Protein Concentration In Lavage Fluid Research Articles

Senkyunolide I protect against lung injury via inhibiting formation of neutrophil extracellular trap in a murine model of cecal ligation and puncture

BackgroundSenkyunolide I (SEI), a component of a Chinese herb named Ligusticum Chuanxiong hort, which is included in the formulation of Xuebijing Injection, a medication used to treat sepsis in China. Our previous study showed that SEI was protective against sepsis-associated encephalopathy and the present study was performed to investigate the role of SEI in sepsis-induced lung injury in a murine model of cecal ligation and puncture (CLP). MethodsSEI (36 mg/kg in 200 μl) or vehicle was administered immediately after CLP surgery. The lung injury was assessed 24 h later by histopathological tests, protein concentration in the bronchoalveolar lavage fluid (BALF), neutrophil recruitment in the lung tissue (myeloperoxidase fluorescence, MPO), pro-inflammatory cytokines and oxidative responses. Platelet activation was detected by CD42d/GP5 immunofluorescence and neutrophil extracellular trap (NET) were determined by immunofluorescence assays and enzyme linked immunosorbent assay (ELISA) of MPO-DNA. In vitro experiments were performed to detect the level of MPO-DNA complex released by SEI-treated neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) or co-cultured with platelets from CLP mice. ResultsSEI administration relieved the injury degree in CLP mice according to the histopathological tests (P < 0.05 compared with DMSO + CLP group). Protein level in the BALF and neutrophil infiltration were remarkably reduced by SEI after CLP surgery (P < 0.05 compared with DMSO + CLP group). TNF-α, IL-1β and IL-6 were decreased in the plasma and lung tissues from CLP mice treated with SEI (P < 0.05 compared with DMSO + CLP group). The phosphorylation of JNK, ERK, p38 and p65 were all inhibited by SEI (P < 0.05 compared with DMSO + CLP group). Immunofluorescence of MPO showed that neutrophil number was significantly lower in SEI treated CLP mice than in vehicle treated CLP mice (P < 0.05). The CD42d/GP5 staining suggested that platelet activation was significantly reduced and the NET level in the lung tissue and plasma was greatly attenuated by SEI treatment (P < 0.05 compared with DMSO + CLP group). In vitro experiments showed that the MPO-DNA level stimulated by PMA was significantly reduced by SEI treatment (P < 0.05 compared with DMSO treatment). Co-culture neutrophils with platelets from CLP mice resulted in higher level of MPO-DNA complex, while SEI partly reversed such effects of platelet on NET formation. ConclusionsSEI was protective against lung injury induced by CLP in mice. The NET formation was significantly reduced by SEI treatment, which might be involved in the mechanism of the protective effect.

The SARS-CoV-2 spike protein subunit S1 induces COVID-19-like acute lung injury in Κ18-hACE2 transgenic mice and barrier dysfunction in human endothelial cells.

Acute lung injury (ALI) leading to acute respiratory distress syndrome is the major cause of COVID-19 lethality. Cell entry of SARS-CoV-2 occurs via the interaction between its surface spike protein (SP) and angiotensin-converting enzyme-2 (ACE2). It is unknown if the viral spike protein alone is capable of altering lung vascular permeability in the lungs or producing lung injury in vivo. To that end, we intratracheally instilled the S1 subunit of SARS-CoV-2 spike protein (S1SP) in K18-hACE2 transgenic mice that overexpress human ACE2 and examined signs of COVID-19-associated lung injury 72 h later. Controls included K18-hACE2 mice that received saline or the intact SP and wild-type (WT) mice that received S1SP. K18-hACE2 mice instilled with S1SP exhibited a decline in body weight, dramatically increased white blood cells and protein concentrations in bronchoalveolar lavage fluid (BALF), upregulation of multiple inflammatory cytokines in BALF and serum, histological evidence of lung injury, and activation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways in the lung. K18-hACE2 mice that received either saline or SP exhibited little or no evidence of lung injury. WT mice that received S1SP exhibited a milder form of COVID-19 symptoms, compared with the K18-hACE2 mice. Furthermore, S1SP, but not SP, decreased cultured human pulmonary microvascular transendothelial resistance (TER) and barrier function. This is the first demonstration of a COVID-19-like response by an essential virus-encoded protein by SARS-CoV-2 in vivo. This model of COVID-19-induced ALI may assist in the investigation of new therapeutic approaches for the management of COVID-19 and other coronaviruses.

Blocking SphK1/S1P/S1PR1 Signaling Pathway Alleviates Lung Injury Caused by Sepsis in Acute Ethanol Intoxication Mice.

