| Background and Objection:Acute lung injury(ALI)/Aeuterespiratorydistresssyndrome(ARDS) is a common clinical critically disease, which is cause by a variety of risk factors internal or external lung excluding cardiogenic facters (such as severe infection, shock, wound, burn and inhalation of harmful gases et al), leading the damage of pulmonary capillary endothelial cells and alveolar epithelial cells, diffuse interstitial lung or alveolar edema and pulmonary acute inflammation. The major clinical manifestations is respiratory distress and refractory hypoxemia. ALI/ARDS progress rapidly, which can be transformed into multiple organ failure that with poor prognosis and high mortality. The exact mechanism of ALI/ARDS is still not fully understood, which may be related to inflammatory mediators, oxidative stress product, alveolar epithelial and vascular endothelial damage and other factors. As the release of a large number of inflammatory mediators and cytokines lead to the increase in lung microvascular permeability, alveolar epithelial cell damage, surfactant secretion reduction coupled with pulmonary edema, the edema fluid dilution effect, and make the content of surfactant lower, pulmonary surface tension increasesd, lung compliance reduced,alveolar collapsed and the effective ventilation area reduced, inducing serious disorder of the ventilation/perfusion ratio and form progressive hypoxemia. Studies found that anti-inflammatory, antioxidant was the most powerful protection measures in ALI/ARDS clinical treatment. Given anti-inflammatory and comprehensive treatment of ALI/ARDS patients with early would contribute to the inflammation subsided and lung function improvement. Therefore, given timely and effective anti-inflammatory, antioxidant treatment for ALI/ARDS would play an important role in improvement of the oxygenation capacity of the lung tissue, correction hypoxemia and improvements of lung function.The transcription factor NF-E2-related factor (NF-E2-related factor2, Nrf2) is a central regulator of cell response to oxidative stress, is a key factor in the cellular oxidative stress response. Nrf2pays an important role in the anti-inflammatory antioxidant mechanism through the interaction of antioxidant response element (antioxidant response element, ARE). A variety of stimuli and drugs can induce the Nrf2overexpression. When the signaling pathway of Nrf2/ARE is activated, it will regulate the expression of antioxidant proteins and phase Ⅱ detoxifying enzymes, such as Heme oxygenase(HO-1), Thioredoxin protein (Trx), Catalase (CAT), Glutathione-S-transferase enzyme(GST), Epoxide hydrolase, Sulfotransferase, Aldehydeaminotransferase,Oxidoreductase(NQO1),UDP-glucuronosyl-transferase(U GT), Aldehyde dehydrogenase, Glutamate cysteine ligase (GCL), Glutathione synthetase et al. They could clear a variety of carcinogens and other toxic or oxidizing substances out from the body, thereby eliminating the destruction on the DNA and biological functions of proteins and make the body environment stability.NF-κB belongs to the Rel protein family, which is a ubiquitous inducible nuclear transcription factor. When cells induce by external stimuli, NF-κB will out of the cytoplasm and enter to the nucleus, combined with the specific site on the DNA strand, thus regulate the transcription of gene. NF-κB can regulate a variety of cytokines, adhesion molecules and inflammatory mediators, such as TNF-a, IL-1β, IL-6, iNOS, COX-2and ICAM-1. Excessive activation of NF-κB can induce synthesis and release of a large number of inflammatory mediators that cause cascade effect of inflammation and speed up the process of ALI and ARDS. Proinflammatory factor (TNF-α, IL-1β) is the inflammatory mediators that produce earliest and play a central role after the body stress. Proinflammatory factor mainly through enhance activity of neutrophil, generate reactive oxygen species and platelet-activating factor, causing inflammatory damage; excessive releasing of inflammatory mediators and increasing of the production of oxidative stress, which is closely related to the occurrence and development of ALI/ARDS.ICAM-1is an intercellular adhesion molecule of the immunoglobulin superfamily that mainly expressed in endothelial cells and lymphocyte. ICAM-1could mediate monocytes, lymphocytes, neutrophils and activated endothelial cells. ICAM-1participates in a number of important physiological and pathological processes such as immunity, inflammation and tumor metastasis. The study found that mutual adhesion of leukocytes and endothelial cells which mediated by ICAM-1is