Response In Acute Lung Injury Research Articles

Caspase-1-mediated extracellular vesicles derived from pyroptotic alveolar macrophages promote inflammation in acute lung injury.

The occurrence and development of acute lung injury (ALI) involve a variety of pathological factors and complex mechanisms. How pulmonary cells communicate with each other and subsequently trigger an inflammatory cascade remains elusive. Extracellular vesicles (EVs) are a critical class of membrane-bound structures that have been widely investigated for their roles in pathophysiological processes, especially in immune responses and tumor progression. Most of the current knowledge of the functions of EVs is related to functions derived from viable cells (e.g., microvesicles and exosomes) or apoptotic cells (e.g., apoptotic bodies); however, there is limited understanding of the rapidly progressing inflammatory response in ALI. Herein, a comprehensive analysis of micron-sized EVs revealed a mass production of 1-5 μm pyroptotic bodies (PyrBDs) release in the early phase of ALI induced by lipopolysaccharide (LPS). Alveolar macrophages were the main source of PyrBDs in the early phase of ALI, and the formation and release of PyrBDs were dependent on caspase-1. Furthermore, PyrBDs promoted the activation of epithelial cells, induced vascular leakage and recruited neutrophils through delivery of damage-associated molecular patterns (DAMPs). Collectively, these findings suggest that PyrBDs are mainly released by macrophages in a caspase-1-dependent manner and serve as mediators of LPS-induced ALI.

Two-photon ratiometric fluorescent probe for imaging of hypochlorous acid in acute lung injury and its remediation effect

Acute lung injury (ALI) is a pulmonary inflammatory disease with high morbidity and mortality rates. However, owing to the unknown etiology and rapid progression of the disease, the diagnosis of ALI is full of challenges with no effective treatment. Since the inflammatory response and oxidative stress played vital roles in the development of ALI, we herein developed the largest emission cross-shift (△λ = 145 nm) two-photon ratiometric fluorescent probe of TPRS-HOCl with high selectivity and short response time toward hypochlorous acid (HOCl) for exploring the relevance between the degree of ALI and HOCl concentration in the development process of the disease. In addition, the inhibition effect of HOCl during different treatment periods was also evaluated. Moreover, the tendency of imaging results was basically in accordance with that of hematoxylin and eosin (H&E) staining and the treatment effect became better in the early stage when using N-acetylcysteine (NAC), demonstrating the sensitivity of TPRS-HOCl toward ALI response. Thus, TPRS-HOCl has great potential to diagnose ALI in the early stage and guide for effective treatment.

MiR-494-3p alleviates acute lung injury through regulating NLRP3 activation by targeting CMPK2.

Acute lung injury (ALI) is a severe respiratory disorder with a high rate of mortality, and is characterized by excessive cell apoptosis and inflammation. MicroRNAs (miRNAs) play pivotal roles in ALI. This study examined the biological function of miR-494-3p in cell apoptosis and inflammatory response in ALI. For this, mice were injected with lipopolysaccharide (LPS) to generate an in-vivo model of ALI (ALI mice), and WI-38 cells were stimulated with lipopolysaccharide (LPS) to generate an in-vitro model of ALI. We found that miR-494-3p was significantly downregulated in the ALI mice and in the in-vitro model. Overexpression of miR-494-3p inhibited inflammation and cell apoptosis in the LPS-induced WI-38 cells, and improved the symptoms of lung injury in the ALI mice. We then identified cytidine/uridine monophosphate kinase 2 (CMPK2) as a novel target of miR-494-3p in the WI-38 cells. Furthermore, miR-494-3p suppressed cell apoptosis and the inflammatory response in LPS-treated WI-38 cells through targeting CMPK2. The NLRP3 inflammasome is reportedly responsible for the activation of inflammatory processes. In our study, CMPK2 was confirmed to activate the NLRP3 inflammasome in LPS-treated WI-38 cells. In conclusion, miR-494-3p attenuates ALI through inhibiting cell apoptosis and the inflammatory response by targeting CMPK2, which suggests the value of miR-494-3p as a target for the treatment for ALI.

