The Protective Effect And Mechanism Of NEMO-binding Domain Peptide On Lipopolysaccharide-induced Acute Lung Injury In Mice

Background and ObjectionAcute lung injury (ALI) and its more severe form, the acute respiratory distress syndrome (ARDS), are major causes of acute respiratory failure in critically ill patients. The mortality associated with acute respiratory failure from ALI/ARDS is still high at about50%. The exact mechanism of ALI/ARDS is still not fully understood, such as infectious diseases, biochemistry injury, severe trauma and post-traumatic infection, shock, poisoning are major risk factors for ALI. The pathological basis of ALI/ARDS are lung microvascular permeability, alveolar epithelial cell damage, surfactant secretion reduction coupled with pulmonary edema and hyaline membrane formation result in uncontrol inflammatory reaction.An improved understanding of the pathogenesis of ALI/ARDS has led to the assessment of several novel treatment strategies. However, many specific therapies have not proved beneficial. Due to the large number of media involved in ALI inflammation and oxidative damage, and mutual contact, reinforce each other, to achieve satisfactory control effect for a single medium of monoclonal antibodies, antagonists, inhibitors are in the process of treatment. In addition, inflammatory mediators necessary for the body, excessive or inadequate damage inhibitors such as dose and timing is difficult to grasp, often makes such studies in trouble. ALI lung protection research to take anti-inflammatory, antioxidant supplement pulmonary surfactant programs, has been in clinical effect, but after years of study also failed to break the efficacy bottleneck. How to maximize the protection of the immune defense cells of lung structure, the domino effect of interrupting the inflammatory cascade amplification ALI progression fundamentally curb? This requires that we re-examination of the imbalance in the ALI process of inflammation, oxidative damage, and the two interaction mechanisms, looking for its core sites regulate.Toll-like receptor4(TLR4)-Nuclear Factor Kappa B (NF-кB) pathway initiates inflammation critical paths, a variety of factors in infection and oxidative stress induced play an important role in the inflammatory response. NF-кB is widely present in the cytoplasm in a fast response transcription factor located TLR4downstream, closely associated with inflammation and oxidative stress, can start and regulation, including growth factors, transcription factors, cytokines, chemokines, adhesion molecules, the anti-apoptotic protein molecule gene transcription involved in the body’s inflammation, apoptosis, immune response and tumor regulation of physiological and pathological processes. Not only can reduce the inhibition of NF-кB inflammatory damage can also weaken oxidative stress, and reduce tissue damage. Many studies have confirmed that NF-кB activation and ALI closely related. It has been proved that the inhibition of NF-кB activation can be significantly reduced LPS-induced septic shock mortality in mice. Our previous research with other foreign study showed lowered NF-кB activation reduces TNF-α-induced inflammatory response of alveolar epithelial cells, oxidative stress injury and apoptosis. Therefore, we speculate, NF-кB is important node connected ALI inflammatory response and oxidative stress, regulate the activity of NF-кB is down ALI inflammatory response key to reducing oxidative stress injury.Down the current research focus NF-кB activation by inhibiting the activity of IкB kinase (IKK), IKK IкB phosphorylation is a key step for NF-кB activation, classical pathway of NF-кB activation in the inflammatory response process. Specifically reduced the activation of NF-кB in inflammation and oxidation reactions through the inhibition of IKK activity. IKK IKKa, IKKβ and IKKy three, subunit composition, wherein IKKy also known as NEMO, IKK regulatory subunit. The study confirmed the IKK activation classic NF-кB signaling pathway dependent IKKy the integrity, and IKKy vast majority of pro-inflammatory factors necessary to activate NF-кB. The existing for IKK complex of NEMO the polypeptide fragment NBD (NEMO binding domain, NBD), with NEMO binding thereby blocking the assembly of the IKK complex and lowered IKK activity, but how to stabilize the biologically active The IKK inhibitor targeting imported into the lungs, thereby improving the ALI runaway inflammatory response and oxidative damage is the use of IKK inhibitors of difficulty. In the field of pharmacy, drug use liposomes to improve efficacy, a measure to improve drug targeting and reduce adverse drug reactions. In this study, the proposed use of exogenous pulmonary surfactant (Pulmonary Surfactant, PS) encapsulating lipophilic NBD via endotracheal atomized form of targeted pulmonary delivery, improve drug stability, can be greater the possibility to improve the affinity of the NBD lung tissue and to promote NBD enter lung structural cells suits derived PS can supplement