2 HBE caused the withered mortality. siRNA VDUP1 HBE transfection efficiency: about 80%. siRNA VDUP1mRNA interfering efficiency and protein levels were approximately 60% and 50%. Normal group, empty plasmid negative control group, liposome group and siRNAVDUP1 group had no significant effect on cell growth. Hoechest 33342 detect siRNA VDUP1 24h after HBE no significant effect on apoptosis. 50μM, 200μM concentration on the siRNA VDUP1 after two HBE no significant effect on apoptosis. 400μM, 600μM and 1000μM H 2 O 2 treatment groups before and after interference HBE apoptotic percentage was: (22.333 ± 2.081,14.333 ± 2.082, n = 3, P = 0.009 ), (36.000 ± 1.826,25.000 ± 3.000, n = 3, P = 0.002), (44.333 ± 4.041,35.000 ± 3.000, n = 3, P = 0.033). VDUP1 siRNA interference group can significantly reduce HBE apoptosis. 4.VDUP1 involved in H 2 O 2 HBE VEGF expression induced effects and possible mechanisms. ① H 2 O 2 can significantly increase the HBE VEGF expression, Ly294002 (10μM) can completely antagonize this effect. HBE135-E6E7 constitutive expression of VEGF 189 and VEGF 165 two subtypes. VEGF 165 / β-actinmRNA in the normal group, 50μM, 200μM and 600μM H 2 O 2 relative expression was 0.379 ± 0.044,0.791 ± 0.042,0.585 ± 0.133,0.720 ± 0.0213. VEGF 189 / β-actin mRNA relative expression in the four groups were: 0.193 ± 0.018,0.270 ± 0.012,0.205 ± 0.074,0.302 ± 0.035. Relative to the control group, VEGF 165 , VEGF 189 in 50μM H 2 O 2 treatments can significantly l high VEGF expression (n = 6, P <0.001), but not concentration-dependent increases. ELISA results showed: normal group, 50μM H 2 O 2 Treatment and Ly294002 (10μM) after pretreatment plus 50μM H 2 O 2 treatment group, the supernatant VEGF concentrations were: 591.5 ± 9.5 pg / ml, 768.9 ± 21.3 pg / ml, 489.3 ± 10.9 pg / ml. H 2 O 2 treatment group was significantly increased VEGF expression (n = 4, P <0.001), and Ly294002 significantly reduced H 2 O 2 -induced VEGF expression (n = 4, P <0.001), even lower than the normal level (n = 4, P <0.001) ② Ly294002 (10μM) for VDUP1/TRX/Jab1 was no significant impact, immunofluorescence shown to reduce VDUP1 to the cell membrane and nuclear translocation of Jab1, TRX expression and localization of no significant impact. Relative to H 2 O 2 treatment group, Ly294002 pretreatment group VDUP1, TRX was no significant difference in the expression, but Ly294002 treatments VDUP1 gathered to the cell membrane and the nucleus decreased, but TRX membrane localization for no obvious effect. In normal HBE Jab1 dispersed in the cytoplasm, no co-localization with VDUP1. In H 2 O 2 for 24h, Jab1 fluorescence intensity slightly increased, Jab1 significantly to the nucleus or nuclear membrane aggregation seen in the nucleus and VDUP1 obvious co-localization of the situation. Ly294002 treatment, right VDUP1, Jab1 expression of fluorescence intensity did not change significantly, there are still two co-localization. ③ siRNA VDUP1 can slightly increase the H 2 O 2 -induced HBE VEGF expression, but there is no statistical significance. Normal control group, 50μM H 2 O 2 treatment group, empty plasmid transfection 50μM H 2 O 2 After the treatment group and siRNAVDUP1 50μM H 2 O 2 treated supernatant VEGF concentrations were: 587.1 ± 12.1 pg / ml, 740.1 ± 12.8 pg / ml, 748.9 ± 22.5 pg / ml, 759.8 ± 8.4 pg / ml. H 2 O 2 treatment group compared to the normal group were significantly different (n = 4, P <0.001). siRNAVDUP1 after H 2 O 2 treatment group concentration slightly increased, but there was no statistically significant difference (n = 4, P = 0.085) Conclusion: 1.VDUP1 oxidation with asthma stress-related, may be used as one of the signs antioxidant. 2.VDUP1 may regulate oxidative stress and tolerance HBE important factor in HBE oxidative stress signaling pathway plays an important regulatory role, VDUP1 and TRX possible synergies involved in oxidative stress. 3.H 2 O 2 to PI 3 K-dependent promote VEGF expression. PI 3 K and pathways involved in VDUP1 translocation process, oxidative stress HBE model VDUP1, TRX and Jab1 synergies that may exist, but VDUP1 is involved in H 2 O 2 -induced VEGF expression and precise control mechanism needs to be further discussed.

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CLC: > Medicine, health > Internal Medicine > Respiratory system and chest diseases > Trachea and bronchial disease > Bronchial disease > Bronchial asthma
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