MiR-150 and SRPK1 regulate AKT3 expression to participate in LPS-induced inflammatory response.

miR-150 was found to target the 3′-untranslated regions of AKT3, and the AKT pathway was affected by SR protein kinase 1 (SRPK1). However, the expression and significance of miR-150, AKT3 and SRPK1 in acute lung injury (ALI) were not clear. Here, we found that the expression of miR-150 was significantly reduced, while the expression of AKT3 and SRPK1 were markedly increased in LPS-treated A549, THP-1 and RAW 264.7 cells. miR-150 significantly decreased levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, reduced the expression of AKT3, but had no impact on SRPK1 expression compared with the control group in LPS-treated A549, THP-1 and RAW 264.7 cells. AKT3 silencing only reduced the production of pro-inflammatory cytokines and showed no effect on miR-150 and SRPK1 expression. Finally, we observed that miR-150 mimics and/or silencing of SRPK1 decreased the expression of AKT3 mRNA. Besides, over-expression of miR-150 or silencing of SRPK1 also reduced the expression of AKT3 protein, which exhibited the lowest level in the miR-150 mimics plus si-SRPK1 group. However, si-SRPK1 had no effect on miR-150 level. In conclusion, miR-150 and SRPK1 separately and cooperatively participate into inflammatory responses in ALI through regulating AKT3 pathway. Increased miR-150 and silenced SRPK1 may be a novel potential factor for preventing and treating more inflammatory lung diseases.

The mitochondrial redistribution of eNOS is involved in lipopolysaccharide induced inflammasome activation during acute lung injury

Acute lung injury (ALI) is a devastating clinical syndrome with no effective therapies. Inflammasome activation has been reported to play a critical role in the initiation and progression of ALI. The molecular mechanisms involved in regulating the activation of inflammasome in ALI remains unresolved, although increases in mitochondrial derived reactive oxygen species (mito-ROS) are involved. Our previous work has shown that the mitochondrial redistribution of uncoupled eNOS impairs mitochondrial bioenergetics and increases mito-ROS generation. Thus, the focus of our study was to determine if lipopolysaccharide (LPS)-mediated inflammasome activation involves the mitochondrial redistribution of uncoupled eNOS. Our data show that the increase in mito-ROS involved in LPS-mediated inflammasome activation is associated with the disruption of mitochondrial bioenergetics in human lung microvascular endothelial cells (HLMVEC) and the mitochondrial redistribution of eNOS. These effects are dependent on RhoA-ROCK signaling and are mediated via increased phosphorylation of eNOS at Threonine (T)-495. A derivative of the mitochondrial targeted Szeto‐Schiller peptide (SSP) attached to the antioxidant Tiron (T-SSP), significantly attenuated LPS-mediated mito-ROS generation and inflammasome activation in HLMVEC. Further, T-SSP attenuated mitochondrial superoxide production in a mouse model of sepsis induced ALI. This in turn significantly reduced the inflammatory response and attenuated lung injury. Thus, our findings show that the mitochondrial redistribution of uncoupled eNOS is intimately involved in the activation of the inflammatory response in ALI and implicate attenuating mito-ROS as a therapeutic strategy in humans.

MiR-23b Attenuates LPS-Induced Inflammatory Responses in Acute Lung Injury via Inhibition of HDAC2

Inflammatory responses play significant role in infectious etiology-induced acute lung injury (ALI). Histone deacetylase 2 is found to be essential and stimulated in lipopolysaccharide (LPS)-induced ALI by regulating proinflammatory cytokines. miR-23b has been demonstrated to be downregulated in LPS-induced inflammatory injury. In this study, we aimed to explore the interaction between miR-23b and HDAC2 and their function in LPS-induced ALI. LPS treatment was induced on murine alveolar macrophage cell line MH-S. Level of miR-23b and HDAC2 were determined by real-time PCR or Western blot. Proinflammatory cytokines expression and secretion were detected by real-time PCR and ELISA assay. The levels of miR-23b and HDAC2 were manipulated by transient transfection of miRNA mimics, shRNA or overexpression vector. The interaction between miR-23b and HDAC2 were tested by Luciferase reporter assay. LPS treatment inhibited miR-23b expression, while increased HDAC2 level in MH-S cells. Proinflammatory cytokines were stimulated by LPS treatment. Knockdown of HDAC2 or overexpression of miR-23b significantly repressed the expression of proinflammatory cytokines induced by LPS. miR-23b could suppress HDAC2 expression by directly targeting to its mRNA. LPS treatment stimulated the inflammatory responses in macrophages through inhibition of miR-23b, enhanced HDAC2 expression and inducing the expression of its downstream targets TNF-α, IL-6, and IL-1β. Overexpression of miR-23b was sufficient to suppress inflammatory responses by targeting HDAC2, making it a promising therapeutic target to ALI treatment.