Acute ethanol intoxication increases the risk of sepsis and aggravates the symptoms of sepsis and lung injury. Therefore, this study aimed to explore whether sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P)/S1P receptor 1 (S1PR1) signaling pathway functions in lung injury caused by acute ethanol intoxication-enhanced sepsis, as well as its underlying mechanism. The acute ethanol intoxication model was simulated by intraperitoneally administering mice with 32% ethanol solution, and cecal ligation and puncture (CLP) was used to construct the sepsis model. The lung tissue damage was observed by hematoxylin-eosin (H&E) staining, and the wet-to-dry (W/D) ratio was used to evaluate the degree of pulmonary edema. Inflammatory cell counting and protein concentration in bronchoalveolar lavage fluid (BALF) were, respectively, detected by hemocytometer and bicinchoninic acid (BCA) method. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and IL-18 in BALF were detected by their commercial enzyme-linked immunosorbent assay (ELISA) kits. The myeloperoxidase (MPO) activity and expression of apoptosis-related proteins and SphK1/S1P/S1PR1 pathway-related proteins were, respectively, analyzed by MPO ELISA kit and Western blot analysis. The cell apoptosis in lung tissues was observed by TUNEL assay. Acute ethanol intoxication (EtOH) decreased the survival rate of mice and exacerbated the lung injury caused by sepsis through increasing pulmonary vascular permeability, neutrophil infiltration, release of inflammatory factors, and cell apoptosis. In addition, EtOH could activate the SphK1/S1P/S1PR1 pathway in CLP mice. However, PF-543, as a specific inhibitor of SphK1, could partially reverse the deleterious effects on lung injury of CLP mice. PF-543 alleviated lung injury caused by sepsis in acute ethanol intoxication rats by suppressing the SphK1/S1P/S1PR1 signaling pathway.

PD-L1 Regulates Inflammation in LPS-Induced Lung Epithelial Cells and Vascular Endothelial Cells by Interacting with the HIF-1α Signaling Pathway.

Sepsis-induced lung injury was the most common cause of death in patients. This study aimed to investigate whether PD-L1 regulates the inflammation in LPS-induced lung epithelial cells and vascular endothelial cells by interacting with the HIF-1α signaling pathway. Sepsis-induced lung injury mice were constructed by cecal ligation and puncture (CLP) procedure, and lipopolysaccharide (LPS)-induced lung epithelial cells and vascular endothelial cells simulate the sepsis-induced lung injury model in vitro. Hematoxylin-eosin (HE) staining detected the morphological changes of the lung tissues, and immunohistochemistry (IHC) detected the PD-L1 expression in lung tissues. Bicinchoninic acid (BCA) assay determined the protein concentration in bronchial alveolar lavage fluid (BALF). The number of PD-1 (+) cells in blood was detected by flow cytometry. The apoptosis in lung tissues and LPS-induced cells was analyzed by TUNEL assay. The inflammatory factor levels and HIF-1α in lung tissues and LPS-induced cells were analyzed by ELISA. The transfection effects of KD-PDL1 or KD-HIF1A in lung epithelial cells and vascular endothelial cells were confirmed by qRT-PCR analysis. The protein expression related to the PD-L1- and HIF-1α-related pathway was determined by Western blot analysis. As a result, LMT-28, as an IL-6 inhibitor, alleviated lung injury and suppressed the apoptosis and inflammation in lung tissues in BALF and the number of PD-1 (+) cells in blood. Sepsis-induced lung injury activated the PD-L1- and HIF-1α-related pathway, while LMT-28 could not completely inhibit the pathway. In addition, downregulation of PD-L1 or downregulation of HIF-1α suppressed the apoptosis and alleviated the inflammation in LPS-induced lung epithelial cells and vascular endothelial cells. Downregulation of PD-L1 had significant effects on lung epithelial cells but had greater effects on vascular endothelial cells. Downregulation of HIF-1α could decrease PD-L1 expression, and downregulation of PD-L1 could also suppress the protein expression of HIF-1α and related pathways. In conclusion, downregulation of PD-L1 alleviated the inflammation in LPS-induced lung epithelial cells and vascular endothelial cells by suppressing the HIF-1α signaling pathway.

Basement membrane degradation and inflammation play a role in the pulmonary hemorrhage induced by a P-III snake venom metalloproteinase

Snakebite envenoming is a neglected tropical disease affecting millions of people every year, especially in vulnerable rural populations in the developing world. Viperid snakes cause envenomings characterized by a complex pathophysiology which includes local and systemic hemorrhage due to the action of snake venom metalloproteinases (SVMPs). The pathogenesis of SVMP-induced systemic hemorrhage has not been investigated in detail. This study explored the pulmonary hemorrhage induced in a murine model by a P-III SVMP from the venom of Crotalus simus. Histological analysis revealed extravasation in the lungs as early as 15 min after intravenous injection of the toxin, and hemorrhage increased at 360 min. Western blot analysis demonstrated the cleavage of basement membrane (BM) proteins in lung homogenates and in bronchoalveolar lavage fluid, implying an enzymatic disruption of this extracellular matrix structure at the capillary-alveolar barrier. Likewise, alveolar edema was observed, with an increment in protein concentration in the bronchoalveolar lavage fluid, and a neutrophil-rich inflammatory infiltrate was present in the parenchyma of the lungs as part of the inflammatory reaction. Pretreatment of mice with indomethacin, pentoxifylline and an anti-neutrophil antibody resulted in a significant decrease in pulmonary hemorrhage at 360 min. These findings suggest that this P-III SVMP induces acute lung injury through the direct action of this enzyme in the capillary-alveolar barrier integrity, as revealed by BM degradation, and as a consequence of the inflammatory reaction that develops in lung tissue. Our findings provide novel clues to understand the mechanism of action of hemorrhagic SVMPs in the lungs.

