Protein Concentration In Lavage Fluid Research Articles

Boric Acid Alleviates Lipopolysaccharide-Induced Acute Lung Injury in Mice.

Acute lung injury (ALI) poses a significant medical challenge due to its widespread occurrence and high mortality rates. Despite extensive efforts, current clinical interventions for ALI have shown limited success. Inflammation plays a central role within ALI progress, and boric acid (BA) has demonstrated anti-inflammatory properties both in vitro and in vivo. However, its potential to mitigate lipopolysaccharide (LPS)-induced ALI remains an area awaiting exploration in research. To bridge this research gap, we created a mouse model of ALI induced by intraperitoneal LPS injection. We employed a comprehensive set of evaluation criteria, including H&E staining, wet/dry ratio measurement, malondialdehyde (MDA)/superoxide dismutase (SOD) the oxidative stress-related biomarkers, assessment of alveolar edema, hemorrhage, inflammatory cell infiltration, and examination of thickened alveolar septum to quantify lung injury. Additionally, we measured inflammatory cytokine levels using ELISA and assessed Nrf2 and HO-1 expressions through western blotting and quantitative real-time PCR (RT-PCR). ER stress-related markers (GRP78, CHOP) were analyzed through western blot analysis. Our findings revealed that prophylactic treatment with BA effectively attenuated LPS-induced ALI, as supported by improved pathological alterations, decreased total protein concentration in bronchoalveolar lavage fluid (BALF), and reduced pulmonary edema. Furthermore, BA exhibited anti-inflammatory properties by suppressing inflammatory cytokines within the lung tissue. BA ingestion caused upregulation in SOD and a decrease in MDA contents in lung tissue homogenates. BA downregulated the levels of GRP78 and CHOP compared to the LPS group. Remarkably, BA also upregulated transcription and protein expression of Nrf2 and HO-1 compared to the LPS group. In conclusion, our study highlights BA's potential as a novel promising prophylactic agent for LPS-induced ALI, offering avenue for improving clinical management of this condition.

MiR-202-3p Targets Calm1 and Suppresses Inflammation in a Mouse Model of Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is regarded as a type of respiratory failure. Emerging evidence has demonstrated the significant roles of microRNAs in various disorders. Nevertheless, the role of miR-202-3p in ARDS is unclear. Forty male C57BL/6 mice treated with phosphate buffer saline/lipopolysaccharide (PBS/LPS) and administrated with NC/miR-202-3p agomir were divided into four groups. A reverse transcription-quantitative polymerase chain reaction was used to evaluate the level of miR-202-3p, its target genes, and proinflammatory factors. Hematoxylin‑eosin was utilized for histological observation of the lung tissues. The Wet/Dry ratio, myeloperoxidase activity, and total protein concentration in bronchoalveolar lavage fluid were assessed to determine pulmonary edema. Western blotting was used for quantifying protein levels of proinflammatory factors, nuclear factor kappa B (NF-κB), and NLR family pyrin domain containing 3 (NLRP3) signaling-associated proteins. Calmodulin 1 (Calm1) protein expression in murine lung tissues was evaluated by immunohistochemistry. The binding relation between miR-202-3p and Calm1 was assessed by luciferase reporter assay. The results showed that miR-202-3p was lowly expressed in the lung tissues of ARDS mice. Overexpressed miR-202-3p relieved LPS-induced edema, reduced proinflammatory factors, and inactivated NF-κB/NLRP3 signaling in murine lung tissues. Calm1 was targeted by miR-202-3p and displayed a high level of LPS-induced ARDS. In conclusion, miR-202-3p targets Calm1 and suppresses inflammation in LPS-induced ARDS, thereby inhibiting the pathogenesis of ARDS in a mouse model.

Syringic acid attenuates acute lung injury by modulating macrophage polarization in LPS-induced mice