Down-regulation of miR-let-7e attenuates LPS-induced acute lung injury in mice via inhibiting pulmonary inflammation by targeting SCOS1/NF-κB pathway.

Excessive pulmonary inflammatory response is critical in the development of acute lung injury (ALI). Previously, microRNAs (miRNAs) have been recognized as an important regulator of inflammation in various diseases. However, the effects and mechanisms of miRNAs on inflammatory response in ALI remain unclear. Herein, we tried to screen miRNAs in the processes of ALI and elucidate the potential mechanism. Using a microarray assay, microRNA let-7e (let-7e) was chose as our target for its reported suppressive roles in several inflammatory diseases. Down-regulation of let-7e by antagomiR-let-7e injection attenuated LPS-induced acute lung injury. We also found that antagomiR-let-7e could obviously improve the survival rate in ALI mice. Moreover, antagomiR-let-7e treatment reduced the production of proinflammatory cytokines (i.e., TNF-α, IL-1β and IL-6) in bronchoalveolar lavage fluid (BALF) of LPS-induced ALI mice. Luciferase reporter assays confirmed that suppressor of cytokine signaling 1 (SOCS1), a powerful attenuator of nuclear factor kappa B (NF-κB) signaling pathway, was directly targeted and suppressed by let-7e in RAW264.7 cells. In addition, it was further observed that SOCS1 was down-regulated, and inversely correlated with let-7e expression levels in lung tissues of ALI mice. Finally, down-regulation of let-7e suppressed the activation of NF-κB pathway, as evidenced by the reduction of p-IκBα, and nuclear p-p65 expressions in ALI mice. Collectively, our findings indicate that let-7e antagomir protects mice against LPS-induced lung injury via repressing the pulmonary inflammation though regulation of SOCS1/NF-κB pathway, and let-7e may act as a potential therapeutic target for ALI.

Activation of NLRP3 inflammasome up-regulates TREM-1 expression in murine macrophages via HMGB1 and IL-18

NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome and triggering receptor expressed on myeloid cells-1 (TREM-1) are considered critical orchestrators of the inflammatory response in acute lung injury (ALI). However, few assumptions are based on the relationship between them. Here, we investigated the effect of NLRP3 inflammasome activation on the TREM-1 expression in lipopolysaccharide (LPS)-induced ALI and macrophages. We found that inhibition of the NLRP3 inflammasome reduced the TREM-1 expression and pathological lung injury in mice with ALI. Then, primary murine macrophages were used to dissect the underlying mechanistic events of the activation NLRP3 inflammasome involved in the TREM-1 expression. Our results demonstrated that the conditioned medium (CM) from NLRP3 inflammasome-activated-macrophages up-regulated the TREM-1 expression in macrophages, while this effect was reversed by an NLRP3 inflammasome inhibitor MCC950. Furthermore, neutralizing antibodies anti-IL-18 and anti-HMGB1 reduced the TREM-1 expression induced by NLRP3 inflammasome activation. Mechanistically, we found that CM from NLRP3 inflammasome-activated-macrophages increased the level of inhibitor κB kinase protein phosphorylation (p-IκBα) and reactive oxygen species (ROS) content, and promoted IκBα protein degradation in macrophages. While the inhibition of nuclear factor kappa-B (NF-κB) and scavenging ROS eliminated the up-regulation of TREM-1 induced by the NLRP3 inflammasome activation in macrophages. In summary, our study confers NLRP3 inflammasome as a new trigger of TREM-1 signing, which allows additional insight into the pathological of the inflammatory response in ALI.

Protectin DX ameliorates inflammation in sepsis-induced acute lung injury through mediating PPARγ/NF-κB pathway.