Isorhapontigenin alleviates lipopolysaccharide-induced acute lung injury via modulating Nrf2 signaling

Nuclear factor erythroid-2 related factor 2 (Nrf2) is involved in mitigating various oxidative stress- and inflammation-induced diseases, including acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Isorhapontigenin (ISO), from the Chinese herb Gnetum cleistostachyum, exhibits antioxidant and anti-inflammatory properties. In this study, we explored the protective effects of ISO in ALI and its underlying molecular mechanisms. ISO significantly mitigated ALI by reducing the lung wet/dry weight ratio, protein concentration in the bronchoalveolar lavage fluid (BALF), and the levels of myeloperoxidase and malondialdehyde. ISO also improved the superoxide dismutase and glutathione activity in vivo. Moreover, ISO effectively ameliorated the changes in IL-1β, IL-6, and TNF-α concentrations in BALF, prevented IκB degradation, and inhibited the phosphorylation of NF-κB p65 subunit in lung tissues; furthermore, it enhanced the nuclear translocation of Nrf2 and inhibited IL-1β, IL-6, TNF-α, iNOS, COX-2, and ROS production in lipopolysaccharide-treated RAW264.7 cells. The protective effects of ISO in ALI were significantly reversed in ML385-treated RAW264.7 cells and the mouse model, indicating its role in Nrf2-activation. In conclusion, ISO effectively ameliorated lipopolysaccharide-induced ALI by reducing inflammation and oxidative stress, primarily through activation of Nrf2 signaling.

MiR-490 alleviates sepsis-induced acute lung injury by targeting MRP4 in new-born mice.

The aim of this study was to investigate whether the effects of miR-490 on acute lung injury (ALI) induced by sepsis in vitro and in vivo were through targeting multi-drug resistance-associated protein 4 (MRP4). MiR-490 agomir/NC agomir was injected into mice before cecal ligation and puncture (CLP). Pulmonary microvascular endothelial cells (PMVECs) were transfected with or without miR-490 agomir/NC agomir/MRP4/empty vector before lipopolysaccharide (LPS) stimulation. Histopathology, injure score, and Wet/Dry (W/D) of lung tissues were assessed. The number of neutrophils, macrophages and total cells, total protein concentration, TNF-α and IL-1β level in bronchoalveolar lavage fluid (BALF) were measured. The levels of caspase-3, Bcl-2, TNF-α, and IL-1β were measured in MPVECs. Dual-luciferase reporter assay was used to analyze the relationship between MRP4 and miR-490. When compared to the sham group, in CLP mice, the alveolar lung tissue showed significantly hyperemic, alveolar collapse, the W/D ratio was increased, and the injury index was increased. The number of neutrophils, macrophages and total cells, total protein concentration, TNF-α and IL-1β levels were significantly increased in BALF from CLP mice. The levels of TNF-α and IL-1β were significantly increased in lung tissue from CLP mice. Overexpression of miR-490 alleviated lung injury caused by CLP and inhibited inflammation in mice. The levels of TNF-α, IL-1β and caspase-3 were significantly increased, but the level of Bcl-2 was significantly decreased in MPVECs treated with LPS compared to the control group. Overexpression of miR-490 also reversed the increase of TNF-α, IL-1β, cleaved caspase-3 and Bcl-2 caused by LPS in MPVECs. Dual-luciferase reporter assay confirmed that the target gene of miR-490 was MRP4. Besides, overexpression of MRP4 upregulated TNF-α, IL-1β, and cleaved caspase-3, but downregulated the increase of Bcl-2 induced by miR-490 agomir transfection. These data suggested that miR-490 could relieve sepsis-induced acute lung injury in neonatal mice via targeting MRP4.

