The microRNA miR-17 regulates lung FoxA1 expression during lipopolysaccharide-induced acute lung injury

Acute lung injury (ALI) is a severe pulmonary disease that causes a high number of fatalities worldwide. Studies have shown that FoxA1 expression is upregulated during ALI and may play an important role in ALI by promoting the apoptosis of alveolar type II epithelial cells. However, the mechanism of FoxA1 overexpression in ALI is unclear. In this study, an in vivo murine model of ALI and alveolar type II epithelial cells injury was induced using lipopolysaccharide (LPS). LPS upregulated FoxA1 in the lung tissue of the in vivo ALI model and in LPS-challenged type II epithelial cells. In contrast, miR-17 was significantly downregulated in these models. After miR-17 antagomir injection, the expression of FoxA1 was significantly increased in ALI mice. MiR-17 mimics could significantly inhibit FoxA1 mRNA and protein expression, whereas the miR-17 inhibitor could significantly increase FoxA1 mRNA and protein expression in LPS-induced type II epithelial cells. Thus, our results suggest that the downregulation of miR-17 expression could lead to FoxA1 overexpression in ALI.     

Mucin1 relieves acute lung injury by inhibiting inflammation and oxidative stress

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a kind of diffuse inflammatory injury caused by various factors, characterized by respiratory distress and progressive hypoxemia. It is a common clinical critical illness. The aim of this study was to investigate the effect and mechanism of the Mucin1 (MUC1) gene and its recombinant protein on lipopolysaccharide (LPS)-induced ALI/ARDS. We cultured human alveolar epithelial cell line (BEAS-2B) and used MUC1 overexpression lentivirus to detect the effect of MUC1 gene on BEAS-2B cells. In addition, we used LPS to induce ALI/ARDS in C57/BL6 mice and use hematoxylin and eosin (H&E) staining to verify the effect of their modeling. Recombinant MUC1 protein was injected subcutaneously into mice. We examined the effect of MUC1 on ALI/ARDS in mice by detecting the expression of inflammatory factors and oxidative stress molecules in mouse lung tissue, bronchoalveolar lavage fluid (BALF) and serum. Overexpression of MUC1 effectively ameliorated LPS-induced damage to BEAS-2B cells. Results of H&E staining indicate that LPS successfully induced ALI/ARDS in mice and MUC1 attenuated lung injury. MUC1 also reduced the expression of inflammatory factors (IL-1β, TNF-α, IL-6 and IL-8) and oxidative stress levels in mice. In addition, LPS results in an increase in the activity of the TLR4/NF-κB signaling pathway in mice, whereas MUC1 decreased the expression of the TLR4/NF-κB signaling pathway. MUC1 inhibited the activity of TLR4/NF-κB signaling pathway and reduced the level of inflammation and oxidative stress in lung tissue of ALI mice.Key words: Mucin1, acute lung injury, inflammation, oxidative stress, TLR4/NF-κB     

Inhaled nitric oxide attenuates acute lung injury via inhibition of nuclear factor-kappa B and inflammation.

The effect of inhaled nitric oxide (NO) on inflammatory process in acute lung injury (ALI) is unclear. The aims of this study were to 1) examine whether inhaled NO affects the biochemical lung injury parameters and cellular inflammatory responses and 2) determine the effect of inhaled NO on the activation of nuclear factor-kappa B (NF-kappa B) in lipopolysaccharide (LPS)-induced ALI. Compared with saline controls, rabbits treated intravenously with LPS showed increases in total protein and lactate dehydrogenase in the bronchoalveolar lavage (BAL) fluid, indicating ALI. LPS-treated animals with NO inhalation (LPS-NO) showed significant decreases in these parameters. Neutrophil numbers in the BAL fluid, the activity of reactive oxygen species in BAL cells, and the levels of interleukin (IL)-1 beta and IL-8 in alveolar macrophages were increased in LPS-treated animals. In contrast, neutrophil numbers and these cellular activities were substantially decreased in LPS-NO animals, compared with LPS-treated animals. NF-kappa B activation in alveolar macrophages from LPS-treated animals was also markedly increased, whereas this activity was effectively blocked in LPS-NO animals. These results suggest that inhaled NO attenuates LPS-induced ALI and pulmonary inflammation. This attenuation may be associated with the inhibition of NF-kappa B activation.     

