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.     

Elevation of KL-6, a lung epithelial cell marker,in plasma and epithelial lining fluid in acute respiratory distress syndrome

KL-6 is a pulmonary epithelial mucin more prominently expressed on the surface membrane of alveolar type II cells when these cells are proliferating, stimulated, and/or injured. We hypothesized that high levels of KL-6 in epithelial lining fluid and plasma would reflect the severity of lung injury in patients with acute lung injury (ALI). Epithelial lining fluid was obtained at onset (day 0) and day 1 of acute respiratory distress syndrome (ARDS)/ALI by bronchoscopic microsampling procedure in 35 patients. On day 0, KL-6 and albumin concentrations in epithelial lining fluid were significantly higher than in normal controls (P < 0.001), and the concentrations of KL-6 in epithelial lining fluid (P < 0.002) and in plasma (P < 0.0001) were higher in nonsurvivors than in survivors of ALI/ARDS. These observations were corroborated by the immunohistochemical localization of KL-6 protein expression in the lungs of nonsurvivors with ALI and KL-6 secretion from cultured human alveolar type II cells stimulated by proinflammatory cytokines. Because injury to distal lung epithelial cells, including alveolar type II cells, is important in the pathogenesis of ALI, the elevation of KL-6 concentrations in plasma and epithelial lining fluid could be valuable indicators for poor prognosis in clinical ALI.     

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.     

NEDD4 attenuates phosgene‐induced acute lung injury through the inhibition of Notch1 activation

Phosgene gas leakage can cause life‐threatening acute lung injury (ALI), which is characterized by inflammation, increased vascular permeability, pulmonary oedema and oxidative stress. Although the downregulation of neuronal precursor cell‐expressed developmentally downregulated 4 (NEDD4) is known to be associated with inflammation and oxidative damage, its functions in phosgene‐induced ALI remain unclear. In this study, rats with phosgene‐induced ALI were intravenously injected with NEDD4‐overexpressing lentiviruses to determine the functions of NEDD4 in this inflammatory condition. NEDD4 expression was decreased in the lung parenchyma of phosgene‐exposed control rats, whereas its expression level was high in the NEDD4‐overexpressing rats. Phosgene exposure increased the wet‐to‐dry lung weight ratio, but NEDD4 abrogated this effect. NEDD4 overexpression attenuated phosgene‐induced lung inflammation, lowering the high lung injury score (based on total protein, inflammatory cells and inflammatory factors in bronchoalveolar lavage fluid) and also reduced phosgene‐induced oxidative stress and cell apoptosis. Finally, NEDD4 was found to interact with Notch1, enhancing its ubiquitination and thereby its degradation, thus attenuating the inflammatory responses to ALI. Therefore, we demonstrated that NEDD4 plays a protective role in alleviating phosgene‐induced ALI, suggesting that enhancing the effect of NEDD4 may be a new approach for treating phosgene‐induced ALI.     

Induction of acute respiratory distress syndrome in rats by lipopolysaccharide and its effect on oxidative stress and antioxidant status in lung

Acute lung injury (ALI) or its severe form, acute respiratory distress syndrome (ARDS) is an important cause of mortality in the human population. Despite significant advances made, the mortality associated with ALI remains unchanged. The objective of the present study was to evaluate the role of oxidative stress, alveolar antioxidant status and multiple organ injury in ARDS induced by lipopolysaccharide (LPS) in rats. Rats were divided into 4 groups, group I control rats were given saline intraperitoneally, whereas groups II, III and IV (LPS-treated) rats received an intraperitoneal injection of LPS (10 mg/kg body weight) and sacrificed after various time intervals. In LPS-treated rats, we observed increased levels of oxidative products, decreased levels of antioxidants in lung tissues and increased levels of serum marker enzymes, suggesting multiple organ injury. Bronchoalveolar lavage fluid (BALF) neutrophil content and protein concentration in LPS-treated rats were significantly elevated in a time-dependent manner. Histological studies revealed neutrophil influx and diffused alveolar damage in LPS-administered rats. These results clearly suggested that increased oxidant levels led to oxidative stress, antioxidant deficiency attenuating lung inflammation and tissue damage. LPS administration resulted in multiple organ failure, leading to increased mortality.     

miR‐34b‐5p inhibition attenuates lung inflammation and apoptosis in an LPS‐induced acute lung injury mouse model by targeting progranulin