the key to aggregation, activation and releasing inflammatory mediators of leukocyte, then aggravating the inflammatory cascade which plays an important role in ALI.Ebselen(Ebs)[2-phenyl-1,2-benzisoselenazol-3(2H)-one] is a class of small molecule organic selenium compounds. Initially studies found that the toxicity of Ebs was low while the characteristics were stable. Then in the testing of its activity found that these substances could enhance the antioxidant capacity of the organization and had the activity of anti-tumor and anti-microbial. Ebs is the best medicine of the currently accepted role of simulation glutathione peroxidase (GPX). Ebs has a considerable effect in the anti-inflammatory, anti-tumor, anti-atherosclerosis, treatment of myocardial and cerebral ischemia and disorders of immune system. Exceptionally for induced lung injury model in a variety of factors, the Ebs shows a strong protective effect. Ebs can induce expression of Nrf2and its downstream gene HO-1and Trx in a variety of disease models. But there is no study about the effect of Ebs in the endo toxin induced acute lung injury that induced by intravenous injection of LPS.This study is to observe the dynamic expression of Nrf2in the endotoxin lung injury, clarify the role of Nrf2in the ALI. We investigate whether the Ebs regulate the Nrf2and induce the expression of downstream antioxidant gene, and then develop the treatment of antopxidation, providing a new way of thinking and methods for the clinical treatment of ALI.Chapter I. Establishment of endotoxin-induced acute lung injury model and the expression of Nrf2Objective To build endotoxin-induced lung injury model, and observe the dynamic expression of the Nrf2, inflammatory factors and oxidative stress products, exploring the response mechanism of body that accepted endotoxin-stimulated.Methods Thirty six male SD rats weight180-220g were divided into two groups: Normal control group (n=18) and model group (n=18). Given intravenous injection of LPS into the caudal vein of the model group (5mg/kg) and intravenous injection of saline into the caudal vein of the normal control group. After modeling for6h,24h,72h, the animals were sacrificed in batches. Taken six animals in each group at each time point and collect the serum, bronchoalveolar lavage fluid (BALF) and lung tissue. Observed and compared the arterial blood gas, lung wet and dry ratio, protein content in BALF, lung histopathology, inflammatory cytokines (TNF-α, IL-1β) in the blood, markers of oxidative stress in lung tissue (MDA, SOD, MPO) and the expression of antioxidant transcription factor Nrf2in each group at each time point. To compare differences by analysis of variance of factorial design and then do Multiple comparisons. P<0.05was considered statistically significantResult1. The changes of PaO2in each group:Time and the group does not exist an interaction effect (F=2.253, P=0.123), group C at three time points, PaO2between (91.10±3.55) mmHg and (93.27±3.45) mmHg, and there were no obvious changes in those groups (F=0.756, P=0.487); while the LPS group tended to increase, between (72.60±6.20) mmHg and (78.93±5.03) mmHg, but the difference was still no statistically significant (F=1.702, P=0.218). The PaO2of rats in LPS group was all lower than in the control group at the same time point from T1, and the difference between the groups was statistically significant (T1,T2, P=0.000, T3P=0.001).2. The changes of PaCO2in each group:Time and the drug does not exist an interaction effect (F=2.566, P=0.094), group C at three time points, PaO2between (45.82±3.21) mmHg and (56.58±4.05) mmHg, and there were no obvious changes in those groups (F=0.358, P=0.705); while the LPS group tended to increase, between (56.67±2.55) mmHg and (53.37±3.80) mmHg, but the difference was still no statistically significant (F=3.988, P=0.051). The PaCO2of rats in LPS group was all higher than in the control group at the same time point from T1, and the difference between the groups was statistically significant(T1P=0.000, T2P=0.002, T3P=0.024).3.The lung wet weight/dry weight ratioin of each group (W/D):Time and the group does not exist an interaction effect (F=0.819, P=0.451), group C at three time points, W/D between (3.88±0.17) and (4.11±0.27), and there were no obvious changes in those groups (F=1.153, P=0.252); while the LPS group tended to decrease, between (5.35±0.26) and (5.11±0.34), but the difference was still no statistically significant (F=1.014, P=0.383).The (W/D) of rats in LPS group was all higher than in the control group at the same time point