Oroxylin-A Rescues LPS-Induced Acute Lung Injury via Regulation of NF-κB Signaling Pathway in Rodents

Background and Purpose

Successful drug treatment for sepsis-related acute lung injury (ALI) remains a major clinical problem. This study was designed to assess the beneficial effects of post-treatment of oroxylin A (OroA), a flavonoid, in ameliorating lipopolysaccharides (LPS)-induced lung inflammation and fatality.

Experimental Approach

Rats were injected with LPS (10 mg/kg, iv) to induce ALI, and OroA was given (15 mg/kg, iv) 1 hr or 6 hrs after LPS challenge. Twenty four hrs after LPS challenge, biochemical changes in the blood and lung tissues, and morphological/histological alterations in the lung associated with inflammation and injury were examined. Therapeutic effect of OroA was assessed by measuring the survival rate in endotoxemic mice.

Key Results

LPS (10 mg/kg, iv) significantly altered WBC counts, elevated plasma tumor necrosis factor (TNF)-α and nitric oxide (NO), increased pulmonary edema, thickened alveolar septa, and decreased survival rate. These changes were ameliorated by OroA (15 mg/kg, iv) administered 1 hr or 6 hrs after LPS challenge. This post-treatment also significantly attenuated LPS-induced activation of nuclear factor-κB (NF-κB) and the release of high mobility group box 1 (HMGB1) in lung tissues. Furthermore, post-treatment with OroA (60 mg/kg, ip) administered 1 hr or 6 hrs after LPS challenge in mice significantly increased survival rate.

Conclusion and Implication

OroA administered after induction of ALI by LPS significantly prevent and revere lung tissues injuries with increased survival rate. Positive post-treatment effects of OroA suggest that OroA is a potentially useful candidate for managing lung inflammation in LPS-induced endotoxemia and septic shock.     

Inhibiting toll-like receptor 4 signaling ameliorates pulmonary fibrosis during acute lung injury induced by lipopolysaccharide: an experimental study

Background

Toll-like receptor 4 (TLR4) is essential in lipopolysaccharide (LPS)-induced fibroblast activation and collagen secretion in vitro. However, its effects on the process of lung fibroblast activation and fibrosis initiation during LPS induced acute lung injury (ALI) remain unknown. The goal of the present study was to determine the effect of inhibiting TLR4 on LPS-induced ALI and fibrosis in vivo.

Methods

The ALI model was established by intraperitoneal injection of LPS in mice. TLR4-small hairpin RNA (shRNA) lentivirus was injected intravenously into the mice to inhibit TLR4 expression. mRNA and protein levels were detected by real-time PCR and Western-blot analysis, respectively. The contents of the C-terminal propeptide of type I procollagen (PICP) in bronchoalveolar lavage fluid (BALF) were detected by ELISA, and the degree of fibrosis was detected by van Gieson collagen staining, the hydroxyproline assay, and alpha smooth muscle actin (α-SMA) immunohistochemical staining.

Results

Overexpression of TLR4, type I procollagen, alpha-SMA, and p-AKT in murine pulmonary tissue after intraperitoneal injection of LPS at 72 hours and 28 days were detected. Moreover, the degree of fibrosis was shown to increase by ELISA analysis of PICP in BALF, van Gieson collagen staining, the hydroxyproline assay, and α-SMA immunohistochemical staining. All of these changes were alleviated by intravenous infection with TLR4-shRNA lentivirus.

Conclusions

Inhibiting TLR4 signaling could ameliorate fibrosis at the early stage of ALI induced by LPS.     

Inhibition of Pyk2 blocks lung inflammation and injury in a mouse model of acute lung injury

Background

Proline-rich tyrosine kinase 2 (Pyk2) is essential in neutrophil degranulation and chemotaxis in vitro. However, its effect on the process of lung inflammation and edema formation during LPS induced acute lung injury (ALI) remains unknown. The goal of the present study was to determine the effect of inhibiting Pyk2 on LPS-induced acute lung inflammation and injury in vivo.