Previous reports have demonstrated that the newly identified lipid mediator protectin DX (PDX) could effectively attenuate multiple organ injuries in sepsis. The aim of our study was to clarify whether PDX could improve acute lung injury (ALI) induced by sepsis and elucidate the relevant potential mechanism. After inducing sepsis by the cecal ligation and puncture approach, mice were treated with a high or low dose of PDX. Pathological changes in the pulmonary tissue were analyzed by hematoxylin-eosin staining, and lung injury score was evaluated. Lung permeability and edema were assessed by lung wet/dry ratio, and protein and cellular load of the bronchoalveolar lavage fluid (BALF). Inflammatory cytokine levels in BALF were measured by ELISA and the expression of PPARγ in the lung tissue was analyzed by immunoblotting. The results suggested that PDX could diminish the inflammatory response in lung tissue after sepsis by upregulating PPARγ and inhibiting the phosphorylation and activation of NF-κB p65. PDX treatment lowered the levels of pro-inflammation cytokines IL-1β, IL-6, TNF-α, and MCP-1, and the levels of anti-inflammatory cytokine IL-10 was increased in the BALF. It also improved lung permeability and reduced lung injury. Furthermore, the protective effect of PDX on lung tissue could be reversed by GW9662, a specific PPAR-γ antagonist. Taken together, our study indicated that PDX could ameliorate the inflammatory response in ALI by activating the PPARγ/NF-κB pathway in a mouse model of sepsis.

SB203580 protects against inflammatory response and lung injury in a mouse model of lipopolysaccharide‑induced acute lung injury.

Acute lung injury (ALI) is characterized by acute hypoxic respiratory failure, pulmonary edema and inflammatory infiltration. ALI has a high mortality rate (~30%) in the clinical setting; therefore, focusing on the treatment of lung edema and inflammatory responses in ALI is of significance. The present study investigated the effect of the p38 mitogen‑activated protein kinase (p38MAPK) inhibitor, SB203580, on lung edema and inflammatory responses in ALI invivo. A mouse model of ALI was established to assess the effect of SB203580 on edema, proinflammatory cytokine production, and the expression of interferon regulatory factor 5 (IRF5) and inducible nitric oxide synthase (iNOS) in lung tissues using immunoblotting, immunohistochemistry, immunofluorescence, hematoxylin and eosin staining, and ELISA. SB203580 inhibited LPS‑induced lung injury and proinflammatory cytokine expression, including tumor necrosis factor‑α and interleukin‑1β. SB203580 also downregulated LPS‑induced IRF5 and iNOS expression, which are widely used as markers of proinflammatory macrophages. Collectively, the present study demonstrated that SB203580 protected against inflammatory responses and lung injury by inhibiting lung edema and downregulating proinflammatory mediators in LPS‑induced lung injury.

MicroRNA‑17 contributes to the suppression of the inflammatory response in lipopolysaccharide‑induced acute lung injury in mice via targeting the toll‑like receptor 4/nuclear factor‑κB pathway.

Acute lung injury (ALI) is a common lung disease with a high mortality rate, which is characterized by an excessive uncontrolled inflammatory response. MicroRNA (miR)-17 has previously emerged as a novel regulatory molecule of inflammatory response in various complex diseases; however, the anti-inflammatory action and associated molecular mechanisms of miR-17 in ALI have not been fully elucidated. The aim of the present study was to investigate the role of miR-17 in the inflammatory response in ALI and to elucidate the potential underlying mechanism. Using a lipopolysaccharide (LPS)-induced ALI mouse model, it was observed that miR-17 was significantly downregulated in lung tissues compared with the control group. In this model, ectopic expression of miR-17 attenuated lung pathological damage, reduced lung wet/dry ratio and lung permeability, and increased survival rate in ALI mice. In addition, agomiR-17 injection significantly suppressed LPS-induced inflammation, as evidenced by a reduction in the activity of myeloperoxidase and the production of interleukin (IL)-6, IL-1β and tumor necrosis factor-α in lung tissues. Of note, toll-like receptor (TLR) 4, an upstream regulator of the nuclear factor (NF)-κB inflammatory signaling pathway, was directly targeted by miR-17, and its translation was suppressed by miR-17 in vitro and in vivo. Using an LPS-induced RAW264.1 macrophage injury model, it was observed that miR-17 overexpression suppressed the pro-inflammatory effect of LPS, while these inhibitory effects were markedly abrogated by TLR4 overexpression. In addition, TLR4 knockdown by si-TLR4 mimicked the effects of miR-17 overexpression on LPS-induced cytokine secretion in the in vitro model. Further experiments revealed that miR-17 significantly reduced the expression of key proteins in the NF-κB pathway, including IKKβ, p-IκBα and nuclear p-p65, and suppressed the NF-κB activity in ALI mice. Collectively, these results indicated that miR-17 protected mice against LPS-induced lung injury via inhibiting inflammation by targeting the TLR4/NF-κB pathway; therefore, miR-17 may serve as a potential therapeutic target for ALI.