the loss of alveolar surfactant inflammation or oxidative stress to collaborative to reach correct the ALI pathological damage, maintain normal The purpose of respiratory function. In summary, the purpose of the present study are as followed:1To compare three methods of lipopolysaccharides administration for inducing ALI in mice by evaluating inflammatory reaction and pathologica change. Select the most appropriate mouse model that easy operate and close to the pathological features of ALI.2To investigate the effect of NBD with different doses pretreatment on the inflamm atory reaction and oxidative stress in lung of mice with ALI when determine the best mouse model.3To research the effect of NBD combination PS pretreatment on excessive inflamm atory reaction and mechanisms in mice with ALI, when select the appropriate dose of the NBD.Method1Comparison of three mouse models of acute lung injury induced by different administration of LPS12weeks of age, SPF grade male BLAB/c mice were anesthesiaed by intraperitoneal injectioning with0.1mL chloral hydrate anesthesia. LPS solution100μL(lmg/mL) was intratracheally atomized (ITA group), intratracheally instilled (ITI group) and intraperitoneally injected (IPA group) to induce ALI in mice. normal control (NC group) were spraied100μL air by intratracheal atomizaed. Evans Blue instead of LPS was intratracheally administered in the two manners to observe the liquid distribution in the lungs. After LPS administration2hours, the mice were sacrificed cut the abdominal aorta afert anesthesiaed, and the lungs were removed to determine the lung coefficient and the histological changes were evaluated by HE staining. The concentration of TNF-a and IL-1β in Bronchoalveolar Lavage Fluid (BALF) with mice were detected by ELISA. Transcription intensity of TNF-a and IL-1β mRNA in lung tissue were detected by Real-time RT-PCR. Phosphorylation level of IкB-a and NF-кB p65in lung tissue were investigated by Western Blotting.2Effect of NBD pretreatment on excessive inflammation and oxidative stress in mice with ALI induced by LPS 12weeks of age, SPF male BLAB/c mice were randomly divided into control group, model group, NBD negative control group (N-NBD), NBD low-dose group (S-NBD), NBD medium dose group (M-NBD) and NBD high dose group (L-NBD), each group contain15numbers. Control group mice were anesthesiaed by intraperitoneal injectioning with0.1mL chloral hydrate anesthesia and spraied100μL air by intratracheal atomizaed. Model mice were atomized intratracheally with LPS100μL (lmg/mL). For the four experimental groups, the mice were atomized intratracheally with2,6,10μg/50μl/mouse of NBD and with6μg/50μl of Antennapedia-NBD as negative control, respectively and treated for30min, then followed by intratracheal atomization again of LPS50μl (2mg/mL).After2h, the mice were sacrificed by sodium pentobarbital and samples were collected. After LPS administration2hours, the mice were sacrificed cut the abdominal aorta afert anesthesiaed and the lungs were removed to determine the lung coefficient and the histological changes were evaluated by HE staining. The concentration of TNF-a and IL-1β in Bronchoalveolar Lavage Fluid (BALF) with mice were detected by ELISA. Transcription intensity of TNF-a, IL-1β, NOX1, NOX2and NOX4mRNA in lung tissue were detected by Real-time RT-PCR. IкB-α, NOX1, NOX2, NOX4protein content and IкB-α, NF-кB p65protein phosphorylation levels in lung tissue were investigated by Western Blotting. The concentration of Superoxide dismutase (SOD), total antioxidant capacity (T-AOC) and the concentration of malondialdehyde (MDA) in lung of mouse were detected by colorimetric.3Effect of NBD combination PS pretreatment on excessive inflammation action in mice with ALI induced by LPS12weeks of age, SPF male BLAB/c mice were randomly divided into into control group, model group, PS group, PS+(N-NBD) group, PS+NBD group and NBD group, each group contain15numbers. Control group mice were anesthesiaed by intraperitoneal injectioning with0.1mL chloral hydrate anesthesia and spraied100μL air by intratracheal atomizaed. Model mice were atomized intratracheally with LPS100μL (lmg/mL). PS and NBD groups mice were atomized intratracheally with corresponding dose monotherapy PS and NBD50μL for0.5h, then followed by intratracheal atomization again of LPS50μl (2mg/mL). PS+(N-NBD) group and PS+NBD group mice were atomized intratracheally with effect dose PS+(N-NBD) and PS+NBD mixed formulations50μL for0.5h, then followed by intratracheal atomization again of LPS50μl (2mg/mL). After LPS administration2hours, The animals in each group were sacrificed. Then Pulmonary histological changes were evaluated by hematoxylin-eosin stain. The concentration of TNF-a and IL-1β in Bronchoalveolar Lavage Fluid (BALF) with mice were detected by ELISA. Transcription level of TNF-a and IL-1β mRNA in lung tissue were detected by Real-time RT-PCR. Phosphorylation of IxB-alpha, NF-kappa B, P38MAPK, ERK1/2and JNK in lung tissue were determined by Western