Methods

C57BL6 mice were given either 10 mg/kg LPS or saline intratracheally. Inhibition of Pyk2 was effected by intraperitoneal administration TAT-Pyk2-CT 1 h before challenge. Bronchoalveolar lavage analysis of cell counts, lung histology and protein concentration in BAL were analyzed at 18 h after LPS treatment. KC and MIP-2 concentrations in BAL were measured by a mouse cytokine multiplex kit. The static lung compliance was determined by pressure-volume curve using a computer-controlled small animal ventilator. The extravasated Evans blue concentration in lung homogenate was determined spectrophotometrically.

Results

Intratracheal instillation of LPS induced significant neutrophil infiltration into the lung interstitium and alveolar space, which was attenuated by pre-treatment with TAT-Pyk2-CT. TAT-Pyk2-CT pretreatment also attenuated 1) myeloperoxidase content in lung tissues, 2) vascular leakage as measured by Evans blue dye extravasation in the lungs and the increase in protein concentration in bronchoalveolar lavage, and 3) the decrease in lung compliance. In each paradigm, treatment with control protein TAT-GFP had no blocking effect. By contrast, production of neutrophil chemokines MIP-2 and keratinocyte-derived chemokine in the bronchoalveolar lavage was not reduced by TAT-Pyk2-CT. Western blot analysis confirmed that tyrosine phosphorylation of Pyk2 in LPS-challenged lungs was reduced to control levels by TAT-Pyk2-CT pretreatment.

Conclusions

These results suggest that Pyk2 plays an important role in the development of acute lung injury in mice and that pharmacological inhibition of Pyk2 might provide a potential therapeutic strategy in the pretreatment for patients at imminent risk of developing acute lung injury.     

Anti-inflammatory effects of novel curcumin analogs in experimental acute lung injury

Background

Acute lung injury (ALI) and its most severe form acute respiratory distress syndrome (ARDS) have been the leading cause of morbidity and mortality in intensive care units (ICU). Currently, there is no effective pharmacological treatment for acute lung injury. Curcumin, extracted from turmeric, exhibits broad anti-inflammatory properties through down-regulating inflammatory cytokines. However, the instability of curcumin limits its clinical application.

Methods

A series of new curcumin analogs were synthesized and screened for their inhibitory effects on the production of TNF-α and IL-6 in mouse peritoneal macrophages by ELISA. The evaluation of stability and mechanism of active compounds was determined using UV-assay and Western Blot, respectively. In vivo, SD rats were pretreatment with c26 for seven days and then intratracheally injected with LPS to induce ALI. Pulmonary edema, protein concentration in BALF, injury of lung tissue, inflammatory cytokines in serum and BALF, inflammatory cell infiltration, inflammatory cytokines mRNA expression, and MAPKs phosphorylation were analyzed. We also measured the inflammatory gene expression in human pulmonary epithelial cells.

Results

In the study, we synthesized 30 curcumin analogs. The bioscreeening assay showed that most compounds inhibited LPS-induced production of TNF-α and IL-6. The active compounds, a17, a18, c9 and c26, exhibited their anti-inflammatory activity in a dose-dependent manner and exhibited greater stability than curcumin in vitro. Furthermore, the active compound c26 dose-dependently inhibited ERK phosphorylation. In vivo, LPS significantly increased protein concentration and number of inflammatory cells in BALF, pulmonary edema, pathological changes of lung tissue, inflammatory cytokines in serum and BALF, macrophage infiltration, inflammatory gene expression, and MAPKs phosphorylation . However, pretreatment with c26 attenuated the LPS induced increase through ERK pathway in vivo. Meanwhile, compound c26 reduced the LPS-induced inflammatory gene expression in human pulmonary epithelial cells.