MNK as a potential pharmacological target for suppressing LPS-induced acute lung injury in mice

Acute lung injury (ALI) or its more severe form, known as acute respiratory distress syndrome (ARDS), is characterized by an initial exudative phase, expression of proinflammatory mediators, activation of inflammatory leukocytes, and impairment of the lung endothelium and epithelium. Despite numerous, novel therapeutic strategies have been developed regarding the pathophysiology of ALI, current treatment is mainly supportive, as specific therapies have not been established in the past few decades. The MAP kinase-interacting kinases (MNK1 and MNK2) are serine threonine kinases which are activated by mitogen-activated protein kinases (MAPKs), regulate protein synthesis by phosphroylating eukaryotic translation initiation factor 4E (eIF4E). Although studies have shown that MAPKs pathway is involved in anti-inflammatory and preventing tissue injury processes, the role of MNKs in ALI has, until now, remained relatively unexplored. Here, we investigated whether partial inhibition of MAPKs pathway by targeting MNKs was effective in the prevention and treatment of ALI. C57BL6 mice were pretreated with MNK1 and MNK2 inhibitor (CGP57380, 30 mg/kg) for 30 min and then challenged with 5 mg/kg LPS for 6 h. The results showed that pretreatment with CGP57380 not only significantly attenuated LPS-induced lung wet/dry ratio, as well as protein content, total cells and neutrophils in bronchoalveolar lavage fluid (BALF), but also decreased the production of pro-inflammatory mediators such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and keratinocyte-derived chemoattractant (KC). In addition, CGP57380 was observed to significantly suppress LPS-stimulated phosphorylation of eIF4E and MAPKs in the mouse bone marrow-derived macrophages (BMDMs). The involvement of MNK2 in lung injury was further evident by MNK2 knockout mice. MNK2 deficiency resulted in the attenuated lung histopathological changes, as also reflected by reductions in neutrophil counts, and the less LPS-induced the production of IL-6, TNF-α and KC in mouse BALF. Taken together, these findings demonstrated for the first time that MNK inhibition could effectively reduce the LPS-induced ALI in mice, suggesting a novel and potential application for MNK-based therapy to treat this serious disease.

PLCε1 mediates one-lung ventilation injury by regulating the p38/RhoA/NFκB activation loop

BackgroundPhospholipase C epsilon-1 (PLCε1) might be a novel and potential target in treating inflammatory conditions. In the present study, we aimed to clarify whether PLCε1 is involved in lung injury caused by one-lung ventilation (OLV) and to elucidate the potential molecular mechanism of PLCε1-mediated signaling pathway on OLV induced inflammatory response and injury. MethodsMale Sprague-Dawley (SD) rats were divided into wide-type (PLCε1-WT) group and PLCε1-KO group, and were treated with OLV for 0.5 h, 1 h, and 2 h respectively. Observation of lung tissue injury in rats was performed by Hematoxylin and eosin (HE) staining and Wet/dry (W/D) radios. In addition, pulmonary microvascular endothelial cells (PMVECs) transfected with PLCε1-si RNA, were stimulated by lipopolysaccharide (LPS). To explore the possible roles of PLCε1 in the OLV induced inflammatory injury and the involved pathway underlying, the lung tissue and bronchoalveolar lavage fluids (BALF) of OLV rats, as well as the PMVECs were prepared for further analysis. Enzyme-linked immunoassay (ELISA) was used to detect the expression of pro-inflammatory factors. The activities of related pathway proteins (NF-κB, phospho-p38, p38, phospho-ERK1/2, ERK1/2, RhoA and ROCK) were also detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. ResultsCompared to the PLCε1-WT rats, PLCε1-KOrats exhibited marked alleviation of lung inflammation as shown by great reduction in lung wet/dry weight ratios, decreases in the expressions of pro-inflammatory mediators, and declines in the number of neutrophils and the protein concentration in bronchoalveolar lavage fluid (BALF). Moreover, the increased expressions of RhoA and NF-κB p65 mRNA induced by OLV were significantly inhibited in PLCε1-KO rats. In LPS treated PMVECs, PLCε1-si RNA transfection ones also showed the decrease expression of proinflammatory mediators, reduction in p38 phosphorylation levels and downregulation of RhoA/ROCK signaling activation. Co-cultured with PLCε1-si RNA and BTRB796 (p38 inhibitors) in LPS-stimulated PMVECs resulted in a significant reduction in RhoA and NF-κB activity. In addition, treatment with either ROCK inhibitor (Y-27632) or dominant negative mutant of RhoA (RhoT19 N) significantly reduced the expression of NF-κB in PLCε1-si RNA treated PMVECs. ConclusionThe results indicated that PLCε1 played an important role in the inflammatory response induced by OLV. Moreover, through promoting p38/RhoA/ROCK activation loop, PLCε1 promoted NF-κB activation and thereby increased the expressions of inflammatory mediators, which induced the PMVECs inflammation and subsequent injury. The results of this study provide a potential therapeutic target for the reduction of inflammatory response in patients with OLV.