Inflammation and apoptosis play important roles in the initiation and progression of acute lung injury (ALI). Our previous study has shown that progranulin (PGRN) exerts lung protective effects during LPS‐induced ALI. Here, we have investigated the potential roles of PGRN‐targeting microRNAs (miRNAs) in regulating inflammation and apoptosis in ALI and have highlighted the important role of PGRN. LPS‐induced lung injury and the protective roles of PGRN in ALI were first confirmed. The function of miR‐34b‐5p in ALI was determined by transfection of a miR‐34b‐5p mimic or inhibitor in intro and in vivo. The PGRN level gradually increased and subsequently significantly decreased, reaching its lowest value by 24 hr; PGRN was still elevated compared to the control. The change was accompanied by a release of inflammatory mediators and accumulation of inflammatory cells in the lungs. Using bioinformatics analysis and RT‐PCR, we demonstrated that, among 12 putative miRNAs, the kinetics of the miR‐34b‐5p levels were closely associated with PGRN expression in the lung homogenates. The gain‐ and loss‐of‐function analysis, dual‐luciferase reporter assays, and rescue experiments confirmed that PGRN was the functional target of miR‐34b‐5p. Intravenous injection of miR‐34b‐5p antagomir in vivo significantly inhibited miR‐34b‐5p up‐regulation, reduced inflammatory cytokine release, decreased alveolar epithelial cell apoptosis, attenuated lung inflammation, and improved survival by targeting PGRN during ALI. miR‐34b‐5p knockdown attenuates lung inflammation and apoptosis in an LPS‐induced ALI mouse model by targeting PGRN. This study shows that miR‐34b‐5p and PGRN may be potential targets for ALI treatments.     

The Hippo–YAP pathway regulates the proliferation of alveolar epithelial progenitors after acute lung injury

Regeneration of pulmonary epithelial cells plays an important role in the recovery of acute lung injury (ALI), which is defined by pulmonary epithelial cell death. However, the mechanism of the regenerative capacity of alveolar epithelial cells is unknown. Using a lung injury mouse model induced by hemorrhagic shock and lipopolysaccharide, a protein mass spectrometry‐based high‐throughput screening and linage tracing technology to mark alveolar epithelial type 2 cells (AEC2s), we analyzed the mechanism of alveolar epithelial cells proliferation. We demonstrated that the expression of Hippo‐yes‐associated protein 1 (YAP1) key proteins were highly consistent with the regularity of the proliferation of alveolar epithelial type 2 cells after ALI. Furthermore, the results showed that YAP1+ cells in lung tissue after ALI were mainly Sftpc lineage‐labeled AEC2s. An in vitro proliferation assay of AEC2s demonstrated that AEC2 proliferation was significantly inhibited by both YAP1 small interfering RNA and Hippo inhibitor. These findings revealed that YAP functioned as a key regulator to promote AEC2s proliferation, with the Hippo signaling pathway playing a pivotal role in this process.     

The role of Cold‐Inducible RNA‐binding protein in respiratory diseases

Cold‐inducible RNA‐binding protein (CIRP) is a stress‐response protein that is expressed in various types of cells and acts as an RNA chaperone, modifying the stability of its targeted mRNA. Intracellular CIRP could also be released into extracellular space and once released, extracellular CIRP (eCIRP) acts as a damage‐associated molecular pattern (DAMP) to induce and amplify inflammation. Recent studies have found that eCIRP could promote acute lung injury (ALI) via activation of macrophages, neutrophils, pneumocytes and lung vascular endothelial cells in context of sepsis, haemorrhagic shock, intestinal ischemia/reperfusion injury and severe acute pancreatitis. In addition, CIRP is also highly expressed in the bronchial epithelial cells and its expression is upregulated in the bronchial epithelial cells of patients with chronic obstructive pulmonary diseases (COPD) and rat models with chronic bronchitis. CIRP is a key contributing factor in the cold‐induced exacerbation of COPD by promoting the expression of inflammatory genes and hypersecretion of airway mucus in the bronchial epithelial cells. Besides, CIRP is also involved in regulating pulmonary fibrosis, as eCIRP could directly activate and induce an inflammatory phenotype in pulmonary fibroblast. This review summarizes the findings of CIRP investigation in respiratory diseases and the underlying molecular mechanisms.     