Conclusions

These results suggest that the novel curcumin analog c26 has remarkable protective effects on LPS-induced ALI in rat. These effects may be related to its ability to suppress production of inflammatory cytokines through ERK pathway. Compound c26, with improved chemical stability and bioactivity, may have the potential to be further developed into an anti-inflammatory candidate for the prevention and treatment of ALI.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0199-1) contains supplementary material, which is available to authorized users.     

Acute Lung Injury and Acute Kidney Injury Are Established by Four Hours in Experimental Sepsis and Are Improved with Pre,but Not Post,Sepsis Administration of TNF-α Antibodies

Introduction

Acute kidney injury (AKI) and acute lung injury (ALI) are serious complications of sepsis. AKI is often viewed as a late complication of sepsis. Notably, the onset of AKI relative to ALI is unclear as routine measures of kidney function (BUN and creatinine) are insensitive and increase late. In this study, we hypothesized that AKI and ALI would occur simultaneously due to a shared pathophysiology (i.e., TNF-α mediated systemic inflammatory response syndrome [SIRS]), but that sensitive markers of kidney function would be required to identify AKI.

Methods

Sepsis was induced in adult male C57B/6 mice with 5 different one time doses of intraperitoneal (IP) endotoxin (LPS) (0.00001, 0.0001, 0.001, 0.01, or 0.25 mg) or cecal ligation and puncture (CLP). SIRS was assessed by serum proinflammatory cytokines (TNF-α, IL-1β, CXCL1, IL-6), ALI was assessed by lung inflammation (lung myeloperoxidase [MPO] activity), and AKI was assessed by serum creatinine, BUN, and glomerular filtration rate (GFR) (by FITC-labeled inulin clearance) at 4 hours. 20 µgs of TNF-α antibody (Ab) or vehicle were injected IP 2 hours before or 2 hours after IP LPS.

Results

Serum cytokines increased with all 5 doses of LPS; AKI and ALI were detected within 4 hours of IP LPS or CLP, using sensitive markers of GFR and lung inflammation, respectively. Notably, creatinine did not increase with any dose; BUN increased with 0.01 and 0.25 mg. Remarkably, GFR was reduced 50% in the 0.001 mg LPS dose, demonstrating that dramatic loss of kidney function can occur in sepsis without a change in BUN or creatinine. Prophylactic TNF-α Ab reduced serum cytokines, lung MPO activity, and BUN; however, post-sepsis administration had no effect.

Conclusions

ALI and AKI occur together early in the course of sepsis and TNF-α plays a role in the early pathogenesis of both.     

Protective effects of intratracheally administered quercetin on lipopolysaccharide-induced acute lung injury

Background

Acute respiratory distress syndrome (ARDS) can result in a life-threatening form of respiratory failure, and established, effective pharmacotherapies are therefore urgently required. Quercetin is one of the most common flavonoids found in fruits and vegetables, and has potent anti-inflammatory and anti-oxidant activities. Quercetin has been demonstrated to exhibit cytoprotective effects through the induction of heme oxygenase (HO)-1. Here, we investigated whether the intratracheal administration of quercetin could suppress lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice as well as the involvement of HO-1 in quercetin’s suppressive effects.

Methods

Mouse model of ALI were established by challenging intratracheally LPS. The wet lung-to-body weight ratio, matrix metalloproteinase (MMP)-9 activities, and pro-inflammatory cytokine productions, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in bronchoalveolar lavage fluid (BALF) were examined in ALI mice with or without quercetin pretreatment. We also examined the effects of quercetin on LPS stimulation in the mouse alveolar macrophage cell line, AMJ2-C11 cells.

Results

Intratracheal administration of quercetin decreased the wet lung-to-body weight ratio. Moreover, quercetin decreased MMP-9 activity and the production of pro-inflammatory cytokines in BALF cells activated by LPS in advance. We determined the expression of quercetin-induced HO-1 in mouse lung, e.g., alveolar macrophages (AMs), alveolar and bronchial epithelial cells. When AMJ2-C11 cells were cultured with quercetin, a marked suppression of LPS-induced pro-inflammatory cytokine production was observed. The cytoprotective effects were attenuated by the addition of the HO-1 inhibitor SnPP. These results indicated that quercetin suppressed LPS-induced lung inflammation, and that an HO-1-dependent pathway mediated these cytoprotective effects.