Blotting.4StatisticallyData are expressed as means±SD. One-way ANOVA was used for comparison among the different groups. When the ANOVA was significant, post Hoc testing of differences between groups was performed using the least significant difference (LSD) test, p<0.05was considered significant.Result1Comparison of three mouse models of acute lung injury induced by different administration of LPS1.1Blue-stained area of the lung tissue of mice atomization Evans blue solution1.1endotracheal more scattered, spotty or patchy distribution at each lobe. Evans blue solution by intratracheal instillation of blue-stained area of the lung tissue of mice are more concentrated and focused on both sides of the main pipe, was patchily distributed, some specimens of Evans blue solution is only distributed in individual lobes, the rest of blue dye were bot been seen.1.2Wet and dry weight ratio of each group of mice lung tissue differences statistically significant difference (F=39.512, P=0.000). LPS administration after2h ITA group, ITI group and the the IPI group of mice lung wet/dry weight ratio was significantly higher than that of NC group, while the ITA group, ITI group and the the IPI group of mice lung wet/dry weight proportion of the difference was not statistically significant1.3Each group of mice lung pathology score difference was statistically significant (F=80.84, P=0.000). ITA group, ITI group and the IPI group of mouse lung pathology score were significantly higher than those in the control group, the ITA group of, and ITI mice lung pathology score significantly higher than the IPI group, while the ITA group and ITI mice lung pathology score difference was not statistically significant. NC group mice alveolar structure is clear, thin alveolar wall, interstitial lung inflammatory cells infiltration. ITA mice each lobe diffuse alveolar septal thickening and alveolar wall destruction, interstitial lung inflammatory cell infiltration and hemorrhage, varying degrees of destruction of the vascular bed. ITI mice destruction of part of the lobe structure, part of the lobe structure is relatively complete, lobe lesions uneven between. IPI mice were mild alveolar structure, the degree of destruction of the vascular bed, and inflammatory cell infiltration.1.4The difference of concentration of TNF-α and IL-1β in BALF with mice was statistically significant. The ITA group in the ITI group, and IPI mice BALF TNF-a and IL-1β concentration increased compared with NC group, the difference was statistically significant. In the ITA group, and ITI mice BALF TNF-a concentrations were significantly higher than the IPI group, while the ITA group was significantly higher than the ITI group. 1.5The difference of transcription level of TNF-a and IL-1β mRNA was statistically significant. ITA group, ITI group and the the IPI group of mice lung tissue TNF-a and IL-1βmRNAtranscriptional level higher than those of the NC group difference was statistically significant. ITA group and ITI group of mouse lung tissue TNF-a mRNA transcription were significantly higher than the IPI group, while the ITA group was significantly higher than the ⅡT group.1.6Each group of mice lung tissue IкB-α protein content and IкB-α, NF-кB p65protein phosphatase level difference was statistically significant. ITA Group, ITI Group and IPI group mouse lung tissue IкB-α protein content than those in the NC group reduced the difference was statistically significant. ITA group, ITI group and the the IPI group of mice lung tissue IкB-α, NF-кB of p65protein phosphorylation levels compared with NC group increased, the difference was statistically significant. IкB-a protein content of the ITA mice lung tissue were significantly lower than the ITI group and IPI group, the ITI group was significantly lower than the IPI group. ITA mice lung tissue IкB-α protein phosphorylation levels significantly higher than the ITI group and IPI group. the ITI group was significantly higher than the IPI group. ITA group and ITI group lung tissue of mice of NF-кBp65protein phosphorylation were significantly higher than the IPI group, while the ITA and ITI group, the difference was not statistically significant.2Effect of NBD pretreatment on excessive inflammation and oxidative stress in mice with ALI induced by LPS2.1Control mice alveolar structure clear, thin alveolar wall, interstitial lung inflammatory cells infiltration. Model group and NBD negative control group mice lobe diffuse alveolar septal thickening, alveolar wall destruction and alveolar collapse, interstitial lung inflammatory cell infiltration and hemorrhage, vascular bed varying degrees of damage. NBD pretreatment in high doses to low0.5h, can significantly reduce the LPS-induced lung tissue injury, and the NBD lung tissue protective effect in a concentration-dependent enhancement. Each group of mice lung pathology score difference was statistically significant (F=69.594, P=0.000).2h after administration of LPS in mouse lung pathology score higher than that of the control group, the difference