Effects of over-expressing heat shock protein 60 on marrow mesenchymal stem cells in the treatment of phosgene-induced acute lung injury

Objective: To investigate the effect of heat shock protein 60 (HSP60) overexpression on the ability of bone marrow mesenchymal stem cells (MSCs) and its therapeutic effect on rats with phosgene induced acute lung injury. Methods: HSP60 was transfected into MSCs by adenovirus. Western blot was used to measure the expressions of HSP60 before and after transfection. CCK-8 assay was used to detect the activity of MSCs, flow cytometry was used to detect the apoptotic ability of MSCs, and Transwell assay was used to observe the migration ability of MSCs. Sixty SPF grade male SD rats were randomly divided into control group, phosgene exposure group (inhalation of phosgene for 5 min) , MSCs group (phosgene exposure, MSCs treatment group) and transfected MSCs group (phosgene exposure, overexpression of HSP60 MSCs treatment group) . The pathological changes of lung were observed by lung pathological section, lung wet dry ratio, the degree of pulmonary edema, the total cell count and total protein content of alveolar lavage fluid, the inflammatory changes of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in BALF and serum were observed. The data were analyzed by Graphpad Prism 8.0 software. Paired comparisons were performed by non paired t-test. One way ANOVA was used for comparison between groups. Results: The proliferation ability of MSCs transfected with HSP60[A= (0.69±0.05) ] was significantly higher than that of MSCs not transfected with HSP60[A= (0.27±0.02) ] (P<0.05) . Compared with the phosgene exposure group, the pulmonary edema and inflammatory factor infiltration of MSCs group and MSCs transfected group were reduced. However, compared with MSCs group, the degree of pulmonary edema in MSCs transfected group was significantly improved, the levels of inflammatory factors IL-6 and TNF-α were significantly decreased, and the total protein content and total cell count in bronchoalveolar lavage fluid were less (P<0.05) . Conclusion: MSCs transfected with HSP60 can enhance the ability of proliferation, anti apoptosis, migration and the curative effect in rats with phosgene induced acute lung injury.

Moesin Is a Novel Biomarker of Endothelial Injury in Sepsis.

Objective Increased vascular permeability and inflammation are principal hallmark of sepsis. Moesin (MSN) is a membrane-associated cytoskeleton protein and crucial for the vascular endothelial function. This study is aimed at evaluating the role of MSN in endothelial injury during the process of sepsis. Methods Serum MSN in septic patients was measured by ELISA. BALB/c mice were injected with different doses of lipopolysaccharide (LPS) or underwent cecal ligation and single or double puncture (CLP) to mimic sublethal and lethal sepsis. After treatment, their serum MSN and PCT levels, wet to dry lung weights (W/D ratio), bronchoalveolar lavage fluid (BALF) protein concentrations, and lung injury scores were measured. The impact of MSN silencing on LPS-altered Rock1/myosin light chain (MLC), NF-κB, and inflammatory factors in human microvascular endothelial cells (HMECs), as well as monolayer HMEC permeability, was tested in vitro. Results Compared with healthy controls, serum MSN increased in septic patients and was positively correlated with SOFA scores and serum PCT levels in septic patients. LPS injection significantly increased serum the MSN and PCT expression, BALF protein levels, and W/D ratio, and the serum MSN levels were positively correlated with serum PCT, lung W/D ratio, and lung injury scores in mice. Similar results were obtained in the way of CLP modelling. LPS enhanced MSN, MLC, NF-κB phosphorylation, increased Rock1 expression, and inflammatory factors release in the cultured HMECs, while MSN silencing significantly mitigated the LPS-induced Rock1 and inflammatory factor expression, NF-κB, and MLC phosphorylation as well as the monolayer hyperpermeability in HMECs. Conclusions Increased serum MSN contributes to the sepsis-related endothelium damages by activating the Rock1/MLC and NF-κB signaling and may be a potential biomarker for evaluating the severity of sepsis.

YAP expression in endothelial cells prevents ventilator-induced lung injury.

Ventilator-induced lung injury is associated with an increase in mortality in patients with respiratory dysfunction, although mechanical ventilation is an essential intervention implemented in the intensive care unit. Intrinsic molecular mechanisms for minimizing lung inflammatory injury during mechanical ventilation remain poorly defined. We hypothesize that Yes-associated protein (YAP) expression in endothelial cells protects the lung against ventilator-induced injury. Wild-type and endothelial-specific YAP-deficient mice were subjected to a low (7 mL/kg) or high (21 mL/kg) tidal volume (VT) ventilation for 4 h. Infiltration of inflammatory cells into the lung, vascular permeability, lung histopathology, and the levels of inflammatory cytokines were measured. Here, we showed that mechanical ventilation with high VT upregulated YAP protein expression in pulmonary endothelial cells. Endothelial-specific YAP knockout mice following high VT ventilation exhibited increased neutrophil counts and protein content in bronchoalveolar lavage fluid, Evans blue leakage, and histological lung injury compared with wild-type littermate controls. Deletion of YAP in endothelial cells exaggerated vascular endothelial (VE)-cadherin phosphorylation, downregulation of vascular endothelial protein tyrosine phosphatase (VE-PTP), and dissociation of VE-cadherin and catenins following mechanical ventilation. Importantly, exogenous expression of wild-type VE-PTP in the pulmonary vasculature rescued YAP ablation-induced increases in neutrophil counts and protein content in bronchoalveolar lavage fluid, vascular leakage, and histological lung injury as well as VE-cadherin phosphorylation and dissociation from catenins following ventilation. These data demonstrate that YAP expression in endothelial cells suppresses lung inflammatory response and edema formation by modulating VE-PTP-mediated VE-cadherin phosphorylation and thus plays a protective role in ventilator-induced lung injury.