Conclusions

Our findings indicated that quercetin suppressed LPS-induced lung inflammation, and that an HO-1-dependent pathway mediated these cytoprotective effects. Intratracheal administration of quercetin will lead to new supportive strategies for cytoprotection in these serious lung conditions.     

Niacinamide mitigated the acute lung injury induced by phorbol myristate acetate in isolated rat's lungs

Background

Phorbol myristate acetate (PMA) is a strong neutrophil activator and has been used to induce acute lung injury (ALI). Niacinamide (NAC) is a compound of B complex. It exerts protective effects on the ALI caused by various challenges. The purpose was to evaluate the protective effects of niacinamide (NAC) on the PMA-induced ALI and associated changes.

Methods

The rat''s lungs were isolated in situ and perfused with constant flow. A total of 60 isolated lungs were randomized into 6 groups to received Vehicle (DMSO 100 μg/g), PMA 4 μg/g (lung weight), cotreated with NAC 0, 100, 200 and 400 mg/g (lung weight). There were 10 isolated lungs in each group. We measured the lung weight and parameters related to ALI. The pulmonary arterial pressure and capillary filtration coefficient (K_fc) were determined in isolated lungs. ATP (adenotriphosphate) and PARP [poly(adenosine diphophate-ribose) polymerase] contents in lung tissues were detected. Real-time PCR was employed to display the expression of inducible and endothelial NO synthases (iNOS and eNOS). The neutrophil-derived mediators in lung perfusate were determined.

Results

PMA caused increases in lung weight parameters. This agent produced pulmonary hypertension and increased microvascular permeability. It resulted in decrease in ATP and increase in PARP. The expression of iNOS and eNOS was upregulated following PMA. PMA increased the neutrophil-derived mediators. Pathological examination revealed lung edema and hemorrhage with inflammatory cell infiltration. Immunohistochemical stain disclosed the presence of iNOS-positive cells in macrophages and endothelial cells. These pathophysiological and biochemical changes were diminished by NAC treatment. The NAC effects were dose-dependent.

Conclusions

Our results suggest that neutrophil activation and release of neutrophil-derived mediators by PMA cause ALI and associated changes. NO production through the iNOS-producing cells plays a detrimental role in the PMA-induced lung injury. ATP is beneficial, while PARP plays a deteriorative effect on the PMA-induced ALI. NAC exerts protective effects on the inflammatory cascade leading to pulmonary injury. This B complex compound may be applied for clinical usage and therapeutic regimen.     

Low tidal volume protects pulmonary vasomotor function from “second-hit” injury in acute lung injury rats

Background

Sepsis could induce indirect acute lung injury(ALI), and pulmonary vasomotor dysfunction. While low tidal volume is advocated for treatment of ALI patients. However, there is no evidence for low tidal volume that it could mitigate pulmonary vasomotor dysfunction in indirect ALI. Our study is to evaluate whether low tidal volume ventilation could protect the pulmonary vascular function in indirect lipopolysaccharide (LPS) induced acute lung injury rats.

Methods

An indirect ALI rat model was induced by intravenous infusion of LPS. Thirty rats (n = 6 in each group) were randomly divided into (1)Control group; (2) ALI group; (3) LV group (tidal volume of 6mL/kg); (4) MV group (tidal volume of 12mL/kg); (5)VLV group (tidal volume of 3mL/kg). Mean arterial pressure and blood gas analysis were monitored every 2 hours throughout the experiment. Lung tissues and pulmonary artery rings were immediately harvested after the rats were bled to be killed to detect the contents of endothelin-1 (ET-1), endothelial nitric oxide synthase (eNOS) and TNF-α. Acetylcholine (Ache)-induced endothelium-dependent and sodium nitroprusside (SNP)-induced endothelium-independent relaxation of isolated pulmonary artery rings were measured by tensiometry.