Downregulated microRNA-27b attenuates lipopolysaccharide-induced acute lung injury via activation of NF-E2-related factor 2 and inhibition of nuclear factor κB signaling pathway

Acute lung injury (ALI) is a life-threatening, diffuse heterogeneous lung injury characterized by acute onset, pulmonary edema, and respiratory failure. Lipopolysaccharide (LPS) is a leading cause for ALI and when administered to a mouse it induces a lung phenotype exhibiting some of the clinical characteristics of human ALI. This study focused on investigating whether microRNA-27b (miR-27b) affects ALI in a mouse model established by LPS-induction and to further explore the underlying mechanism. After model establishment, the mice were treated with miR-27b agomir, miR-27b antagomir, or D-ribofuranosylbenzimidazole (an inhibitor of nuclear factor-E2-related factor 2 [Nrf2]) to determine levels of miR-27b, Nrf2, nuclear factor kappa-light-chain-enhancer of activated B cells nuclear factor κB (NF-κB), p-NF-κB, and heme oxygenase-1 (HO-1). The levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) in bronchoalveolar lavage fluid (BALF) were determined. The results of luciferase activity suggested that Nrf2 was a target gene of miR-27b. It was indicated that the Nrf2 level decreased in lung tissues from ALI mice. The downregulation of miR-27b decreased the levels of IL-1β, IL-6, and TNF-α in BALF of ALI mice. Downregulated miR-27b increased Nrf2 level, thus enhancing HO-1 level along with reduction of NF-κB level as well as the extent of NF-κB phosphorylation in the lung tissues of the transfected mice. Pathological changes were ameliorated in LPS-reduced mice elicited by miR-27b inhibition. The results of this study demonstrate that downregulated miR-27b couldenhance Nrf2 and HO-1 expressions, inhibit NF-κB signaling pathway, which exerts a protective effect on LPS-induced ALI in mice.     

Beneficial effects of ApoA-I on LPS-induced acute lung injury and endotoxemia in mice

High density lipoprotein (HDL) binds lipopolysaccharide (LPS) and neutralizes its toxicity. The aim of our study was to investigate the effects of Apolipoprotein (ApoA-I), the major apolipoprotein of HDL, on LPS-induced acute lung injury (ALI) and endotoxemia. BALB/c mice were challenged with LPS, followed by ApoA-I or saline administration for 24h. The mice were then sacrificed and histopathological analysis of the lung was performed. We found that ApoA-I could attenuate LPS-induced acute lung injury and inflammation. To investigate the mechanisms, we measured tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) levels in the serum and bronchoalveolar lavage (BAL) fluid and found that ApoA-I could significantly inhibit LPS-induced increases in the IL-1beta and TNF-alpha levels in serum (P<0.05, respectively), as well as in the IL-1beta, TNF-alpha, and IL-6 levels in BAL fluid (P<0.01 and P<0.05, P<0.05, respectively). Moreover, we evaluated the effect of ApoA-I on the mortality of L-929 cells which were attacked by LPS-activated peritoneal macrophages. We found that ApoA-I could significantly inhibit the LPS-induced cell death in a dose-dependent fashion. Furthermore, we investigated in vivo the effects of ApoA-I on the mortality rate and survival time after LPS administration and found that ApoA-I significantly decreased the mortality (P<0.05) and increased the survival time (P<0.05). In summary, the results suggest that ApoA-I could effectively protect against LPS-induced endotoxemia and acute lung damage. The mechanism might be related to inhibition of inflammatory cytokine release from macrophages.     

Spred-2 Deficiency Exacerbates Lipopolysaccharide-Induced Acute Lung Inflammation in Mice

Background

Acute respiratory distress syndrome (ARDS) is a severe and life-threatening acute lung injury (ALI) that is caused by noxious stimuli and pathogens. ALI is characterized by marked acute inflammation with elevated alveolar cytokine levels. Mitogen-activated protein kinase (MAPK) pathways are involved in cytokine production, but the mechanisms that regulate these pathways remain poorly characterized. Here, we focused on the role of Sprouty-related EVH1-domain-containing protein (Spred)-2, a negative regulator of the Ras-Raf-extracellular signal-regulated kinase (ERK)-MAPK pathway, in lipopolysaccharide (LPS)-induced acute lung inflammation.