from T1, and the difference between the groups was statistically significant(T1, T2, T3P=0.000).4. The BALF protein in each groups (mg/L):Time and the group does not exist an interaction effect (F=2.573, P=0.094), group C at three time points, BALF protein between (186.19±23.86) mg/L and (192.77±28.58) mg/L, and there were no obvious changes in those groups (F=0.101,P=0.906); while the LPS group tended to decrease, between (378.43±32.75) and (321.97±31.44). The difference had statistically significant (F=4.503, P=0.031). The BALF protein of rats in LPS group was all higher than in the control group at the same time point from T1, and the difference between the groups was statistically significant (T1, T2, T3P=0.000).5. The lung histopathological changes in each groups: Light microscopy showed the changes of lung histopathology in each group of three time points were small. The structure of lung tissue in group C was clear, without inflammatory cell infiltration in alveolar spaces, alveolar wall is thin, and the stromal vascular was not expansion; while the bronchial epithelial was also integrity. The alveolar structure of the LPS group were extensive destruction, accompany to the lung tissue edema and pulmonary interstitial widening, serous exudate, a large number of red blood cells and neutrophils in alveolar space.6. The MDA expression of the lung tissue in each groups (nmol/mg):Time and the group does not exist an interaction effect (F=2.755, P=0.080), group C at three time points, MDA between (7.01±2.52) nmol/mg and (7.44±2.10) nmol/mg; and there were no obvious changes in those groups (F=0.048, P=0.954); while the LPS group tended to decrease, between (26.47±4.80) and (19.36±3.68) nmol/mg. The difference was no statistically significant (F=3.112,,P=0.076). The MDA expression of the rats lung tissue in LPS group was all higher than in the control group at the same time point from T1, and the difference between the groups was statistically significant(T1, T3P=0.000, T3P=0.003).7. The SOD activity of the lung tissue in each groups (U/mg):Time and the group does not exist an interaction effect(F=2.864, P=0.073), group C at three time points, SOD activity between(193.07±17.39) U/mg and (203.60±16.89)U/mg; and there were no obvious changes in those groups (F=0.657, P=0.533); while the LPS group tended to increase, between (93.08±12.53) U/mg and (115.70±13.64) U/mg. The difference had statistically significant (F=4.211±P=0.037). The SOD activity of the rat lung tissue in LPS group was all lower than in the control group at the same time point from T1, and the difference between the groups was statistically significant (T1, T2, T3P=0.000).8.The MPO activity of rat lung tissue in each groups (U/mg):Time and the group does not exist an interaction effect (F=1.713, P=0.198), group C at three time points, MPO activity between (2.17±0.52) U/g and (2.02±0.78)U/g; and there were no obvious changes in those groups (F=0.091,.P=0.914); while the LPS group tended to decrease, between (7.78±1.59) U/g and (6.30±1.03) U/g. The difference had statistically significant (F=1.686, P=0.221).The MPO activity of rat lung tissue in LPS group was all higher than in the control group at the same time point from T1, and the difference between the groups was statistically significant (T1, T2, T3P=0.000).9.The TNF-α expression in the serum of each groups (ng/L):Time and the group exist an interaction effect (F=4.909, P=0.015), group C at three time points, TNF-α between (93.43±20.69)pg/ml and (90.53±19.99) pg/ml; and there were no obvious changes in those groups (F=0.031, P=0.970); while the LPS group tended to decrease, between (379.05±41.85) pg/ml and (298.95±39.52) pg/ml, the difference had statistically significant (F=6.554, P=0.010). The TNF-α expression in the rats serum of LPS group was all higher than in the control group at the same time point from T1, and the difference between the groups was statistically significant (T1, T2, T3P=0.000).lO.The IL-1β in the serum of each groups (ng/L):Time and the group does not exist an interaction effect (F=3.284, P=0.052), group C at three time points, IL-1β between (81.76±20.43) pg/ml and (74.23±20.10)pg/ml; and there were no obvious changes in those groups (F=0.239, P=0.790); while the LPS group tended to decrease, between (344.89±46.94) pg/ml and (270.38±41.71) pg/ml, the difference had statistically significant (F=4.046, P=0.041). The IL-1β in the rats serum of LPS group was all higher than in the control group at the same time point from T1, and the difference between the groups was statistically significant (T1, T2, T3P=0.000).11. The relative expression of Nrf2of Lung tissue in each groups:Time and the group does not exist an interaction effect (F=2.682, P=0.109). There was no significant difference at each time point in Group C (F=0.273, P=0.770), while the LPS group peaked at T2, but the difference was no statistically significant (F=2.114, P=0.202). The relative expression of Nrf2of rats lung tissue in LPS group was all higher than in the control group at the same time point from T1, and the difference between the groups was statistically significant (T1P=0.013, T2P=0.011, T3P=0.025).Conclusion Tail vein injection5mg/kg of LPS could induce oxygenation obstacles, aggravate interstitial pulmonary edema, oxidative stress and inflammatory response after6h、24h、72h, while the most obvious in6h; And could be able to activate the antioxidant system of body, increase the expression of Nrf2, but not obvious with time changes. So in this study we sew that use5mg/kg of LPS can successfully induce the ALI rat model, which could provide ALI animal model for further research. Chapter Ⅱ. The protective effects and mechanisms of Ebselen to endotoxin induced acute lung injuryObjective Through the establishment of endotoxin induced acute lung injury model, observe the influence of Ebs on inflammatory factors and oxidative stress of endotoxic acute lung injury. By studying the antioxidant transcription factor Nrf2and its downstream antioxidant genes HO-1, Trx-1, as well as NF-κB, ICAM-1, et al, clarifying the protective effects and mechanisms of Ebs on endotoxin-induced acute lung injury.Methods Forty eight male SD rats weight180-220g were divided into six groups (n=8):normal control (Group C), model group (LPS), Low-dose of Ebs group (EbsL Group) medium dose group (EbsM Group) High dose group (EbsH Group) and Dexamethasone group (DEX Group). Ebs and DEX Dissolved in a solvent according to the literature method. Group C:Tail vein injection of identical size of saline and intraperitoneal injection of solvent. LPS Group:Tail vein injection of LPS5mg/kg and intraperitoneal injection of solvent; EbsL Group:Tail vein injection of LPS5mg/kg and intraperitoneal injection of Ebselen7.5mg/kg; EbsM Group:Tail vein injection of LPS5mg/kg and intraperitoneal injection of Ebselen15mg/kg; EbsH Group:Tail vein injection of LPS5mg/kg and intraperitoneal injection of Ebselen30mg/kg; DEX Group:Tail vein injection of Dexamethasone5mg/kg; After modeling for6h, the animals were sacrificed in batches and collected the serum,bronchoalveolar lavage fluid (BALF) and lung tissue. We observed and compared the arterial blood gas, lung wet and dry ratio, protein content in BALF, lung histopathology and the expression of antioxidant factors (HO-1,Trx-1), transcription factor (Nrf2, NF-κB), adhesion molecule (ICAM-1). Result1. The changes of PaO2for SD rats in each group:By One-way ANOVA test, there had statistically significant differences between groups (F=15.517, P=0.000). The LPS group was lower than the control group, there was significant difference between the groups (P=0.000); The EbsL group compared with LPS Group, there was no significant difference between the groups (P=0.060); The PaO2of EbsM Group (P=0.041), EbsH Group (P=0.003)and Dex Group (P=0.000) are all higher than the LPS Group, there was significant difference between the groups. Ebselen could mitigate the lower PaO2of SD rats in ALI.2. The changes of PaCO2for SD rats in each group:By One-way ANOVA test, there had statistically significant differences between groups (F=13.815, P=0.000). The LPS group was higher than the control group, there was significant difference between the groups (P=0.000); The EbsL group (P=0.332) and EbsM group (P=0.070) compared with LPS Group, there was no significant difference between the groups; The PaCO2of EbsH Group and Dex Group are lower than the LPS Group, there was significant difference between the groups (P=0.000); Ebselen could make the PaCO2of SD rats lower in acute lung injury, which indicate that Ebselen could improve the oxygen exchange barriers of SD rats caused by ALI.3. The changes of lung wet weight/dry weight ratioin in each group (W/D):By One-way ANOVA test, there had statistically significant differences between groups (F=17.682, P=0.000). The LPS group was higher than the control group, there was significant difference between the groups (P=0.000); The EbsL group (P=0.336) and EbsM group (P=0.087) compared with LPS Group, there was no significant difference between the groups; The (W/D) of EbsH Group(P=0.003) and Dex Group (P=0.000) are lower than the LPS