Results

There was no difference within groups concerning blood pressure, PaCO₂ and SNP-induced endothelium-independent relaxation of pulmonary artery rings. Compared with MV group, LV group significantly reduced LPS-induced expression of ET-1 level (113.79 ± 7.33pg/mL vs. 152.52 ± 12.75pg/mL, P < 0.05) and TNF-α (3305.09 ± 334.29pg/mL vs.4144.07 ± 608.21pg/mL, P < 0.05), increased the expression of eNOS (IOD: 15032.05 ± 5925.07 vs. 11454.32 ± 6035.47, P < 0.05). While Ache (10^-7mol/L-10^-4mol/L)-induced vasodilatation was ameliorated 30% more in LV group than in MV group.

Conclusions

Low tidal volume could protect the pulmonary vasodilative function during indirect ALI by decreasing vasoconstrictor factors, increasing expressions of vasodilator factors in pulmonary endothelial cells, and inhibiting inflammation injuries.     

Sonic Hedgehog Signaling: Evidence for Its Protective Role in Endotoxin Induced Acute Lung Injury in Mouse Model

Objective

To investigate the protective role of the sonic hedgehog (SHH) signaling associated with a lipopolysaccharide (LPS)-induced acute lung injury (ALI) in a mouse model.

Methods

Male BALB/c mice were randomly divided into four groups: control, LPS, LPS-cyclopamine group and cyclopamine group. ALI was induced by LPS ip injection (5 mg/kg). The sonic hedgehog inhibitor cyclopamine (50 mg/kg) was given to the LPS-cyclopamine group at 30 min after LPS injection as well as normal mice as control. Lung injury was observed histologically in hematoxylin and eosin (HE) stained tissue sections, semi-quantified by lung tissue injury score, and the lung tissue mass alteration was measured by wet to dry weight ratio (W/D). mRNA expression levels of TNF-α, SHH, Patched (PTC) and GLI1 in lung tissue were studied with real time quantitative PCR (RT-PCR), while the protein expression of SHH and GLI1 was determined by western blot analysis.

Results

Lung tissue injury score, thickness of alveolar septa, W/D, and TNF-α mRNA expression levels were significantly higher in the ALI mice than the normal mice (P<0.05). The mRNA expression levels of SHH, PTC, and GLI1 in the ALI mice were significantly higher at 12h and 24h after LPS injection, but not at the 6h time point. Protein production of SHH and GLI1 at 6h, 12h, and 24h in the lungs of ALI mice significantly increased, in a time-dependent manner, compared with that in normal mice. Cyclopamine alone has no effect on pathological changes in normal mice. Intervention with cyclopamine in ALI mice led to a reduction in mRNA levels of SHH, PTC, and GLI1 as well as SHH and GLI1 protein levels; meanwhile, the pathological injury scores of lung tissues, thickness of alveolar septa, W/D, and mRNA expression levels of TNF-α increased compared with mice receiving LPS only.

Conclusion

The SHH signaling pathway was activated in response to LPS-induced ALI, and up-regulation of SHH expression could alleviate lung injury and be involved in the repair of injured lung tissue.     

Role of TLR4-p38 MAPK-Hsp27 signal pathway in LPS-induced pulmonary epithelial hyperpermeability

Background

The breakdown of alveolar barrier dysfunction contributes to Lipopolysaccharide stimulated pulmonary edema and acute lung injury. Actin cytoskeleton has been implicated to be critical in regulation of epithelial barrier. Here, we performed in vivo and in vitro study to investigate role of TLR4-p38 MAPK-Hsp27 signal pathway in LPS-induced ALI.

