| Asthma is a chronic respiratory disease characterized by chronic airway inflammation, airway hyperresponsiveness, airway remolding, which brings great harm to human life and health. Researches show that there is about 300 million people with asthma worldwide, and there is about 27 million asthma patients in China, which is increasing.Presently, most scholars believe that the environmental causes of asthma should be divided into two categories that pathogenic factor TRIGGER and predisposing factor CONTRIBUTOR, pathogenic factor refers to factors that caused the first attack of asthma, called "Trigger" and it is the main cause of both the occurrence and development of asthma; predisposing factor is that hidden asthma remobilization or acute asthma inducing from patients suffering from asthma which is one of the integrated predisposing factors playing an important role in the promotion and further development of asthma recurrence. In the above two categories of factors, some factors such as allergens, irritant gases and harmful gases, occupational factors, viruses, food and drugs, not only can lead to the occurrence of asthma, but also making significantly influence in asthma development. However, we should be clear that all the environmental factors of asthma is not the only factor of determining asthma illness, the body quality of asthma atopic patients is also very important. Individual patient allergies and the:external environment is the double impact of asthma risk factors.At present, there are many environmental factors involved in the occurrence and development of asthma. And it is found that toluene diisocyanate (TDI) attracts extensive attention. TDI is a highly reactive industrial chemical characterized by the presence of highly reactive N=C=O groups, which daily used in home and architectural decoration and with widespread use in the manufacture of polyurethane foams, Polyurethane elastomers, palnt and plastics. It is currently the most common cause of occupational asthma. Due to the wide distribution of TDI, many workers are regularly exposed to TDI,5-15% of those develop asthma, while the global incidence rate of asthma in the general population is 0.1-8%, China 0.11-2.03%. The clinical manifestations and pathological changes of TDI-induced asthma is basically the same as allergic asthma. But the pathogenesis responsible for the disease is still ambiguous. The current studies demonstrate that there are two major aspects involved in TDI-induced asthma. Firstly, the human bronchial epithelial cells(HBE)as target cells stimulated by TDI may result in abnormal cell structure and effect the release of biological active cytokines, which plays an important role in TDI-induced asthma; Secondly, like other low-molecular-weight chemicals, TDI is not antigenic per se (it is a hapten), but it is spontaneously reactive with a variety of proteins, such as albumin, keratin, and ciliated cell tubulin, and forms immune conjugates like TDI-human serum albumin (TD1-HSA). As a antigenic substance, TDI-HSA penetrates into the submucosal tissue before presented by antigen presenting cells, which will produce active materials from these activities. Together with the cytokines produced by HBE, these active materials activates lymphocytes (including Thl and Th2), neutrophils and other inflammatory cells that leads to a series of inflammatory reactions, so the study of the trigger and maintenance mechanism is the key step of diagnosing and treating TDI-induced asthma.Oxidative stress has been involved in nature history of asthma, airway inflammation, airway hyper-responsiveness, enhanced airway microvascular permeability and tissue injury and remodeling. Nevertheless, study on the mechanism of oxidative stress in asthma is making slowly progress. In addition, a long-recognized property of HBE is function as a complex physical barrier that defends against exposures to potentially harmful inhaled substances and microbial pathogens. It is now clearly that HBE also play crucial roles in initiating and augmenging airway host defense mechanisms. HBE in people with asthma are often in contact with exogenous and endogenous oxidative stress conditions. Antioxidant deficiency in HBE will result in the activation of innate immunity and acquired immunity, which fully indicated that the redox state in HBE play an initiative and critical role in asthma.Studies prove that intracellular glutathione (GSH) of HBE is reduced following exposure to TDI, that significantly changes the redox balance of these cells. TDI exposure induces laryngo-tracheal eosinophilia, which can be ameliorated by supplementation with antioxidant vitamins in guinea pigs. All of these suggest that oxidative stress is closely related to the development of TDI-induced asthma.Our previous studies have proved that TDI-HSA significantly increases the permeability coefficients of 16HBE monolayers. Although there are few researches on the relationship between oxidative stress and airway epithelial permeability, evidence shows that oxidative stress can enhance intestinal epithelial permeability. Therefore, we speculate that TDI may also enhance airway epithelial permeability through a oxidative stress-mediated pathway, which penetrate into the submucosal tissue in regulating the pathological process. In view of TDI and oxidative stress play an important role in the pathogenesis of asthma, and on the basis of preliminary studies, we take HBE as the main research cell and reactive oxygen species (ROS) as the main detection factor, and want explore the roles of oxidative stress in epithelial permeability, which may provide a new experimental evidence of pathogenesis and therapeutic targets for TDI-induced asthma.The first section of this research:Objective:To investigate