Regulatory Role of STAT3-PLSCR2 Axis in Antiviral and Inflammatory Response

Abstract Cytokines are key factors for initiating inflammatory response and among them IFN-I is one of the most versatile because of its dual roles in antiviral and inflammatory activities. While STAT1 and STAT2 are critical for triggering antiviral response, the signaling events downstream of IFN-I receptor contributing to inflammation have yet to be defined. Here we demonstrate that STAT3 and one of its interacting partners PLSCR2 negatively regulate IFN-I-mediated antiviral and inflammatory response. Enhanced expression of IFN-I-stimulated genes (ISGs) is found in cells either absence of STAT3 or PLSCR2, leading to increased resistance to viral infection in vitro and in vivo. Moreover, STAT3 can cooperate with PLSCR2 to suppress IFN-I response. Interestingly, GSEA analysis for IFN-I-stimulated cells lacking PLSCR2 versus WT control displays an inflammatory signature with increased expression of inflammatory cytokines and chemokines, their receptors and TLRs. Stimulation of TLR ligands, including LPS, R848 or CpG, also results in enhanced expression of IFN-I and inflammatory cytokines and chemokines in PLSCR2KO cells as opposed to WT control. PLSCR2KO mice are also more susceptible to LPS-induced endotoxic shock in vivo with increased serum TFN. Similarly, PLSCR2KO mice also display increased acute lung injury in response to intratracheal administration of LPS with increased infiltration of macrophages and neutrophils and production of inflammatory cytokines and chemokines. Enhanced TLR response is also found in STAT3KO BM-derived macrophages. Therefore, these results demonstrate that STAT3 coordinates with PLSCR2 to regulate antiviral and inflammatory response and define a novel axis in modulating IFN-I-mediated response.

MiR-342 attenuates lipopolysaccharide-induced acute lung injury via inhibiting MAPK1 expression.

Micro RNA (miRNA) and mitogen-activated protein kinase (MAPK) are reported as the crucial regulators of inflammatory responses in acute lung injury (ALI). This study will explore the role of the miR-342/MAPK1 axis in regulation of lipopolysaccharide (LPS)-induced ALI. We found that miR-342 was down-regulated in LPS-induced A549 cells compared with the control group with DMSO, accompanied by elevated inflammatory cytokines and apoptosis. Over-expression of miR-342 reduced LPS-induced inflammatory responses and apoptosis in LPS-stimulated A549 cells, and had a protective role in LPS-treated mice with ALI by decreasing levels of inflammatory cytokines, improving survival of mice with ALI, and ameliorating the lung permeability. Dual-luciferase reporter gene assay demonstrated that miR-342 regulated the expression of MAPK1 by directly targeting its 3' untranslated region (3'-UTR). Mechanistically, MAPK1 silencing abrogated LPS-induced inflammatory injury in A549 cells, and partially enhanced the protective effect of miR-342. Therefore, miR-342 attenuates LPS-induced ALI by targeting MAPK1 expression, thereby protecting against A549 cell injury induced by LPS and lung injury of mice with ALI.