Methods

For in vivo studies, 6–8-week-old C57 mice were used, Bronchoalveolar lavage Fluid /Blood fluorescent ratio, wet-to-dry lung weight ratio, as well as protein concentrations and neutrophil cell counts in BALF were detected as either directly or indirectly indicators of pulmonary alveolar barrier dysfunction. And hematoxylin and eosin staining was performed to estimate pulmonary injury. The in vitro explorations of transepithelial permeability were achieved through transepithelial electrical resistance measurement and testing of FITC-Dextran transepithelial flux in A549. In addition, cytoskeletal rearrangement was tested through F-actin immunostaining. And SB203580 was used to inhibit p38 MAPK activation, while siRNA was administered to genetically knockdown specific protein.

Results

We showed that LPS triggered activation of p38 MAPK, rearrangement of cytoskeleton which resulted in severe epithelial hyperpermeability and lung edema. A549 pretreated with TLR4 siRNA、p38 MAPK siRNA and its inhibitor SB203580 displayed a lower permeability and fewer stress fibers formation after LPS stimulation, accompanied with lower phosphorylation level of p38 MAPK and Hsp27, which verified the involvement of TLR4-p38 MAPK-Hsp27 in LPS-evoked alveolar epithelial injury. Inhibition of p38 MAPK activity with SB203580 in vivo attenuated pulmonary edema formation and hyperpermeability in response to LPS.

Conclusions

Our study demonstrated that LPS increased alveolar epithelial permeability both in vitro and in vivo and that TLR4- p38 MAPK- Hsp27 signal pathway dependent actin remolding was involved in this process.

     

Efficacy and Safety of Inhaled Carbon Monoxide during Pulmonary Inflammation in Mice

Background

Pulmonary inflammation is a major contributor to morbidity in a variety of respiratory disorders, but treatment options are limited. Here we investigate the efficacy, safety and mechanism of action of low dose inhaled carbon monoxide (CO) using a mouse model of lipopolysaccharide (LPS)-induced pulmonary inflammation.

Methodology

Mice were exposed to 0–500 ppm inhaled CO for periods of up to 24 hours prior to and following intratracheal instillation of 10 ng LPS. Animals were sacrificed and assessed for intraalveolar neutrophil influx and cytokine levels, flow cytometric determination of neutrophil number and activation in blood, lung and lavage fluid samples, or neutrophil mobilisation from bone marrow.

Principal Findings

When administered for 24 hours both before and after LPS, inhaled CO of 100 ppm or more reduced intraalveolar neutrophil infiltration by 40–50%, although doses above 100 ppm were associated with either high carboxyhemoglobin, weight loss or reduced physical activity. This anti-inflammatory effect of CO did not require pre-exposure before induction of injury. 100 ppm CO exposure attenuated neutrophil sequestration within the pulmonary vasculature as well as LPS-induced neutrophilia at 6 hours after LPS, likely due to abrogation of neutrophil mobilisation from bone marrow. In contrast to such apparently beneficial effects, 100 ppm inhaled CO induced an increase in pulmonary barrier permeability as determined by lavage fluid protein content and translocation of labelled albumin from blood to the alveolar space.

Conclusions

Overall, these data confirm some protective role for inhaled CO during pulmonary inflammation, although this required a dose that produced carboxyhemoglobin values close to potentially toxic levels for humans, and increased lung permeability.     

Mesenchymal Stromal (Stem) Cell Therapy Fails to Improve Outcomes in Experimental Severe Influenza

Rationale

Severe influenza remains a major public health threat and is responsible for thousands of deaths annually. Increasing antiviral resistance and limited effectiveness of current therapies highlight the need for new approaches to influenza treatment. Extensive pre-clinical data have shown that mesenchymal stromal (stem) cell (MSC) therapy can induce anti-inflammatory effects and enhance repair of the injured lung. We hypothesized that MSC therapy would improve survival, dampen lung inflammation and decrease acute lung injury (ALI) in a murine model of severe influenza.