Methods

Wild-type (WT) mice and Spred-2^−/− mice were exposed to intratracheal LPS (50 µg in 50 µL PBS) to induce pulmonary inflammation. After LPS-injection, the lungs were harvested to assess leukocyte infiltration, cytokine and chemokine production, ERK-MAPK activation and immunopathology. For ex vivo experiments, alveolar macrophages were harvested from untreated WT and Spred-2^−/− mice and stimulated with LPS. In in vitro experiments, specific knock down of Spred-2 by siRNA or overexpression of Spred-2 by transfection with a plasmid encoding the Spred-2 sense sequence was introduced into murine RAW264.7 macrophage cells or MLE-12 lung epithelial cells.

Results

LPS-induced acute lung inflammation was significantly exacerbated in Spred-2^−/− mice compared with WT mice, as indicated by the numbers of infiltrating leukocytes, levels of alveolar TNF-α, CXCL2 and CCL2 in a later phase, and lung pathology. U0126, a selective MEK/ERK inhibitor, reduced the augmented LPS-induced inflammation in Spred-2^−/− mice. Specific knock down of Spred-2 augmented LPS-induced cytokine and chemokine responses in RAW264.7 cells and MLE-12 cells, whereas Spred-2 overexpression decreased this response in RAW264.7 cells.

Conclusions

The ERK-MAPK pathway is involved in LPS-induced acute lung inflammation. Spred-2 controls the development of LPS-induced lung inflammation by negatively regulating the ERK-MAPK pathway. Thus, Spred-2 may represent a therapeutic target for the treatment of ALI.     

Angiogenic growth factors in the pathophysiology of a murine model of acute lung injury

Capillary leakage and alveolar edema are hallmarks of acute lung injury (ALI). Neutrophils and serum macromolecules enter alveoli, promoting inflammation. Vascular endothelial growth factor (VEGF) causes plasma leakage in extrapulmonary vessels. Angiopoietin (Ang)-1 and -4 stabilize vessels, attenuating capillary leakage. We hypothesized that VEGF and Ang-1 and -4 modulate vessel leakage in the lung, contributing to the pathogenesis of ALI. We examined a murine model of lipopolysaccharide (LPS)-induced ALI. C57BL/6 and 129/J mice were studied at baseline and 24, 48, and 96 h after single or multiple doses of aerosolized LPS. Both strains exhibited time- and dose-dependent increases in inflammation and a deterioration of lung mechanics. Bronchoalveolar lavage (BAL) protein levels increased significantly, suggesting capillary leakage. Increased BAL neutrophil and total protein content correlated with time-dependent increased tissue VEGF and decreased Ang-1 and -4 levels, with peak VEGF and minimum Ang-1 and -4 expression after 96 h of LPS challenge. These data suggest that changes in the balance between VEGF and Ang-1 and -4 after LPS exposure may modulate neutrophil influx, protein leakage, and alveolar flooding during early ALI.     

Protectin DX promotes epithelial injury repair and inhibits fibroproliferation partly via ALX/PI3K signalling pathway

Acute respiratory distress syndrome/acute lung injury (ARDS/ALI) is histologically characterized by extensive alveolar barrier disruption and excessive fibroproliferation responses. Protectin DX (PDX) displays anti‐inflammatory and potent inflammation pro‐resolving actions. We sought to investigate whether PDX attenuates LPS (lipopolysaccharide)‐induced lung injury via modulating epithelial cell injury repair, apoptosis and fibroblasts activation. In vivo, PDX was administered intraperitoneally (IP) with 200 ng/per mouse after intratracheal injection of LPS, which remarkedly stimulated proliferation of type II alveolar epithelial cells (AT II cells), reduced the apoptosis of AT II cells, which attenuated lung injury induced by LPS. Moreover, primary type II alveolar cells were isolated and cultured to assess the effects of PDX on wound repair, apoptosis, proliferation and transdifferentiation in vitro. We also investigated the effects of PDX on primary rat lung fibroblast proliferation and myofibroblast differentiation. Our result suggests PDX promotes primary AT II cells wound closure by inducing the proliferation of AT II cells and reducing the apoptosis of AT II cells induced by LPS, and promotes AT II cells transdifferentiation. Furthermore, PDX inhibits transforming growth factor‐β₁ (TGF‐β₁) induced fibroproliferation, fibroblast collagen production and myofibroblast transformation. Furthermore, the effects of PDX on epithelial wound healing and proliferation, fibroblast proliferation and activation partly via the ALX/ PI3K signalling pathway. These data present identify a new mechanism of PDX which targets the airway epithelial cell and fibroproliferation are potential for treatment of ARDS/ALI.     