HMGB1‐induced vascular hyperpermeability requires β‐catenin phosphorylation

Endothelial hyperpermeability is a hallmark of acute lung injury in response to sepsis. The imbalance between adherence junction (AJ) mediated cell‐cell adherence forces and stress fiber driven contractile forces contributes to increased endothelial permeability. Here, we spotlight the effects of β‐catenin Y654 andY142 phosphorylation on HMGB1‐mediated endothelial barrier leakage. Our results showed that phospho‐deficiencies at both β‐catenin Y654 and Y142 ameliorated pulmonary vascular dysfunction in male C57 mice receiving a cecal ligation and puncture operation. In vitro analysis indicated that high mobility group box‐1 protein (HMGB1) triggered β‐catenin Y654 and Y142 phosphorylation, causing β‐catenin translocation and adherence junction (AJ) disruptions as well as cytoskeleton rearrangement. In addition, β‐catenin Y654 dephosphorylation attenuated HMGB1‐mediated dissociation of VE‐cadherin/β‐catenin and, hence, partially prevented endothelial hyperpermeability. β‐catenin Y142 dephosphorylation abolished HMGB1‐induced uncoupling of β‐catenin and α‐catenin, suppressed cytoskeletal reassembly and, hence, alleviated endothelial hyperpermeability. Further investigation demonstrated that RAGE and Src were required for β‐catenin Y654 phosphorylation in response to HMGB1, while FAK was responsible for HMGB1‐triggered β‐catenin Y142 phosphorylation. In sum, this study revealed the role of β‐catenin Y654 and Y142 phosphorylation in HMGB1‐mediated endothelial hyperpermeability through dysregulation between adherence and contractile forces. This result advances understanding of the mechanisms underlying pulmonary vascular hyperpermeability in sepsis.Support or Funding InformationThis work was supported by National Natural Science Foundation of China (81670076, 31871183); Guangdong Natural Science Foundation (2017A030313462); General Program from Natural Science Foundation of China (81370226, 81170297); The Team project of Natural Science Foundation of Guangdong, China (S2013030013217); Grant of NSFC Guangdong Joint Foundation of China (U1601225); Key Scientific and Technological Program of Guangzhou City (201607020016); Special Funds for the Cultivation of Guangdong College Students’ Scientific and Technological Innovation [pdjh2017a0095].

Marrow-derived mesenchymal stem cells regulate the inflammatory response and repair alveolar type II epithelial cells in acute lung injury of rats.

ObjectiveWe investigated the effect of untransplantable bone marrow-derived mesenchymal stem cells (BMSCs) in acute lung injury (ALI) and whether BMSCs attenuate damage of lipopolysaccharide (LPS) to alveolar type II epithelial cells (AECIIs).MethodsALI models were prepared by nebulizing LPS and then BMSCs were infused 1 hour later. We observed histopathological changes of lung tissue and evaluated inflammatory exudation by the wet/dry weight ratio, bronchiolar lavage fluid cell count, and protein concentration determination. Inflammatory and vascular factors were detected by immunohistochemistry and western blotting. For in vitro experiments, AECIIs were stimulated with 10 μg/mL LPS for 4 hours and then BMSCs were seeded in transit inserts to co-culture for 24 hours. The activity of AECIIs was detected.ResultsIn the LPS + BMSCs group, histopathological examination showed that the degree of lung injury was significantly reduced compared with the LPS group. Protein expression of inflammatory and vascular factors was significantly lower with treatment. Optical density values and cell viability of the LPS + BMSCs group were significantly higher than those of the LPS group.ConclusionsUntransplanted-BMSCs can inhibit the inflammatory response in ALI and promote repair of AECIIs. This might be due to substances secreted by BMSCs and interaction between these substances.

A sesquiterpenoid from Ligularia pleurocaulis modulated macrophages polarisation through TLR4 pathway

An eremophilane-type sesquiterpenoid (EPS), 3-oxo-eremophila-1,7(11)-dien-12,8β-olide, has been isolated from anti-inflammatory folk herbs, Ligularia pleurocaulis. The aim of present study is to explore protective effects of EPS on lipopolysaccharide (LPS)-induced inflammatory responses in acute lung injury (ALI). EPS treatments (40 and 80 mg/kg) significantly ameliorated LPS-stimulated pathological changes in lungs. Furthermore, in vivo and in vitro mechanism studies suggest that EPS exerts its protective effects on LPS-induced ALI by regulating macrophage polarisation via suppression of TLR4/MyD88-mediated MAPK and NF-κB signaling pathways, and EPS may be useful for the prevention on ALI in the clinical setting.