Methods

C57Bl/6 mice were infected with influenza A/PuertoRico/8/34 (mouse-adapted H1N1) or influenza A/Mexico/4108/2009 (swine-origin pandemic H1N1) and administered human or mouse MSCs via the tail vein, either pre- or post- infection. MSC efficacy was evaluated as both an independent and adjunctive treatment strategy in combination with the antiviral agent, oseltamivir. Weight loss and survival were monitored. Inflammatory cells, cytokine/chemokines (IFN-γ, CXCL10, CCL2 and CCL5) and markers of ALI (total protein and IgM), were measured in bronchoalveolar lavage fluid and lung parenchyma.

Results

Administration of murine MSCs or human MSCs in a prophylactic or therapeutic regimen failed to improve survival, decrease pulmonary inflammation/inflammatory cell counts or prevent ALI in influenza virus-infected mice. MSCs administered in combination with oseltamivir also failed to improve outcomes.

Conclusions

Despite similarities in the clinical presentation and pathobiology of ALI and severe influenza, our findings suggest that MSC therapy may not be effective for prevention and/or treatment of acute severe influenza.     

1α,25-Dihydroxyvitamin D3 Ameliorates Seawater Aspiration-Induced Acute Lung Injury via NF-κB and RhoA/Rho Kinase Pathways

Introduction

Inflammation and pulmonary edema are involved in the pathogenesis of seawater aspiration-induced acute lung injury (ALI). Although several studies have reported that 1α,25-Dihydroxyvitamin D₃ (calcitriol) suppresses inflammation, it has not been confirmed to be effective in seawater aspiration-induced ALI. Thus, we investigated the effect of calcitriol on seawater aspiration-induced ALI and explored the probable mechanism.

Methods

Male SD rats receiving different doses of calcitriol or not, underwent seawater instillation. Then lung samples were collected at 4 h for analysis. In addition, A549 cells and rat pulmonary microvascular endothelial cells (RPMVECs) were cultured with calcitriol or not and then stimulated with 25% seawater for 40 min. After these treatments, cells samples were collected for analysis.

Results

Results from real-time PCR showed that seawater stimulation up-regulated the expression of vitamin D receptor in lung tissues, A549 cells and RPMVECs. Seawater stimulation also activates NF-κB and RhoA/Rho kinase pathways. However, we found that pretreatment with calcitriol significantly inhibited the activation of NF-κB and RhoA/Rho kinase pathways. Meanwhile, treatment of calcitriol also improved lung histopathologic changes, reduced inflammation, lung edema and vascular leakage.

Conclusions

These results demonstrated that NF-κB and RhoA/Rho kinase pathways are critical in the development of lung inflammation and pulmonary edema and that treatment with calcitriol could ameliorate seawater aspiration-induced ALI, which was probably through the inhibition of NF-κB and RhoA/Rho kinase pathways.     

Creation of Lung-Targeted Dexamethasone Immunoliposome and Its Therapeutic Effect on Bleomycin-Induced Lung Injury in Rats

Objective

Acute lung injury (ALI), is a major cause of morbidity and mortality, which is routinely treated with the administration of systemic glucocorticoids. The current study investigated the distribution and therapeutic effect of a dexamethasone(DXM)-loaded immunoliposome (NLP) functionalized with pulmonary surfactant protein A (SP-A) antibody (SPA-DXM-NLP) in an animal model.

Methods

DXM-NLP was prepared using film dispersion combined with extrusion techniques. SP-A antibody was used as the lung targeting agent. Tissue distribution of SPA-DXM-NLP was investigated in liver, spleen, kidney and lung tissue. The efficacy of SPA-DXM-NLP against lung injury was assessed in a rat model of bleomycin-induced acute lung injury.

Results

The SPA-DXM-NLP complex was successfully synthesized and the particles were stable at 4°C. Pulmonary dexamethasone levels were 40 times higher with SPA-DXM-NLP than conventional dexamethasone injection. Administration of SPA-DXM-NLP significantly attenuated lung injury and inflammation, decreased incidence of infection, and increased survival in animal models.

