Inflammatory Response In Acute Lung Injury Research Articles

Vitamin D Binding Protein: A Potential Factor in Geriatric COVID-19 Acute Lung Injury.

Previous research indicated that vitamin D binding protein (VDBP) is an independent multifunctional protein that plays a vital role in acute inflammatory and tissue damage. However, its role in acute lung injury (ALI) due to coronavirus disease 2019 (COVID-19) is unclear, and studies are lacking. This study intends to investigate the difference in serum VDBP levels in COVID-19 patients with ALI or without ALI and further explore the role of VDBP in the inflammatory response of ALI through cellular models. The serum was collected from COVID-19 patients, and the concentration of serum VDBP was detected. Construct a VDBP gene-silencing plasmid and transfect it into human alveolar epithelial A549 cells. After 72 hours of lipopolysaccharide (LPS) intervention, The inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were detected, and cell counting kit-8 (CCK-8) assay was used to detect cell viability. Flow cytometry was used to detect cell apoptosis. The serum concentration of VDBP was significantly higher in COVID-19 with ALI (P < 0.05). Correlation analysis indicated serum VDBP positively correlated with leukocyte (r=0.329, P = 0.002), c-reaction protein (r = 0.470, P < 0.001), serum amyloid A (r = 0.900, P < 0.001), procalcitonin (r = 0.670, P < 0.001), and interleukin6 (r = 0.452, P < 0.001). Simultaneously, the logistic regression analysis showed that increased serum VDBP was an independent risk factor for ALI in COVID-19 patients (OR 1.003 95% CI 1.001-1.006, P = 0.002). In human alveolar epithelial A549 cells, after LPS intervention, the inflammatory factor IL-1β and TNF-A significantly reduced in the VDBP gene silencing group compared to the negative control (NC) group (P < 0.05). The cell viability of the VDBP gene silencing group was significantly increased compared to the NC group, and the cell apoptosis rate was significantly reduced (P < 0.05). In COVID-19 patients, acute lung injury may lead to increased serum concentration of VDBP. VDBP plays a vital role in promoting inflammatory response and apoptosis of bronchial epithelial cells.

Impact of lipopolysaccharide-induced acute lung injury in aged mice

Study Aim: As the geriatric population rapidly expands, there has been a concurrent increase in elderly admissions to intensive care units (ICUs). Acute lung injury (ALI) is a prevalent reason for these admissions and carries poorer survival rates for the aged population compared to younger counterparts. The aging lung is subject to physiological, cellular, and immunological changes. However, our understanding of how aging impacts the clinical progression of ALI is limited. This study explored the effect of aging using a murine model of ALI. Methods: Female C57BL/6J mice, aged 7–8 wk (young) and 18 months (aged), were divided into four groups: young controls, aged controls, young with ALI (YL), and aged with ALI (AL). ALI was induced via intratracheal administration of lipopolysaccharide (LPS, 0.5 mg/kg). The animals were euthanized 72 h after LPS exposure. Results: The AL group exhibited a significantly increased wet/dry ratio compared to the other three groups, including the YL group. The bronchoalveolar lavage (BAL) fluid in the AL group had more cells overall, including more neutrophils, than the other groups. Inflammatory cytokines in BAL fluid showed similar trends. Histological analyses demonstrated more severe lung injury and fibrosis in the AL group than in the other groups. Increased transcription of senescence-associated secretory phenotype markers, including PAI-1 and MUC5B, was more prominent in the AL group than in the other groups. This trend was also observed in BAL samples from humans with pneumonia. Conclusions: Aging may amplify lung damage and inflammatory responses in ALI. This suggests that physicians should exercise increased caution in the clinical management of aged patients with ALI.

LncRNA NEAT1: A novel regulator associated with the inflammatory response in acute respiratory distress syndrome