A COX-2/sEH dual inhibitor PTUPB alleviates lipopolysaccharide-induced acute lung injury in mice by inhibiting NLRP3 inflammasome activation.

Rationale: Dysregulation of arachidonic acid (ARA) metabolism results in inflammation; however, its role in acute lung injury (ALI) remains elusive. In this study, we addressed the role of dysregulated ARA metabolism in cytochromes P450 (CYPs) /cyclooxygenase-2 (COX-2) pathways in the pathogenesis of lipopolysaccharide (LPS)-induced ALI in mice.Methods: The metabolism of CYPs/COX-2-derived ARA in the lungs of LPS-induced ALI was investigated in C57BL/6 mice. The COX-2/sEH dual inhibitor PTUPB was used to establish the function of CYPs/COX-2 dysregulation in ALI. Primary murine macrophages were used to evaluate the underlying mechanism of PTUPB involved in the activation of NLRP3 inflammasome in vitro.Results: Dysregulation of CYPs/COX-2 metabolism of ARA occurred in the lungs and in primary macrophages under the LPS challenge. Decrease mRNA expression of Cyp2j9, Cyp2j6, and Cyp2j5 was observed, which metabolize ARA into epoxyeicosatrienoic acids (EETs). The expressions of COX-2 and soluble epoxide hydrolase (sEH), on the other hand, was significantly upregulated. Pre-treatment with the dual COX-2 and sEH inhibitor, PTUPB, attenuated the pathological injury of lung tissues and reduced the infiltration of inflammatory cells. Furthermore, PTUPB decreased the pro-inflammatory factors, oxidative stress, and activation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome in LPS-induced ALI mice. PTUPB pre-treatment remarkably reduced the activation of macrophages and NLRP3 inflammasome in vitro. Significantly, both preventive and therapeutic treatment with PTUPB improved the survival rate of mice receiving a lethal dose of LPS.Conclusion: The dysregulation of CYPs/COX-2 metabolized ARA contributes to the uncontrolled inflammatory response in ALI. The dual COX-2 and sEH inhibitor PTUPB exerts anti-inflammatory effects in treating ALI by inhibiting the NLRP3 inflammasome activation.

Keratinocyte Growth Factor-2 Reduces Inflammatory Response to Acute Lung Injury Induced by Oleic Acid in Rats by Regulating Key Proteins of the Wnt/β-Catenin Signaling Pathway.

Reducing inflammation can effectively relieve acute lung injury (ALI). Objective. To test whether keratinocyte growth factor-2 (KGF-2) can reduce oleic acid-induced inflammation in ALI of rats and explore its possible mechanism. Methods. 45 Sprague-Dawley rats were randomly divided into control group, ALI group, and ALI + KGF-2 group. The animal model of acute lung injury was established by injecting 0.1 mL/kg oleic acid into the tail vein of rats. Rats in the control group were injected with equal volume of normal saline (NS). Each group needs pretreatment 72 hours before the preparation of the acute lung injury model. The control group and ALI group were instilled with 5 ml/kg NS through the airway, and the same amount of KGF-2 was instilled in the ALI + KGF-2 group. It takes 8 hours to successfully prepare the ALI model. Observe the pathological changes of lung tissue through light microscopy, ultrastructural changes through electron microscopy, and the lung wettability/dry weight (w/d) ratio and lung permeability index (LPI). By detecting changes in inflammatory factors in lung tissue and changes in the number of BALF cells, the changes in inflammation in each group were observed. The expressions of Wnt5a, β-catenin, and APC in lung tissue were detected by immunohistochemistry and Western blot. The changes of key proteins in Wnt/β-catenin signaling pathway in the lung tissue of each group were observed. Result. Compared with the ALI group, after KGF-2 pretreatment, the degree of lung injury was reduced, the expression of inflammatory factors was reduced, and the number of red blood cells and white blood cells in BALF was reduced. It can also be observed that the expression of Wnt5a, β-catenin, and APC, a key protein in the Wnt/β-catenin signaling pathway, is reduced. The analysis showed that the number of inflammatory factors, red blood cells, and white blood cells in BALF was positively correlated with the expression of Wnt5a, β-catenin, and APC. Conclusion. KGF-2 may reduce the inflammatory response in ALI induced by oleic acid by regulating key proteins in the Wnt/β-catenin signaling pathway.