Immunosuppressive effect of mesenchymal stem cells on lung and gut CD8+ T cells in lipopolysaccharide‐induced acute lung injury in mice

ObjectivesAcute lung injury (ALI) not only affects pulmonary function but also leads to intestinal dysfunction, which in turn contributes to ALI. Mesenchymal stem cell (MSC) transplantation can be a potential strategy in the treatment of ALI. However, the mechanisms of synergistic regulatory effects by MSCs on the lung and intestine in ALI need more in‐depth study.Materials and methodsWe evaluated the therapeutic effects of MSCs on the murine model of lipopolysaccharide (LPS)‐induced ALI through survival rate, histopathology and bronchoalveolar lavage fluid. Metagenomic sequencing was performed to assess the gut microbiota. The levels of pulmonary and intestinal inflammation and immune response were assessed by analysing cytokine expression and flow cytometry.ResultsMesenchymal stem cells significantly improved the survival rate of mice with ALI, alleviated histopathological lung damage, improved intestinal barrier integrity, and reduced the levels of inflammatory cytokines in the lung and gut. Furthermore, MSCs inhibited the inflammatory response by decreasing the infiltration of CD8⁺ T cells in both small‐intestinal lymphocytes and Peyer''s patches. The gut bacterial community diversity was significantly altered by MSC transplantation. Furthermore, depletion of intestinal bacterial communities with antibiotics resulted in more severe lung and gut damages and mortality, while MSCs significantly alleviated lung injury due to their immunosuppressive effect.ConclusionsThe present research indicates that MSCs attenuate lung and gut injury partly via regulation of the immune response in the lungs and intestines and gut microbiota, providing new insights into the mechanisms underlying the therapeutic effects of MSC treatment for LPS‐induced ALI.     

Fas inhibition attenuates lipopolysaccharide-induced apoptosis and cytokine release of rat type II alveolar epithelial cells

The aim of this study is to investigate whether silencing of Fas could have an influence on type II alveolar epithelial cell (AEC) apoptosis and inflammatory cytokine production, which prevents alveolar healing after acute lung injury (ALI). Rat primary type II AECs were isolated by elastase cell dispersion and IgG panning. The cells were transfected with Fas-specific small interfering RNA (siRNA) followed by treatment with lipopolysaccharide (LPS), Fas ligand (FasL) or both. The effects of siRNA-mediated silencing of Fas on LPS-induced apoptosis and cytokine release were then assessed. Notably, LPS, either alone or together with FasL, significantly stimulated type II AEC apoptosis and the release of tumor necrosis factor-alpha (TNF-α) and monocyte chemoattractant protein 1 (MCP-1) (P < 0.05 versus the control without treatment). Moreover, the effects exerted by both LPS and FasL were considerably counteracted by pretreatment with Fas-siRNA (P < 0.05 versus treatment with LPS and FasL). In conclusion, inhibition of Fas can diminish LPS-induced apoptosis and inflammatory cytokine production in type II AECs, and Fas specific siRNAs may have therapeutic potentials for intervention of ALI/ARDS.     

Alveolar Type II Epithelial Cell Dysfunction in Rat Experimental Hepatopulmonary Syndrome (HPS)

The hepatopulmonary syndrome (HPS) develops when pulmonary vasodilatation leads to abnormal gas exchange. However, in human HPS, restrictive ventilatory defects are also observed supporting that the alveolar epithelial compartment may also be affected. Alveolar type II epithelial cells (AT2) play a critical role in maintaining the alveolar compartment by producing four surfactant proteins (SPs, SP-A, SP-B, SP-C and SP-D) which also facilitate alveolar repair following injury. However, no studies have evaluated the alveolar epithelial compartment in experimental HPS. In this study, we evaluated the alveolar epithelial compartment and particularly AT2 cells in experimental HPS induced by common bile duct ligation (CBDL). We found a significant reduction in pulmonary SP production associated with increased apoptosis in AT2 cells after CBDL relative to controls. Lung morphology showed decreased mean alveolar chord length and lung volumes in CBDL animals that were not seen in control models supporting a selective reduction of alveolar airspace. Furthermore, we found that administration of TNF-α, the bile acid, chenodeoxycholic acid, and FXR nuclear receptor activation (GW4064) induced apoptosis and impaired SP-B and SP-C production in alveolar epithelial cells in vitro. These results imply that AT2 cell dysfunction occurs in experimental HPS and is associated with alterations in the alveolar epithelial compartment. Our findings support a novel contributing mechanism in experimental HPS that may be relevant to humans and a potential therapeutic target.     