Validation of novel hub genes and molecular mechanisms in acute lung injury using an integrative bioinformatics approach.

Acute lung injury (ALI) is an inflammatory pulmonary disease that can easily develop into serious acute respiratory distress syndrome, which has high morbidity and mortality. However, the molecular mechanism of ALI remains unclear, and few molecular biomarkers for diagnosis and treatment have been identified. In this study, we aimed to identify novel molecular biomarkers using a bioinformatics approach. Gene expression data were obtained from the Gene Expression Omnibus database, co-expressed differentially expressed genes (CoDEGs) were identified using R software, and further functional enrichment analyses were conducted using the online tool Database for Annotation, Visualization, and Integrated Discovery. A protein-protein interaction network was established using the STRING database and Cytoscape software. Lipopolysaccharide (LPS)-induced ALI mouse model was constructed and verified. The hub genes were screened and validated in vivo. The transcription factors (TFs) and miRNAs associated with the hub genes were predicted using the NetworkAnalyst database. In total, 71 CoDEGs were screened and found to be mainly involved in the cytokine-cytokine receptor interactions, and the tumor necrosis factor and malaria signaling pathways. Animal experiments showed that the lung injury score, bronchoalveolar lavage fluid protein concentration, and wet-to-dry weight ratio were higher in the LPS group than those in the control group. Real-time polymerase chain reaction analysis indicated that most of the hub genes such as colony-stimulating factor 2 (Csf2) were overexpressed in the LPS group. A total of 20 TFs including nuclear respiratory factor 1 (NRF1) and two miRNAs were predicted to be regulators of the hub genes. In summary, Csf2 may serve as a novel diagnostic and therapeutic target for ALI. NRF1 and mmu-mir-122-5p may be key regulators in the development of ALI.

Diammonium glycyrrhizinate lipid ligand ameliorates lipopolysaccharide-induced acute lung injury by modulating vascular endothelial barrier function.

The aim of the present study was to investigate the effects of diammonium glycyrrhizinate lipid ligand (DGLL) treatment on acute lung injury (ALI) and pulmonary edema induced by lipopolysaccharide (LPS) in Sprague-Dawley rats. Rats orally received 30, 60 and 120 mg/kg DGLL. After 1 h, the rat ALI model was established by LPS (10 mg/kg) intraperitoneal injection. After 6 h, lung injury was evaluated using hematoxylin and eosin staining techniques. Pulmonary edema was evaluated using lung wet-dry weight ratio, protein concentrations in the bronchoalveolar lavage fluid (BALF) and Evans blue (EB) extravasation in lung tissue. The expression levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in lung tissues were measured using ELISA. Myeloperoxidase (MPO) expression levels were detected by immunohistochemical staining. Western blotting was used to measure the expression level changes of intercellular adhesion molecule (ICAM)-1, as well as adherent and tight junction proteins, including vascular endothelial (VE)-cadherin, zonula occludens (ZO)-1, occludin and junctional adhesion molecule (JAM)-1 that were associated with pulmonary inflammation and microvascular permeability. DGLL treatment significantly alleviated ALI induced by LPS, which was demonstrated by reduction of MPO-positive cells and expression levels of TNF-α, IL-1β and ICAM-1 in rat lung tissues. In addition, DGLL abrogated LPS-induced pulmonary edema, decreased the protein concentration in BALF and reduced EB extravasation. DGLL also reversed the reduced expression of VE-cadherin and tight junction proteins, including ZO-1, occludin and JAM-1 in the lung tissues caused by LPS. In conclusion, DGLL exhibits a protective effect on LPS-induced rat ALI, which is associated with the inhibition of inflammatory cell infiltration and microvascular barrier disruption. The present results provide a theoretical basis for the application of DGLL for the potential clinical treatment of ALI.

Dexmedetomidine targets miR-146a and participates in the progress of chronic obstructive pulmonary disease in vivo and in vitro.