Thalidomide reduces lipopolysaccharide/zymosan-induced acute lung injury in rats

Pharmacological therapies targeting fulminant lung inflammation in acute lung injury (ALI) need to be improved. We evaluated the effect of thalidomide, a chemical modulating both acute and chronic inflammation, on ALI induced by intravenous administration of lipopolysaccharide (LPS) and zymosan in male Sprague-Dawley rats. Injection of LPS and zymosan induced significant lung inflammation, as evidenced by increased neutrophil sequestration in lung tissue as well as enhanced nitric oxide metabolite (NO _x ^– ) production in the serum and bronchoalveolar lavage (BAL) fluid. Lactate dehydrogenase (LDH) activity and protein concentration in BAL fluid were significantly increased after administration of LPS and zymosan. Pulmonary microvascular permeability was determined using the Evans blue retention method, which showed a significant increase in microvascular permeability after LPS and zymosan administration, indicating the development of ALI. Animals that received thalidomide (100 mg/kg) 2 h prior to LPS injection had significantly reduced pulmonary NO _x ^– production, pulmonary microvascular permeability, and LDH activity and protein concentration in BAL fluid. We therefore conclude that thalidomide ameliorates lung inflammation and reduces ALI induced by combined LPS and zymosan administration in rats.     

PCTR1 improves pulmonary edema fluid clearance through activating the sodium channel and lymphatic drainage in lipopolysaccharide-induced ARDS

Acute respiratory distress syndrome (ARDS) is a lethal clinical syndrome characterized by damage of the epithelial barriers and accumulation of pulmonary edema fluid. Protectin conjugates in tissue regeneration 1 (PCTR1), an endogenously produced lipid mediator, are believed to exert anti-inflammatory and pro-resolution effects. PCTR1 (1 µg/kg) was injected at 8 hr after lipopolysaccharide (LPS; 14 mg/kg) administration, and the rate of pulmonary fluid clearance was measured in live rats at 1 hr after PCTR1 treatment. The primary type II alveolar epithelial cells were cultured with PCTR1 (10 nmol/ml) and LPS (1 μg/ml) for 8 hr. PCTR1 effectively improved pulmonary fluid clearance and ameliorated morphological damage and reduced inflammation of lung tissue, as well as improved the survival rate in the LPS-induced acute lung injury (ALI) model. Moreover, PCTR1 markedly increased sodium channel expression as well as Na, K-ATPase expression and activity in vivo and in vitro. In addition, PCTR1i also upregulated the expression of LYVE-1 in vivo. Besides that, BOC-2, HK7, and LY294002 blocked the promoted effect of PCTR1 on pulmonary fluid clearance. Taken together, PCTR1 upregulates sodium channels' expression via activating the ALX/cAMP/P-Akt/Nedd4-2 pathway and increases Na, K-ATPase expression and activity to promote alveolar fluid clearance. Moreover, PCTR1 also promotes the expression of LYVE-1 to recover the lymphatic drainage resulting in the increase of lung interstitial fluid clearance. In summary, these results highlight a novel systematic mechanism for PCTR1 in pulmonary edema fluid clearance after ALI/ARDS, suggesting its potential role in a therapeutic approach for ALI/ARDS.     