Keratinocyte Growth Factor Gene Delivery via Mesenchymal Stem Cells Protects against Lipopolysaccharide-Induced Acute Lung Injury in Mice

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high morbidity and mortality, and have no specific therapy. Keratinocyte growth factor (KGF) is a critical factor for pulmonary epithelial repair and acts via the stimulation of epithelial cell proliferation. Mesenchymal stem cells (MSCs) have been proved as good therapeutic vectors. Thus, we hypothesized that MSC-based KGF gene therapy would have beneficial effects on lipopolysaccharide(LPS)-induced lung injury. After two hours of intratracheal LPS administration to induce lung injury, mice received saline, MSCs alone, empty vector-engineered MSCs (MSCs-vec) or KGF-engineered MSCs (MSCs-kgf) via the tail vein. The MSCs-kgf could be detected in the recipient lungs and the level of KGF expression significantly increased in the MSCs-kgf mice. The MSC-mediated administration of KGF not only improved pulmonary microvascular permeability but also mediated a down-regulation of proinflammatory responses (reducing IL-1β and TNF-α) and an up-regulation of anti-inflammatory responses (increasing cytokine IL-10). Furthermore, the total severity scores of lung injury were significantly reduced in the MSCs-kgf group compared with the other three groups. The underlying mechanism of the protective effect of KGF on ALI may be attributed to the promotion of type II lung epithelial cell proliferation and the enhancement of surfactant synthesis. These findings suggest that MSCs-based KGF gene therapy may be a promising strategy for ALI treatment.     

Mononuclear Phagocyte-Derived Microparticulate Caspase-1 Induces Pulmonary Vascular Endothelial Cell Injury

Lung endothelial cell apoptosis and injury occurs throughout all stages of acute lung injury (ALI/ARDS) and impacts disease progression. Lung endothelial injury has traditionally been focused on the role of neutrophil trafficking to lung vascular integrin receptors induced by proinflammatory cytokine expression. Although much is known about the pathogenesis of cell injury and death in ALI/ARDS, gaps remain in our knowledge; as a result of which there is currently no effective pharmacologic therapy. Enzymes known as caspases are essential for completion of the apoptotic program and secretion of pro-inflammatory cytokines. We hypothesized that caspase-1 may serve as a key regulator of human pulmonary microvascular endothelial cell (HPMVEC) apoptosis in ALI/ARDS. Our recent experiments confirm that microparticles released from stimulated monocytic cells (THP1) induce lung endothelial cell apoptosis. Microparticles pretreated with the caspase-1 inhibitor, YVAD, or pan-caspase inhibitor, ZVAD, were unable to induce cell death of HPMVEC, suggesting the role of caspase-1 or its substrate in the induction of HPMVEC cell death. Neither un-induced microparticles (control) nor direct treatment with LPS induced apoptosis of HPMVEC. Further experiments showed that caspase-1 uptake into HPMVEC and the induction of HPMVEC apoptosis was facilitated by caspase-1 interactions with microparticulate vesicles. Altering vesicle integrity completely abrogated apoptosis of HPMVEC suggesting an encapsulation requirement for target cell uptake of active caspase-1. Taken together, we confirm that microparticle centered caspase-1 can play a regulator role in endothelial cell injury.     

Effects of hyperoxia on transdifferentiation of primary cultured typeII alveolar epithelial cells from premature rats

Hyperoxia exposure is a significant risk factor for the impaired alveolarization characteristic of bronchopulmonary dysplasia. Type II alveolar epithelial cells (AECIIs) may serve as "alveolar stem cells" to transdifferentiate into type I alveolar epithelial cells (AECIs). Here, we show that hyperoxia is capable of inducing transdifferentiation of AECIIs in premature rats in vitro. Hyperoxia-induced transdifferentiation was characterized by typical morphological changes, inhibition of cellular proliferation, decline in expression rate of Ki67, accumulation of cells in the G(1) phase of the cell cycle, increased expression of AECI-specific protein aquaporin 5, and decreased expression of AECII-associated protein surfactant protein C. These results suggest that hyperoxia may induce transdifferentiation of AECIIs into AECIs and the transdifferentiation may be responsible for repairing early lung injury.     