Group, there was significant difference between the groups; Ebselen could significantly reduce the pulmonary edema of SD rats caused by ALI.4. The changes of BALF protein in each groups:By One-way ANOVA test, there had statistically significant differences between groups (F=53.602, P=0.000). The LPS group was higher than the control group, there was significant difference between the groups (P=0.000); The EbsL group (P=0.246) and EbsM group (P=0.066) compared with LPS Group, there was no significant difference between the groups; The BALF protein of EbsH Group and Dex Group are lower than the LPS Group, there was significant difference between the groups (.P=0.000); Ebselen could significantly reduce the BALF protein of SD rats caused by acute lung injury, which indicate that Ebselen could reduce the increasing permeability of pulmonary vascular of SD rats caused by ALI.5. The lung histopathological changes in each groups:The alveolar structure of Group C is clear under the light microscopy, the alveolar wall was thin, there is no inflammatory cell infiltration between alveolar wall and interstitial. In LPS group, the perivascular space was widened, lung interval widened, which could see a large number of red blood cells and neutrophils, part of the lung septal thinning, fracture. In the EbsL group and EbsM group, the alveolar structure was partially destroyed while lung interval widened, besides there are amount of exudation of red blood cells and inflammatory cell infiltration in the alveolar space. In the EbsH group and DEX group, the alveolar structure remains relatively complete while lung interval widened slightly, besides the red blood cells and inflammatory cell infiltration was significantly reduced in the alveolar space.6.The changes of MDA in each groups:By One-way ANOVA test, there had statistically significant differences between groups (F=7.149, P=0.000). The LPS group was higher than the control group, there was significant difference between the groups (P=0.000); The EbsM group (P=0.027), EbsH group (P=0.009) and Dex group (P=0.001) are all lower than LPS Group, there was significant difference between the groups; while the MDA of EbsL Group compared to LPS Group, there was no significant difference between the groups (P=0.187); Ebselen could reduce the MDA of lung tissue for SD rats with ALI, indicate that Ebselen can reduce the generation of oxygen free radicals of SD rats that caused by endotoxin-induced acute lung injury, then reduce the oxidative stress of lung tissue.7.The changes of SOD in each groups:By One-way ANOVA test, there had statistically significant differences between groups (F=13.064, P=0.000). The LPS group was lower than the control group, there was significant difference between the groups (P=0.000); The EbsM group (P=0.010), EbsH group(P=0.001) and Dex group (P=0.000) are all higher than LPS Group, there was significant difference between the groups, while the SOD of EbsL Group compared to LPS Group, there was no significant difference between the groups (P=0.275); Ebselen could increase the SOD levels in lung tissue of rats with ALI, reduce the oxidative stress of lung tissue.8. The changes of MPO in rats lung tissue of each groups:By One-way ANOVA test, there had statistically significant differences between groups (F=8.947, P=0.000). The LPS group was higher than the control group, there was significant difference between the groups (P=0.000); The EbsH group (P=0.013), Dex group (P=0.001) are lower than LPS Group, there was significant difference between the groups, while the MPO of EbsM Group (P=0.158) and EbsL Group (P=0.059) compared to LPS Group, there was no significant difference between the groups. Ebselen could reduce the MPO level in lung tissue of rats with ALI. Ebselen reduce the degree of aggregation of neutrophils in the lung tissue of endotoxin-induced acute lung injury.9. The changes of TNF-a in rats lung tissue of each groups:By One-way ANOVA test, there had statistically significant differences between groups (F=43.971, P=0.000). The LPS group was higher than the control group, there was significant difference between the groups (P=0.000); The EbsM group (P=0.001), EbsH group (P=0.000) and Dex group (P=0.000) are all lower than LPS Group, there was significant difference between the groups, while the TNF-a of EbsL Group compared to LPS Group, there was no significant difference between the groups (P=0.118); Ebselen could reduce the TNF-a level of rats lung tissue that caused by acute lung injury, which indicate that Ebselen reduce the proinflammatory media TNF-a in lung