was statistically significant. Low Medium High three doses of NBD by intratracheal the atomization pretreatment0.5h after the mouse lung pathology score compared with the model was significantly lower. Low Medium High three doses NBD pretreatment group between mouse lung pathology score in a concentration-dependent reduction in the difference was statistically significant.2.2The difference of concentration of TNF-α and IL-1β in BALF with mice was statistically significant. LPS by intratracheal the atomization administration after2h in BALF TNF-α and IL-1β concentrations compared with the control group was significantly higher. Low Medium High three doses of NBD by intratracheal the atomization pretreatment0.5h after three groups of mice BALF TNF-a and IL-1β concentration decreased, compared with the model group and the difference was statistically significant. Low Medium High the three dose NBD pretreatment group in BALF TNF-α concentrations in a concentration-dependent reduction in the difference was statistically significan. The NBD high-dose group and NBD in dose mice BALF IL-1β concentration were significantly lower than NBD low-dose group, while the the NBD high-dose group and middle dose group had no significant difference.2.3The difference of transcription level of TNF-α and IL-1β mRNA was statistically significant. LPS the atomization administered by intratracheal2h after mouse lung tissue TNF-α and IL-1β mRNA transcription level than those in the control group was significantly higher (P<0.05). Low in the high-dose NBD by intratracheal the atomization pretreatment0.5h, the group mouse lung tissue TNF-α and IL-1β mRNA transcription level compared with model group decreased, the difference was statistically significant (P<0.05), low in the third year mouse lung tissue TNF-a and IL-1β mRNA transcription level between the two doses NBD pretreatment group in a concentration-dependent reduction in the difference was statistically significant (P<0.05).2.4Mouse lung tissue IкB-α protein content and IкBα, NF-кB p65protein phosphatase level difference was statistically significant. LPS the atomization administered by intratracheal after2h, IкB-α protein content of the lung tissue of mice compared with the control group was significantly reduction in IкB-α, NF-кB p65protein phosphorylation levels compared with the control group were significantly liter high. Low Medium High three doses of NBD by intratracheal atomization pretreatment0.5h after three groups of mice lung tissue IкB-α protein content compared with model group increased IкB-α and NF-кB p65phosphorylation levels compared with model group reduced, the difference was statistically significant. Low Medium High the three dose NBD pretreatment group IкB-α protein content of mouse lung tissue showed no significant difference, while Low Medium High the three dose NBD pretreatment group between the lung tissue of mice IкB-α and NF-кB p65protein phosphorylation levels in a concentration-dependent reduction in the difference was statistically significant.2.5Groups total protein in the mouse lung tissue SOD, T-AOC and MDA concentration difference was statistically significant. LPS the atomization administered by intratracheal after2h SOD and T-AOC concentration of total protein in the lung tissue of mice was significantly lower than in the control group, compared with the control group, the MDA concentration increased significantly. NBD middle dose group and high dose group of SOD and T-AOC concentration enhanced compared with the model group, the difference was statistically significant, and the NBD model group, low dose group difference was not statistically significant. Low Medium High three doses NBD pretreatment group MDA concentration compared with the model group decreased, the difference was statistically significant. NBD middle dose group and high dose group lower concentration of MDA the NBD-dose group significantly reduced, while the difference was not statistically significant between the NBD in dose and high dose group.2.6Groups of mouse lung tissue NOX1, NOX2and NOX4mRNA transcription level difference was statistically significant. LPS the atomization administered by intratracheal after2h, elevated than the normal control group of mice lung tissue NOX1, NOX2and NOX4mRNA transcriptional level, the difference was statistically significant. Low Medium High three doses of NBD by intratracheal the atomization pretreatment0.5h after, the three groups of mice lung tissue NOX1, NOX2and NOX4mRNA transcription level compared with the model group decreased, the difference was statistically significant. The NBD high dose group and NBD middle dose group NOX1, NOX2and NOX4mRNA transcripts of water on average lower than NBD low dose group, the difference was statistically significant, while the concentration of IL-1β NBD high dose group and NBD in dose group was no significant difference.2.7Groups of mice lung tissue NOX1, NO2and NOX4protein expression level of the difference was statistically significant. LPS