HMGB1 participates in LPS‑induced acute lung injury byactivating the AIM2 inflammasome in macrophagesand inducing polarization of M1 macrophages via TLR2, TLR4, and RAGE/NF‑κB signaling pathways.

High mobility group box 1 (HMGB1), a crucial proinflammatory cytokine, was reported to activate the absent in melanoma 2 (AIM2) inflammasome, which are both essential in acute lung injury (ALI). However, their interaction mechanism has remained elusive. Macrophages are known to express the AIM2 inflammasome and the main receptors [receptor for advanced glycation end products (RAGE), Toll-like receptor 2/4 (TLR-2/TLR-4)] of HMGB1 to transmit intracellular signals. The present study aimed to indicate whether HMGB1 participates in the process of lipopolysaccharides (LPS)-induced ALI through activating the AIM2 inflammasome in macrophages, as well as inducing polarization of M1 macrophages via TLR2, TLR4 and RAGE/nuclear factor-κB (NF-κB) signaling pathways. In an in vivo mouse model of LPS-induced ALI, anti-HMGB1, recombinant (r) HMGB1, LPS from Rhodobacter sphaeroides (LPS-RS, TLR2/4 antagonist) or FPS-ZM1 (RAGE antagonist) were administrated. In in vitro studies, bone marrow-derived macrophages from mice primed with LPS were stimulated with or without anti-HMGB1, rHMGB1, LPS-RS, or FPS-ZM1. The findings revealed that anti-HMGB1, LPS-RS and FPS-ZM1 significantly decreased infiltration of inflamematory cells, wet-to-dry ratio, myeloperoxidase activity in the lung, the levels of cytokines, as well as macrophages and neutrophil infiltration in the bronchoalveolar lavage fluid. However, rHMGB1 aggravated the inflammatory response in ALI. Mechanistically, anti-HMGB1, LPS-RS and FPS-ZM1 attenuated activation of TLR2, TLR4, and RAGE/NF-κB signaling pathways and expression of the AIM2 inflammasome in macrophages. However, rHMGB1 enhanced their expression levels and induced polarization of M1 macrophages. These results indicated that HMGB1 could participate in the pathogenesis of ALI by activating the AIM2 inflammasome in macrophages, as well as inducing polarization of M1 macrophages through TLR2, TLR4 and RAGE/NF-κB signaling pathways.

Suppression of NLRP3 and NF-κB signaling pathways by α-Cyperone via activating SIRT1 contributes to attenuation of LPS-induced acute lung injury in mice

α-Cyperone is the volatile oil component in Cyperus rotundus L. The previous reports had shown the inhibition of α-Cyperone on NF-κB signaling in LPS-stimulated RAW264.7 cells. However, it is still unclear whether α-Cyperone could suppress inflammatory response in acute lung injury (ALI) induced by LPS. The aim of this study is to investigate the suppression of α-Cyperone on LPS-induced ALI in mice. In this study, we established the LPS-induced ALI model and compared different doses of α-Cyperone with the control group and LPS group. Accordingly, the following indexes would be compared, including lung wet/dry ratio, MPO activity, inflammatory cell number, histopathological changes, levels of inflammatory cytokines, NF-κB and NLRP3 signaling pathways activation. The results demonstrated that α-Cyperone had the effect on reducing the wet/dry ratio and MPO activity. Furthermore, the increase of inflammatory cells and inflammatory cytokines could be inhibited by α-Cyperone. Meanwhile, α-Cyperone could downregulate NF-κB and NLRP3 signaling pathways. Finally, we found α-Cyperone could up-regulate the expression of SIRT1 and SIRT1 inhibitor could reverse the protective effects of α-Cyperone on ALI. In conclusion, α-Cyperone showed the protective effect on LPS-induced ALI in mice by suppressing the NF-κB and NLRP3 signaling pathways, mainly via up-regulating SIRT1. This provides a potential drug for the treatment of LPS-induced ALI.