tissue of rats with endotoxin-induced ALI.10. The change of IL-1β in rats lung tissue of each groups:By One-way ANOVA test, there had statistically significant differences between groups (F=61.297, P=0.000). The LPS group was higher than the control group, there was significant difference between the groups (P=0.000); The EbsH group (P=0.002) and Dex group (P=0.000) are lower than LPS Group, there was significant difference between the groups, while the IL-1β of EbsM Group (P=0.262) and EbsL Group (P=0.059) compared to LPS Group, there was no significant difference between the groups; Ebselen could reduce the IL-1β level of rats lung tissue that caused by acute lung injury, indicate that Ebselen reduce the proinflammatory media IL-1β in lung tissue of rats with ALI, which play an anti-inflammatory role.11. The changes of expression of protein for Trx-1in rats lung tissue of each groups: By One-way ANOVA test, there had statistically significant differences between groups (F=58.187, P=0.000). The LPS group was higher than the control group, there was significant difference between the groups (P=0.047); The EbsM group (P=0.022) and EbsH group (P=0.045) are higher than LPS group, there was significant difference between the groups, but there was no significant difference between EbsL group (P=0.945) and LPS. Ebselen could increase the expression of Trx-1in rats lung tissue that caused by acute lung injury, indicating that Ebselen can increase the expression of antioxidant factors of Trx-1and inhibit the oxidative stress and inflammatory responses.12. The changes of expression of protein for HO-1in rats lung tissue of each groups: By One-way ANOVA test, there had statistically significant differences between groups (F=126.725, P=0.000). The LPS group was higher than the control group, there was significant difference between the groups (P=0.019); The EbsL group (P=0.000), EbsM group (P=0.000), EbsH group (P=0.000) are higher than LPS group, there was significant difference between the groups. Ebselen could increased the expression of HO-1in rats lung tissue that caused by acute lung injury, indicating that Ebselen improve pulmonary oxidative stress and inflammatory responses by regulating the expression levels of antioxidant factors.13. The changes of expression of protein for ICAM-1in rats lung tissue of each groups:By One-way ANOVA test, there had statistically significant differences between groups (F=101.524, P=0.000). The LPS group was higher than the control group, there was significant difference between the groups (P=0.000); The EbsL group (P=0.021), EbsM group (P=0.000), EbsH group (P=0.000) are lower than LPS group, there was significant difference between the groups. Ebselen could decreased the expression of ICAM-1in rats lung tissue that caused by acute lung injury, indicating that Ebselen can inhibit inflammatory responses by interaction with ICAM-1.14. The changes of expression of protein for Nrf2in rats lung tissue of each groups: By One-way ANOVA test, there had statistically significant differences between groups (F=91.602, P=0.000). The LPS group was higher than the control group, there was significant difference between the groups(P=0.034); The EbsL group (P=0.001), EbsM group (P=0.000), EbsH group (P=0.000) are higher than LPS group, there was significant difference between the groups. Ebselen could increase the expression of Nrf2in rat lung tissue that caused by acute lung injury, and enhance the ability of anti-inflammatory and anti-oxidation.15. The changes of expression of protein for NF-κB in rats lung tissue of each groups: By One-way ANOVA test, there had statistically significant differences between groups(F=46.037, P=0.000). The LPS group was higher than the control group, there was significant difference between the groups (P=0.000); The EbsL group (P=0.011), EbsM group (P=0.000), EbsH group (P=0.000) are lower than LPS group, there was significant difference between the groups. Ebselen can significantly inhibit NF-κB phosphorylation in rat lung tissue of endotoxin-induced ALI, and reduce the inflammatory response.Conclusion Ebselen shows pretective effect in endotoxin-induced lung injury, may be related to the induction of generation of Nrf2in lung tissue, enhancing of the expression of HO-1and Trx-1, thus inhibit the activation of NF-κB in lung tissue, reduce the oxidative stress product of MDA, MPO, the release of inflammatory cytokines TNF-α, IL-1β, and the generation of adhesion molecules ICAM-1that ultimately reduce the damage of lung tissue. |
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