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease and the third leading cause of death in the world. Dexmedetomidine has been reported to effectively inhibit histamine-induced bronchoconstriction. However, the molecular mechanism of dexmedetomidine in COPD has not been found. To explore the role and mechanism of dexmedetomidine in COPD, and to provide theoretical basis for clinical treatment of COPD. The expression of miR-146a was regulated by mimics or inhibitor and the relative expression of apoptotic proteins p53, Bax and Bcl-2 in human bronchial epithelial 16HBE cells was determined by real-time PCR and Western blot. Dexmedetomidine was treated for 16HBE cells and alveolar epithelial type II cells (AEC2), the cell apoptosis was detected by TUNEL and Hoechst33342 staining. A COPD rat model was established by smoking to test the effects of dexmedetomidine on the progression of COPD. The levels of IL-6, IL-1β and TNF-α in serum were measured by ELISA and the protein concentration of bronchoalveolar lavage fluid (BALF) was also detected in dexmedetomidine treated COPD rat model. miR-146a promoted 16HBE cell apoptosis and reduced cell proliferation. Additionally, dexmedetomidine was showed to reduce the 16HBEL cell apoptosis through reducing the expression of miR-146a. Moreover, dexmedetomidine regulated cell apoptosis and cell apoptosis through miR-146a in AEC2 cells. More importantly, dexmedetomidine attenuated the morphology and pathology of COPD rat model. Dexmedetomidine reduced 16HBE cells and AEC2 cell apoptosis and attenuated COPD by down-regulating miR-146a.

Upregulation of endothelial cell-derived exosomal microRNA-125b-5p protects from sepsis-induced acute lung injury by inhibiting topoisomerase II alpha.

ObjectiveEmerging evidence has revealed that exosomal microRNAs (miRNAs) are implicated in human diseases. However, role of exosomal miR-125b-5p in sepsis-induced acute lung injury (ALI) remains further explored. We focused on the effect of exosomal miR-125b-5p on ALI progression via targeting topoisomerase II alpha (TOP2A).MethodsThe ALI mouse models were established by cecal ligation and perforation, which were then treated with miR-125b-5p agomir or overexpressed TOP2A. Next, the pathological structure of ALI mouse lung tissues were observed, miR-125b-5p, TOP2A and vascular endothelial growth factor (VEGF) expression was determined, and the lung water content, inflammatory response, protein content in bronchoalveolar lavage fluid (BALF) and cell apoptosis in ALI mouse lung tissues were assessed. Exosomes were extracted from endothelial cells (ECs) and identified, which were then injected into the modeled mice to observe their roles in ALI. The targeting relationship between miR-125b-5p and TOP2A was confirmed.ResultsMiR-125b-5p was downregulated while TOP2A was upregulated in ALI mice. MiR-125b-5p elevation or ECs-derived exosomes promoted VEGF expression, improved pathological changes and restrained lung water content, inflammatory response, protein content in BALF and cell apoptosis in lung tissues ALI mice. TOP2A overexpression reversed the repressive role of miR-125b-5p upregulation in ALI, while downregulated miR-125b-5p abrogated the effect of ECs-derived exosomes on ALI. TOP2A was confirmed as a direct target gene of miR-125b-5p.ConclusionOur study indicates that ECs-derived exosomes overexpressed miR-125b-5p to protect from sepsis-induced ALI by inhibiting TOP2A, which may contribute to ALI therapeutic strategies.

Protective effect of ginsenoside rd on lipopolysaccharide-induced acute lung injury through its anti-inflammatory and anti-oxidative activity

Background: Inflammation and oxidation stress are key factors in the mechanism of acute lung injury (ALI). Therefore, suppression of the inflammatory response and oxidative stress could be a potential strategy to treat lipopolysaccharide (LPS)-induced ALI. Ginsenoside Rd (Rd), a natural Ginseng extract, alleviates inflammation and oxidative stress in several diseases such as Alzheimer's disease and cerebral ischemia, but its effect on ALI is still unclear. Aims and Objectives: To explore the protective effect of Rd on LPS-induced ALI and explored associated mechanisms. Materials and Methods: Mice were divided into five groups: A sham-operated group, a LPS-induced ALI group, and three LPS groups pretreated with Rd doses of 20, 40, and 80 mg/kg, respectively. The pathological changes of lung, collagen deposition, pulmonary edema, inflammatory cytokine, oxidative stress and the expression levels of TLR4 and NF-κB were detected. Results: The oral administration of Rd dose dependently attenuated histopathologic changes in the lung, lung edema, pulmonary collagen deposition, protein concentration in bronchoalveolar lavage fluid (BALF), myeloperoxidase (MPO) activity, and inflammatory cell infiltration. In addition, Rd suppressed the LPS-induced inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-6, and IL-1 β in BALF. The productions of oxidative stress-related enzymes (catalase, superoxide dismutase, and glutathione peroxidase) in lung tissue were significantly upregulated by Rd administration. However, malondialdehyde and pulmonary MPO activity was reduced in the Rd-pretreated groups when compared with LPS-induced ALI group. Rd treatment also dose dependently suppressed LPS-induced NF-κB activation and TLR4 expression. Conclusion: Overall, these findings provide evidence that Rd pretreatment inhibits LPS-induced ALI through anti-inflammatory and antioxidative actions, suggesting that it could be a promising protective drug for LPS-induced ALI.