Conclusions

The administration of SPA-DXM-NLP to animal models resulted in increased levels of DXM in the lungs, indicating active targeting. The efficacy against ALI of the immunoliposomes was shown to be superior to conventional dexamethasone administration. These results demonstrate the potential of actively targeted glucocorticoid therapy in the treatment of lung disease in clinical practice.     

Proteomic Profiling of Bronchoalveolar Lavage Fluid in Critically Ill Patients with Ventilator-Associated Pneumonia

Rationale

Ventilator-associated pneumonia (VAP) is a common complication in patients with acute lung injury (ALI) and can lead to increased morbidity and mortality. Identifying protein profiles specific to VAP in bronchoalveolar lavage fluid (BALF) may aid in earlier diagnosis, elucidate mechanisms of disease, and identify putative targets for therapeutic intervention.

Methods

BALF was obtained from 5 normal subjects and 30 ALI patients: 14 with VAP (VAP⁺) and 16 without VAP (VAP^–). Each sample underwent shotgun proteomic analysis based on tandem mass spectrometry. Differentially expressed proteins between the groups were identified using statistical methods based on spectral counting. Mechanisms of disease were explored using functional annotation and protein interaction network analysis. Supervised classification algorithms were implemented to discover a proteomic classifier for identifying critically ill patients with VAP.

Results

ALI patients had distinct BALF proteomic profiles compared to normal controls. Within the ALI group, we identified 76 differentially expressed proteins between VAP⁺ and VAP^–. Functional analysis of these proteins suggested activation of pro-inflammatory pathways during VAP. We identified and validated a limited proteomic signature that discriminated VAP⁺ from VAP^– patients comprised of three proteins: S100A8, lactotransferrin (LTF), and actinin 1 (ACTN1).

Conclusions

Combining proteomic with computational analyses is a powerful approach to study the BALF proteome during lung injury and development of VAP. This integrative methodology is a promising strategy to differentiate clinically relevant subsets of ALI patients, including those suffering from VAP.     

Mast Cell Stabilization Alleviates Acute Lung Injury after Orthotopic Autologous Liver Transplantation in Rats by Downregulating Inflammation

Background

Acute lung injury (ALI) is one of the most severe complications after orthotopic liver transplantation. Amplified inflammatory response after transplantation contributes to the process of ALI, but the mechanism underlying inflammation activation is not completely understood. We have demonstrated that mast cell stabilization attenuated inflammation and ALI in a rodent intestine ischemia/reperfusion model. We hypothesized that upregulation of inflammation triggered by mast cell activation may be involve in ALI after liver transplantation.

Methods

Adult male Sprague–Dawley rats received orthotopic autologous liver transplantation (OALT) and were executed 4, 8, 16, and 24 h after OALT. The rats were pretreated with the mast cell stabilizers cromolyn sodium or ketotifen 15 min before OALT and executed 8 h after OALT. Lung tissues and arterial blood were collected to evaluate lung injury_. β-hexosaminidase and mast cell tryptase levels were assessed to determine the activation of mast cells. Tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 in serum and lung tissue were analyzed by enzyme-linked immunosorbent assay. Nuclear factor-kappa B (NF-κB) p65 translocation was assessed by Western blot.

Results

The rats that underwent OALT exhibited severe pulmonary damage with a high wet-to-dry ratio, low partial pressure of oxygen, and low precursor surfactant protein C levels, which corresponded to the significant elevation of pro-inflammatory cytokines, β-hexosaminidase, and tryptase levels in serum and lung tissues. The severity of ALI progressed and maximized 8 h after OALT. Mast cell stabilization significantly inhibited the activation of mast cells, downregulated pro-inflammatory cytokine levels and translocation of NF-κB, and attenuated OALT-induced ALI.

Conclusions

Mast cell activation amplified inflammation and played an important role in the process of post-OALT related ALI.