BackgroundAcute respiratory distress syndrome (ARDS) is a life-threatening condition with an unfavorable prognosis. As the pathogenesis of ARDS remains unclear, we aimed to identify the core genes associated with ARDS and the mechanisms by which competing endogenous RNAs (ceRNAs) regulate the disease’s progression. MethodsThree mRNA microarray datasets (GSE17355, GSE48787, and GSE130936), derived from the Gene Expression Omnibus (GEO) database, were selected. Common differentially expressed genes (DEGs) related to acute lung injury (ALI) were identified and subjected to enrichment analysis. Then, hub genes were figured out through the protein–protein interaction (PPI) network and functional analysis, and targeted miRNAs and lncRNAs were predicted. Finally, the ceRNA networks associated with ALI were constructed and validated experimentally. ResultsA total of 155 upregulated and 93 downregulated DEGs were identified in the three datasets. The TNF signaling pathway and IL-17 signaling pathway were the most enriched pathways. Then, eleven DEGs enriched in the IL-17 signaling pathway were selected as the hub genes. Three miRNAs (mmu-mir-155-5p, mmu-mir-21a-5p, and mmu-mir-122-5p), which were located in the lung tissue and predicted to bind the hub genes at the same time, and two lncRNAs (Neat1 and Tug1), which have binding sites for the aforementioned miRNAs, were filtered. With qPCR verification, we identified a ceRNA network composed of NEAT1, miR-21-5p, MMP9, and CXCL5. NEAT1 knockdown promoted the migration and reduced the expression of pro-inflammatory factor and reactive oxygen species (ROS) in lung epithelial cells. We eventually confirmed that NEAT1/miR-21-5p/CXCL5/MMP9 played a pivotal role in regulating the inflammatory response in ALI. ConclusionThe IL-17 signaling pathway is of great importance in the pathogenesis of ARDS. NEAT1/miR-21-5p is involved in the inflammation of ALI by regulating CXCL5 and MMP9.

Electroacupuncture Preconditioning Alleviates Lipopolysaccharides-Induced Acute Lung Injury by Downregulating LC3-II/I and Beclin 1 Expression.

Our study aimed to investigate the effect of electroacupuncture pretreatment on the inflammatory response and expression levels of LC3-II/I and Beclin 1 using a model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Eighteen male Sprague-Dawley (SD) rats were randomly divided into three groups: normal control group (NC, n = 6), LSP modeling group (LM, n = 6), and electroacupuncture group (EA, n = 6). Rats in the EA group received electroacupuncture pretreatment at bilateral Zusanli (ST36) and Chize (LU5) points for five days (30 min each time daily, frequency; 3 Hz/15 Hz, intensity; 1 mA). Rats in the EA and LM groups were then injected with 5 mg/kg LPS (Beijing, Solarbio Company, concentration; 5 mg/mL) through the tail vein, while those in the NC group were injected with 5 mg/kg saline. The animals were sacrificed six hours after LPS or saline injection through cervical vertebrae by dislocation under deep anesthesia. Orbital blood was collected for the analysis of serum inflammatory factors including interleukin-1β (IL-1β) and transforming growth factor-β (TGF-β). The lower left lung was excised, stained with hematoxylin-eosin (HE), and subjected to histopathological analysis. The mRNA and protein expression of Beclin 1 and LC3 II/I in the lower right lung tissues were detected via RT-qPCR and Western blot analyses, respectively. The results showed that lung injury score was significantly higher in the LM group than that of the NC group (P < 0.01) and EA group (P < 0.01). The IL-1β contents were significantly decreased in the EA group (P < 0.01) than in the LM group. In contrast, the GF-β contents were increased in the EA group significantly when compared with the LM group (P < 0.01). RT-qPCR and Western blot detection showed that the relative gene expression of LC3‐II/I and Beclin 1 was significantly lower in the EA group than in the LM group (P < 0.01). However, the relative protein expression level of LC3‐II/I and Beclin 1 was slightly lower in the EA group than the in LM group (P > 0.05). These results show that electroacupuncture pretreatment reduces the inflammatory response in ALI and can protect lung tissue by inhibiting the gene and protein expression levels of LC3‐II/I and Beclin 1.

Long noncoding RNA Kcnq1ot1 prompts lipopolysaccharide-induced acute lung injury by microRNA-7a-5p/Rtn3 axis

BackgroundLong noncoding RNA (lncRNA)-regulated mechanism in acute lung injury (ALI) has attracted special interests in study researches. We planned to disclose whether KCNQ1 overlapping transcript 1 (Kcnq1ot1) is involved in ALI and its mechanism.MethodsThe lipopolysaccharide (LPS)-induced ALI model was established in mice. Kcnq1ot1, microRNA (miR)-7a-5p and Reticulon 3 (Rtn3) levels were measured in lung tissues of mice. The vector that changed Kcnq1ot1, miR-7a-5p and Rtn3 expression was injected into LPS-treated mice, and pathological damage, fibrosis, apoptosis and inflammatory response were subsequently examined in lung tissues. The relation between Kcnq1ot1 and miR-7a-5p, and that between miR-7a-5p and Rtn3 were identified.ResultsKcnq1ot1 and Rtn3 expression increased while miR-7a-5p expression decreased in LPS-treated mice. Reduced Kcnq1ot1 or elevated miR-7a-5p alleviated pathological damage, fibrosis, apoptosis and inflammatory response in ALI mice, while overexpressed Rtn3 worsened ALI in mice. Downregulation of Rtn3 reversed the exacerbation of miR-7a-5p downregulation in ALI mice. Kcnq1ot1 competitively bound to miR-7a-5p and miR-7a-5p negatively mediated Rtn3 expression.ConclusionOur experiments evidence that silencing Kcnq1ot1 upregulates miR-7a-5p to suppress Rtn3 expression, thereby diminishing LPS-induced ALI.