Protective effects of recombinant 53-kDa protein of Trichinella spiralis on acute lung injury in mice via alleviating lung pyroptosis by promoting M2 macrophage polarization

Trichinella spiralis represents an effective treatment for autoimmune and inflammatory diseases. The effects of recombinant T. spiralis (TS) 53-kDa protein (rTsP53) on acute lung injury (ALI) remain unclear. Here, mice were divided randomly into a control group, LPS group, and rTsP53 + LPS group. ALI was induced in BALB/c mice by LPS (10 mg/kg) injected via the tail vein. rTsP53 (200 µl; 0.4 μg/μl) was injected subcutaneously three times at an interval of 5 d before inducing ALI in the rTsP53+LPS group. Lung pathological score, the ratio and markers of classic activated macrophages (M1) and alternatively activated macrophages (M2), cytokine profiles in alveolar lavage fluid, and pyroptosis protein expression in lung tissue were investigated. RTsP53 decreased lung pathological score. Furthermore, rTsP53 suppressed inflammation by increasing IL-4, IL-10, and IL-13. There was an increase in alveolar M2 macrophage numbers, with an increase in CD206 and arginase-1-positive cells and a decrease in alveolar M1 markers such as CD197 and iNOS. In addition, the polarization of M2 macrophages induced by rTsP53 treatment could alleviate ALI by suppressing lung pyroptosis. RTsP53 was identified as a potential agent for treating LPS-induced ALI via alleviating lung pyroptosis by promoting M2 macrophage polarization.     

Curcumin Modulates the Inflammatory Response and Inhibits Subsequent Fibrosis in a Mouse Model of Viral-induced Acute Respiratory Distress Syndrome

Acute Respiratory Distress Syndrome (ARDS) is a clinical syndrome characterized by diffuse alveolar damage usually secondary to an intense host inflammatory response of the lung to a pulmonary or extrapulmonary infectious or non-infectious insult often leading to the development of intra-alveolar and interstitial fibrosis. Curcumin, the principal curcumoid of the popular Indian spice turmeric, has been demonstrated as an anti-oxidant and anti-inflammatory agent in a broad spectrum of diseases. Using our well-established model of reovirus 1/L-induced acute viral pneumonia, which displays many of the characteristics of the human ALI/ARDS, we evaluated the anti-inflammatory and anti-fibrotic effects of curcumin. Female CBA/J mice were treated with curcumin (50 mg/kg) 5 days prior to intranasal inoculation with 10⁷ pfu reovirus 1/L and daily, thereafter. Mice were evaluated for key features associated with ALI/ARDS. Administration of curcumin significantly modulated inflammation and fibrosis, as revealed by histological and biochemical analysis. The expression of IL-6, IL-10, IFNγ, and MCP-1, key chemokines/cytokines implicated in the development of ALI/ARDS, from both the inflammatory infiltrate and whole lung tissue were modulated by curcumin potentially through a reduction in the phosphorylated form of NFκB p65. While the expression of TGFß1 was not modulated by curcumin, TGFß Receptor II, which is required for TGFß signaling, was significantly reduced. In addition, curcumin also significantly inhibited the expression of α-smooth muscle actin and Tenascin-C, key markers of myofibroblast activation. This data strongly supports a role for curcumin in modulating the pathogenesis of viral-induced ALI/ARDS in a pre-clinical model potentially manifested through the alteration of inflammation and myofibroblast differentiation.     

褪黑素对内毒素致大鼠急性肺损伤的保护作用

目的:研究内毒素(LPS)致大鼠急性肺损伤(ALI)时,p-p38蛋白激酶(p-p38MAPK)在肺组织的表达及褪黑素(MT)对肺组织的保护作用及其机制。方法:将72只健康雄性SD大鼠随机分为3组,每组24只,对照组(Control)、模型组(LPS)和褪黑素干预组(LPS+MT),采用气管内滴注LPS的方法建立大鼠ALI的模型,通过免疫组织化学染色和Western blot技术检测大鼠肺组织中p-p38蛋白激酶的表达变化,并在光镜下观察大鼠肺组织形态学变化。结果:Control组气道和肺组织可见反应极弱的p-p38蛋白激酶阳性细胞,散在分布于气道上皮细胞和肺泡上皮细胞;LPS组p-p38蛋白激酶阳性细胞较对照组明显增多(P0.05或P0.01),主要分布于浸润的炎症细胞、气道上皮细胞、肺泡上皮细胞和血管内皮细胞;LPS+MT组气道和肺组织中阳性细胞数较LPS组明显减少(P0.05或P0.01),Western blot结果与免疫组织化学一致。结论:LPS致大鼠急性肺损伤模型中,肺内炎性、非炎性细胞均有p38MAPK信号通路的激活;MT对急性肺损伤的保护机制可能与其抑制p38 MAPK信号通路的过度激活有关。