the atomization administered by intratracheal2h after lung tissue of mice NOX1, NOX2and NOX4protein expression levels compared with the normal control group increased significantly. NBD middle dose group and high dose group of mouse lung tissue NOX1and NOX2protein expression levels compared with the model group decreased, the difference was statistically significant, NBD low-dose group compared with model group difference was not statistically significant. Low Medium High pretreatment group of three doses of NBD mouse lung tissue NOX4protein expression levels compared with model group decreased, and the difference was statistically significant.3Effect of NBD combination PS pretreatment on excessive inflammation action in mice with ALI induced by LPS3.1The difference of concentration of TNF-a and IL-1β in BALF with mice was statistically significant. The model mice BALF TNF-a and IL-1β concentrations than the control group was significantly higher, and the difference was statistically significant. PS group, NBD group and PS+NBD group of TNF-a and IL-1β concentration compared with the model group decreased, and the difference was statistically significant. PS+NBD group, TNF-a and IL-1β concentration compared to the PS group significantly decreased, while no significant difference between the PS+NBD group with NBD group.3.2Mice lung tissue TNF-a and IL-1β mRNA transcription level difference was statistically significant. Model group, TNF-a and IL-1β mRNA transcription level compared with the control group increased, the difference was statistically significant. PS group, NBD group and PS+NBD group of TNF-a and IL-1β mRNA transcription level compared with model group decreased, and the difference was statistically significant. The PS+NBD group TNF-a mRNA transcription level than those of PS and NBD monotherapy group was significantly lower, IL-1β mRNA transcription levels were only significantly decreased compared to the PS group compared with the NBD group the difference was not statistically significant.3.3Mouse lung tissue IкB-α protein content and IкB-α and of NF-icBp65protein phosphorylation level difference was statistically significant. IкB-α protein content of the model group significantly reduced compared with the control group, while the level of phosphorylation of IкB-α and NF-icBp65increased significantly compared with the control group. PS group, NBD group and PS+NBD group IкB-α protein content increased significantly compared with model group, IicB-a and the NF-кBp65 phosphorylation levels compared with the model group was significantly lower. The PS+NBD group IкB-a protein content compared to the PS group increased significantly, compared with the NBD group difference was not statistically significant. The PS+NBD group IкB-α and of NF-кBp65protein phosphorylation levels than those of the PS and NBD group significantly reduced, but the difference was not statistically significant between the PS group and NBD group.3.4Mice lung tissue P38, ErKl/2and JNK protein phosphorylation level difference was statistically significant. The model group P38, ErKl/2and JNK phosphorylation levels than those in the control group increased, the difference was statistically significant. The the PS group P38phosphorylation levels compared with the model group was significantly reduced, while PS+NBD group and NBD group and the model group, the difference was not statistically significant. PS group ErKl12phosphorylation levels compared with the model group, the difference was not statistically significant, while the PS+NBD group and NBD group compared with the model group was significantly lower. PS group, NBD group and PS+NBD group JNK phosphorylation levels compared with the model group was significantly lower, while the difference was not statistically significant between the PS group, NBD group and PS+NBD group.Conclusion1Compared with the intratracheal instillation and intraperitoneal injection, intratracheal atomization enables LPS more evenly distributed in the lungs, causing a stronger lung tissue inflammation amplification reaction, causing more severe lung tissue damage, and thus closer to the acute lung injury pathological criteria, and endotracheal the atomization LPS has non-invasive surgery, and simple operation is preferred copy ALI mouse model program.2Give a certain dose of NBD pretreatment inhibited LPS induced ALI mouse lung tissue NF-кB signaling pathway positive feedback amplifier, thereby significantly reduced lung tissue inflammation and oxidative stress injury, protecting the cells of the lung tissue structure.3Give PS joint NBD pretreatment, not only can effectively inhibit LPS induced ALI mouse lung tissue NF-кB signaling pathway positive feedback amplifier, thereby significantly reduce the inflammation of the lung tissue injury, and exogenous PS can be an effective complement to the excessive inflammatory response and oxidative stress leading to the pulmonary surface active material, the combined use, have a synergistic anti-inflammatory effect.