Protective effect of ginsenoside Rd on lipopolysaccharide-induced acute lung injury through its anti-inflammatory and anti-oxidative activity

Background: Inflammation and oxidation stress are key factors in the mechanism of acute lung injury (ALI). Therefore, suppression of the inflammatory response and oxidative stress could be a potential strategy to treat lipopolysaccharide (LPS)-induced ALI. Ginsenoside Rd (Rd), a natural Ginseng extract, alleviates inflammation and oxidative stress in several diseases such as Alzheimer's disease and cerebral ischemia, but its effect on ALI is still unclear. Aims and Objectives: To explore the protective effect of Rd on LPS-induced ALI and explored associated mechanisms. Materials and Methods: Mice were divided into five groups: A sham-operated group, a LPS-induced ALI group, and three LPS groups pretreated with Rd doses of 20, 40, and 80 mg/kg, respectively. The pathological changes of lung, collagen deposition, pulmonary edema, inflammatory cytokine, oxidative stress and the expression levels of TLR4 and NF-κB were detected. Results: The oral administration of Rd dose dependently attenuated histopathologic changes in the lung, lung edema, pulmonary collagen deposition, protein concentration in bronchoalveolar lavage fluid (BALF), myeloperoxidase (MPO) activity, and inflammatory cell infiltration. In addition, Rd suppressed the LPS-induced inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-6, and IL-1 β in BALF. The productions of oxidative stress-related enzymes (catalase, superoxide dismutase, and glutathione peroxidase) in lung tissue were significantly upregulated by Rd administration. However, malondialdehyde and pulmonary MPO activity was reduced in the Rd-pretreated groups when compared with LPS-induced ALI group. Rd treatment also dose dependently suppressed LPS-induced NF-κB activation and TLR4 expression. Conclusion: Overall, these findings provide evidence that Rd pretreatment inhibits LPS-induced ALI through anti-inflammatory and antioxidative actions, suggesting that it could be a promising protective drug for LPS-induced ALI.

Estradiol attenuates LPS-induced acute lung injury via induction of aquaporins AQP1 and AQP5

Background Acute lung injury (ALI) is associated with increased inflammation and oxidative stress. Estradiol is produced by the ovaries and is the most active hormone of estrogen. Our aim was to investigate whether estradiol contributes to protect against lipopolysaccharide (LPS)-induced ALI via induction of aquaporins AQP1 and AQP5 and the underlying mechanisms. Methods and results For induction of ALI, LPS was applied once by intraperitoneal injection in SD rats 14 days after oophorectomy. To assess the therapeutic effects of estradiol on LPS-induced ALI, estradiol was subcutaneously injected for 1 h prior to LPS challenge. Estradiol can significantly attenuate the lung edema reflected by decreasing wet-to-dry weight ratio and permeability of lung and total protein concentration of bronchial lavage fluid (BALF). Results of histological detection showed that estradiol attenuated the lung injury reflected by reducing edema, congestion, and thickening pulmonary septal of lung tissues. In addition, estradiol attenuated TNF-α, IL-1β, and IL-6 and oxidative stress in lung tissues. Estradiol was more effective than estradiol associated with ERα antagonist or ERβ antagonist in protecting against LPS-induced ALI in rats. Mechanistically, we investigate whether estradiol regulates the expression of AQP1 and AQP5 in lung tissues. Of interest, estradiol upregulates AQP1 and AQP5 mRNA and protein expression. Taken together, these results demonstrate that estradiol can increase the expression of AQP1 and AQP5, which plays a critical role in ameliorating oxidative stress and downregulating inflammatory responses induced by LPS.Conclusion Therefore, these findings strongly suggest that AQP1 and AQP5 mediate the anti-inflammatory and antioxidant effects of estradiol.

Xuebijing attenuates decompression-induced lung injuries.

The lung is among the primary organs involved in decompression sickness (DCS). Xuebijing (XBJ), a traditional Chinese medicine, has been widely used in the treatment of various acute lung diseases. This study aimed to explore potential benefit of XBJ on lung injuries induced by DCS in a rabbit model. Twenty-four male New Zealand white rabbits underwent a simulated air dive to 50 meters' sea water for 60 min with 2.5 min decompression, and received an intravenous injection of XBJ (5 ml·kg-1) or an equal volume of saline immediately following decompression. DCS signs were monitored for 24 h, and blood was sampled before simulated diving and at 6 h and 12 h following decompression for determination of inflammatory indices. Lung tissues were sampled after euthanasia for histology analysis and lung water content, as well as tumour necrosis factor-α level. Another six rabbits were used as control. XBJ significantly ameliorated lung injuries (lung wet/dry ratio and total protein content in bronchoalveolar lavage fluid), and notably inhibited systemic (serum level of interleukin-1β) and local (tumour necrosis factor-α in bronchoalveolar lavage fluid) inflammation responses. The results strongly suggest the benefits of XBJ on ameliorating DCS lung injuries, which is possibly via inhibiting systemic and local inflammation. XBJ may be a potential candidate for the treatment of decompression-induced lung injuries.