Role of Cholinergic Anti-Inflammatory Pathway in Protecting Sepsis-Induced Acute Lung Injury through Regulation of the Conventional Dendritic Cells.

Background The cholinergic anti-inflammatory pathway connects the immune response system and the nervous system via the vagus nerve. The key regulatory receptor is the α7-subtype of the nicotinic acetylcholine receptor (α7nAChR). Cholinergic anti-inflammatory pathway has been proved to be effective in suppressing the inflammation responses in acute lung injury (ALI). Dendritic cells (DCs), the important antigen-presenting cells, also express the α7nAChR. Past studies have indicated that reducing the quantity of mature conventional DCs and inhibiting the maturation of pulmonary DCs may prove effective for the treatment of ALI. However, the effects of cholinergic anti-inflammatory pathway on maturation, function, and quantity of DCs and conventional DCs in ALI remain unclear. Objective It was hypothesized that cholinergic anti-inflammatory pathway may inhibit the inflammatory response of ALI by regulating maturation, phenotype, and quantity of DCs and conventional DCs. Methods GTS-21 (GTS-21 dihydrochloride), an α7nAchR agonist, was prophylactically administered in sepsis-induced ALI mouse model and LPS-primed bone marrow-derived dendritic cells. The effects of GTS-21 were observed with respect to maturation, phenotype, and quantity of DCs, conventional DCs, and conventional DCs2 (type 2 conventional DCs) and the release of DC-related proinflammatory cytokines in vivo and in vitro. Results The results of the present study revealed that GTS-21 treatment decreased the maturation of DCs and the production of DC-related proinflammatory cytokines in vitro and in sepsis-induced ALI mouse model; it reduced the quantity of CD11c+MHCII+ conventional DCs and CD11c+CD11b+ conventional DCs2 in vivo experiment. Conclusions Cholinergic anti-inflammatory pathway contributes to the reduction in the inflammatory response in ALI by regulating maturation, phenotype, and quantity of DCs, conventional DCs, and conventional DCs2.

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.

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.

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.

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 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.

Inhibition of glycolysis alleviates lipopolysaccharide-induced acute lung injury in a mouse model.

Gluconic metabolic reprogramming, immune response, and inflammation are intimately linked. Glycolysis involves in the pathologic progress in acute and chronic inflammatory diseases. However, the involvement of glycolysis in the acute lung injury (ALI) is still unclear. This study investigated the role of glycolysis in an animal model of ALI. First, we found that lactate content in serum was remarkably increased in ALI patients and a murine model induced by intratracheal administration of lipopolysaccharide (LPS). The key proteins involving in glycolysis were robustly elevated, including HK2, PKM2, and HIF-1α. Intriguingly, inhibition of glycolysis by 2-deoxyglucose (2-DG) pronouncedly attenuated the lung tissue pathological injury, accumulation of neutrophil, oxidative stress, expression of proinflammatory factors in the lung of ALI mice induced by LPS. The 2-DG treatment also strongly suppressed the activation of the NOD-like receptor (NLR) family and pyrin domain-containing protein 3 (NLRP3) inflammasome. Furthermore, we investigated the role of glycolysis in the inflammatory response of primary murine macrophages activated by LPS in vitro. We found that the 2-DG treatment remarkably reduced the expression of proinflammatory factors induced by LPS, including tumor necrosis factor-α messenger RNA (mRNA), pro-interleukin (IL)-1β mRNA, pro-IL-18 mRNA, NLRP3 mRNA, caspase-1 mRNA, and IL-1β protein. Altogether, these data provide a novel link between gluconic metabolism reprogramming and uncontrolled inflammatory response in ALI. This study suggests glycolytic inhibition as an effective anti-inflammatory strategy in treating ALI.