the effect of toluene diisocyanate (TDI) on reactive oxygen species (ROS) production of human bronchial epithelial cells (HBE) in vitro.Methods:1. The TDI-human serum albumin (TDI-HSA) conjugate was prepared by a modified Son's method.2. Methyltetrazolium (MTT) assay was used to assess the 16HBE cell viability under different concentrations of TDI-HSA, groups as follows:The normal control group, TDI-HSA 10ug/ml group, TDI-HSA 20ug/ml group, TDI-HSA 40ug/ml group, TDI-HSA 60ug/ml group, TDI-HSA 80ug/ml group, TDI-HSA 100ug/ml group, TDI-HSA 120ug/ml group, TDI-HSA 150ug/ml group (n=7), stimulated 16HBE for 48 hours, then MTT colorimetric assay detected cell viability.3. Flow cytometry was applied to detect the level of intracellular ROS of 16HBE under the different concentrations of TDI-HSA, groups as follows:The normal control group, TDI-HSA 20ug/ml group, TDI-HSA 60ug/ml group, TDI-HSA 100ug/ml group (n=3). After stimulated for 24h, the cells digested by trypsin was collected, and the level of intracellular ROS of 16HBE cells was detected by flow cytometry with an oxidation-sensitive fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) uploading.4. Methyltetrazolium (MTT) assay was used to assess the 16HBE cell viability under different concentrations of N-acetylcysteine (NAC), groups as follows:The normal control group, NAC 10 mmol/L group, NAC 20 mmol/L group, NAC 50 mmol/L group, NAC 80 mmol/L group (n=6), stimulated 16HBE for 12 hours, then MTT colorimetric assay detected cell viability.5. Selected concentration of TDI-HSA 100 ug/ml, which increased the most of ROS, pretreatment of the cells with antioxidant NAC (50 mmol/L) for 40min. After stimulated for 24h, the cells digested by trypsin was collected, and the level of intracellular ROS of 16HBE cells was detected by flow cytometry with DCFH-DA uploading and observed by fluorescence microscope.6. Statistical methods:SPSS13.0 analysis statistical software was used for date analysis. Data was expressed as mean±SD, One-way analysis of variance (one-way ANOVA) was used to compare the overall mean when the variance was Homogeneity, and LSD method was used for Multiple comparisons among the groups; when the variance was not Homogeneity, Welch method was used to compare the overall mean, Dunnett's T3 was used for Multiple comparisons among the groups. Significance was accepted when P<0.05.Results:1. The effect of different concentrations of TDI-HSA on cell viability of normal human bronchial epithelial cell 16HBE:TDI-HSA with the concentrations of 10 ug/ml,20 ug/ml,40 ug/ml,60 ug/ml,80 ug/ml,100 ug/ml,120 ug/ml for 16HBE did not significantly affect cell viability, while the concentration of 150 ug/ml significantly reduced cell viability (P<0.05).2. The effect of different concentrations of N-acetylcysteine (NAC) on cell viability of normal human bronchial epithelial cell 16HBE:NAC with the concentrations of 10 mmol/L,20 mmol/L,50 mmol/L for 16HBE did not significantly affect cell viability, while the concentration of 80 mmol/L significantly reduced cell viability (P<0.001).3. The effect of different concentrations of TDI-HSA and NAC on intracellular ROS production of 16HBE, results:20 ug/ml group,60 ug/ml group,100 ug/ml group increased intracellular fluorescent intensity compared with the control group, with statistical significance (P<0.05), and the effect showed dose dependent. NAC+100 ug/ml group decreased intracellular ROS production compared with 100 ug/ml treatment group, with statistical significance (P<0.05), and compared to the control group, increased intracellular ROS production, with statistical significance (P<0.05). The green fluorescence in the control cells appear to be very weak, but significantly increased after 24 h treatment of 100 ug/ml TDI-HSA. The green fluorescence filled and stained in cytoplasm while significantly reduced by NAC.Conclusion: TDI-HSA increased the production of ROS in 16HBE under certainconcentrations, which did not affect the cell viability, and the effect showed dosedependent. The 100 ug/ml group increased the most, and can be inhibited byantioxidant NAC. The significant production of ROS in 16HBE treated withTDI-HSA may play an important role in the pathogenesis of TDI-induced asthma.The second section of this research:Objective:To investigate the effect of toluene diisocyanate (TDI) on HBE permeability.Methods:1. Millicell-Electrical Resistance System (Millipore, Bedford, MA) was applied to detect the permeability of cell monolayer, groups as follows:The normal control group, TDI-HSA 100 ug/ml group, NAC+TDI-HSA100 ug/ml.group (n=4). After stimulated for 24 h, the permeability of cell monolayer was assessed by detecting the transepithelial electrical resistance (TEER).2. Statistical methods:SPSS13.0 analysis statistical software was used for date analysis. Data was expressed as mean±SD, One-way analysis of variance (one-way ANOVA) was used to compare the overall mean when the variance was Homogeneity, and LSD method was used for Multiple comparisons among the groups; when the variance was not Homogeneity, Welch method was used to compare the overall mean, Dunnett's T3 was used for Multiple comparisons among the groups. Significance was accepted when P< 0.05.Results:TEER reduced in TDI-HSA 100 ug/ml group comparing with the control group, with statistical significance (P<0.001). NAC+100 ug/ml group enhanced TEER comparing with 100 ug/ml treatment group, with statistical significance (P<0.001).Conclusion:TDI-HSA significantly enhanced 16HBE permeability, and this effect can be inhibited by antioxidant NAC. TDI enhanced the permeability of 16HBE cell monolayer partially through a ROS-mediated pathway, indicating the importance of oxidative stress in TDI-induced pulmonary diseases. |
PDF Full Text Request