Lipopolysaccharide-induced CCN1 production enhances interleukin-6 secretion in bronchial epithelial cells

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a clinical complication caused by primary or secondary lung injury, as well as by systemic inflammation. Researches regarding molecular pathophysiology of ALI/ARDS are immerging with an ultimate aim towards developing prognostic molecular biomarkers and molecule-based therapy. However, the molecular mechanisms concerning ALI/ARDS are still not completely understood. The purpose of the present study was to identify a crucial role of CCN1 in inflammatory microenvironment during ALI/ARDS and focus on a potential communication between CCN1 and interleukin-6 (IL-6) in the airway epithelial cells. Our data illustrated that the expression levels of CCN1 and IL-6 in bronchoalveolar lavage fluid (BALF) in a lipopolysaccharide (LPS)-induced ALI mouse model were significantly elevated and the pulmonary expression of CCN1 was restricted to bronchial epithelial cells. Interestingly, both endogenous and exogenous CCN1 stimulated IL-6 production in vitro. Furthermore, LPS-induced IL-6 production in a bronchial epithelial cell line was blocked by CCN siRNA whereas CCN1 induced by LPS was sensitive to PI3K inhibition. Together, our data indicate a linear signal pathway, LPS-CCN1-IL-6, existing in bronchial epithelial cells after LPS exposure. This finding may represent an additional mechanism and a novel target for development of therapy and biomarker on ALI/ARDS.     

MCTR1 enhances the resolution of lipopolysaccharide‐induced lung injury through STAT6‐mediated resident M2 alveolar macrophage polarization in mice

Acute respiratory distress syndrome (ARDS) is a fatal disease characterized by excessive infiltration of inflammatory cells. MCTR1 is an endogenously pro‐resolution lipid mediator. We tested the hypothesis that MCTR1 accelerates inflammation resolution through resident M2 alveolar macrophage polarization. The mice received MCTR1 via intraperitoneal administration 3 days after LPS stimulation, and then, the bronchoalveolar lavage (BAL) fluid was collected 24 hours later to measure the neutrophil numbers. Flow cytometry was used to sort the resident and recruited macrophages. Post‐treatment with MCTR1 offered dramatic benefits in the resolution phase of LPS‐induced lung injury, including decreased neutrophil numbers, reduced BAL fluid protein and albumin concentrations and reduced histological injury. In addition, the expression of the M2 markers Arg1, FIZZ1, Remlα, CD206 and Dectin‐1 was increased on resident macrophages in the LPS + MCTR1 group. Resident macrophage depletion abrogated the therapeutic effects of MCTR1, and reinjection of the sorted resident macrophages into the lung decreased neutrophil numbers. Finally, treatment with MCTR1 increased STAT6 phosphorylation. The STAT6 inhibitor AS1517499 abolished the beneficial effects of MCTR1. In conclusion, MCTR1 promotes resident M2 alveolar macrophage polarization via the STAT6 pathway to accelerate resolution of LPS‐induced lung injury.     

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.     

Wolf–Hirschhorn syndrome candidate 1 facilitates alveolar macrophage pyroptosis in sepsis-induced acute lung injury through NEK7-mediated NLRP3 inflammasome activation

Sepsis is a complex clinical syndrome with high incidence and mortality. Acute lung injury (ALI) is a common complication of sepsis. At present, there is no effective therapeutic strategy to treat ALI. The SET domain–containing histone methyltransferase Wolf–Hirschhorn syndrome candidate 1 (WHSC1) regulates cancer progression, while its role in sepsis-induced ALI remains unclear. Thus, this study aimed to study the effect of WHSC1 on sepsis-induced ALI and to explore the potential mechanism of action. In the study, LPS treatment induced lung injury. WHSC1 was highly expressed in LPS-induced ALI. Knockdown of WHSC1 attenuated LPS-induced ALI and pyroptosis in vivo. Besides, knockdown of WHSC1 attenuated LPS-induced alveolar macrophage pyroptosis in vitro. Furthermore, NIMA-related kinase-7 (NEK7) expression could be regulated by WHSC1, and NEK7 bound to NLRP3 in alveolar macrophages. Moreover, WHSC1 regulated alveolar macrophage pyroptosis through modulating NEK7-mediated NLRP3 inflammasome activation. In conclusion, WHSC1 was highly expressed in LPS-induced ALI. WHSC1 facilitated alveolar macrophage pyroptosis in sepsis-induced ALI through NEK7-mediated NLRP3 inflammasome activation. WHSC1 may be a valuable target for the therapy of sepsis-induced ALI.