BackgroundAcute lung injury (ALI) is a continuum of lung changes caused by multiple lung injuries, characterized by a syndrome of uncontrolled systemic inflammation that often leads to significant morbidity and death. Anti-inflammatory is one of its treatment methods, but there is no safe and available drug therapy. Syringic acid (SA) is a natural organic compound commonly found in a variety of plants, especially in certain woody plants and fruits. In modern pharmacological studies, SA has anti-inflammatory effects and therefore may be a potentially safe and available compound for the treatment of acute lung injury. PurposeThis study attempts to reveal the protective mechanism of SA against ALI by affecting the polarization of macrophages and the activation of NF-κB signaling pathway. Trying to find a safer and more effective drug therapy for clinical use. MethodsWe constructed the ALI model using C57BL/6 mice by intratracheal instillation of LPS (10 mg/kg). Histological analysis was performed with hematoxylin and eosin (H&E). The wet-dry ratio of the whole lung was measured to evaluate pulmonary edema. The effect of SA on macrophage M1-type was detected by flow cytometry. BCA protein quantification method was used to determine the total protein concentration in bronchoalveolar lavage fluid (BALF). The levels of Interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α in BALF were determined by the ELISA kits, and RT-qPCR was used to detect the expression levels of IL-6, IL-1β and TNF-α mRNA of lung tissue. Western blot was used to detect the expression levels of iNOS and COX-2 and the phosphorylation of p65 and IκBα in the NF-κB pathway in lung tissue. In vitro experiments were conducted with RAW267.4 cell inflammation model induced by 100 ng/ml LPS and A549 cell inflammation model induced by 10 μg/ml LPS. The effects of SA on M1-type and M2-type macrophages of RAW267.4 macrophages induced by LPS were detected by flow cytometry. The toxicity of compound SA to A549 cells was detected by MTT method which to determine the safe dose of SA. The expressions of COX-2 and the phosphorylation of p65 and IκBα protein in NF-κB pathway were detected by Western blot. ResultsWe found that the pre-treatment of SA significantly reduced the degree of lung injury, and the infiltration of neutrophils in the lung interstitium and alveolar space of the lung. The formation of transparent membrane in lung tissue and thickening of alveolar septum were significantly reduced compared with the model group, and the wet-dry ratio of the lung was also reduced. ELISA and RT-qPCR results showed that SA could significantly inhibit the production of IL-6, IL-1β, TNF-α. At the same time, SA could significantly inhibit the expression of iNOS and COX-2 proteins, and could inhibit the phosphorylation of p65 and IκBα proteins. in a dose-dependent manner. In vitro experiments, we found that flow cytometry showed that SA could significantly inhibit the polarization of macrophages from M0 type macrophages to M1-type macrophages, while SA could promote the polarization of M1-type macrophages to M2-type macrophages. The results of MTT assay showed that SA had no obvious cytotoxicity to A549 cells when the concentration was not higher than 80 μM, while LPS could promote the proliferation of A549 cells. In the study of anti-inflammatory effect, SA can significantly inhibit the expression of COX-2 and the phosphorylation of p65 and IκBα proteins in LPS-induced A549 cells. ConclusionSA has possessed a crucial anti-ALI role in LPS-induced mice. The mechanism was elucidated, suggesting that the inhibition of macrophage polarization to M1-type and the promotion of macrophage polarization to M2-type, as well as the inhibition of NF-κB pathway by SA may be the reasons for its anti-ALI. This finding provides important molecular evidence for the further application of SA in the clinical treatment of ALI.

S100A9 exacerbates sepsis-induced acute lung injury via the IL17-NFκB-caspase-3 signaling pathway

BackgroundSepsis-induced acute lung injury (ALI) is associated with considerable morbidity and mortality in critically ill patients. S100A9, a key endothelial injury factor, is markedly upregulated in sepsis-induced ALI; however, its specific mechanism of action has not been fully elucidated. MethodsThe Gene Expression Omnibus database transcriptome data for sepsis-induced ALI were used to screen for key differentially expressed genes (DEGs). Using bioinformatics analysis methods such as Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and protein-protein interaction network analyses, the pathogenesis of sepsis-induced ALI was revealed. Intratracheal infusion of lipopolysaccharide (LPS, 10 mg/kg) induced ALI in wild-type (WT) and S100A9 knockout mice. Multiomics analyses (transcriptomics and proteomics) were performed to investigate the potential mechanisms by which S100A9 exacerbates acute lung damage. Hematoxylin-eosin, Giemsa, and TUNEL staining were used to evaluate lung injury and cell apoptosis. LPS (10 μg/mL)-induced murine lung epithelial MLE-12 cells were utilized to mimic ALI and were modulated by S100A9 lentiviral transfection. The impact of S100A9 on cell apoptosis and inflammatory responses were identified using flow cytometry and PCR. The expression of interleukin (IL)-17–nuclear factor kappa B (NFκB)–caspase-3 signaling components was identified using western blotting. ResultsSix common DEGs (S100A9, S100A8, IFITM6, SAA3, CD177, and MMP9) were identified in the six datasets related to ALI in sepsis. Compared to WT sepsis mice, S100A9 knockout significantly alleviated LPS-induced ALI in mice, with reduced lung structural damage and inflammatory exudation, decreased exfoliated cell and protein content in the lung lavage fluid, and reduced apoptosis and necrosis of pulmonary epithelial cells. Transcriptomic analysis revealed that knocking out S100A9 significantly affected 123 DEGs, which were enriched in immune responses, defense responses against bacteria or lipopolysaccharides, cytokine-cytokine receptor interactions, and the IL-17 signaling pathway. Proteomic analysis revealed that S100A9 knockout alleviated muscle contraction dysfunction and structural remodeling in sepsis-induced ALI. Multiomics analysis revealed that S100A9 may be closely related to interferon-induced proteins with tetratricopeptide repeats and oligoadenylate synthase-like proteins. LPS decreased MLE12 cell activity, accompanied by high expression of S100A9. The expression of IL-17RA, pNFκB, and cleaved-caspase-3 were increased by S100A9 overexpression and reduced by S100A9 knockdown in LPS-stimulated MLE12 cells. S100A9 knockdown decreases transcription of apoptosis-related markers Bax, Bcl and caspase-3, alleviating LPS-induced apoptosis. ConclusionsS100A9 as a key biomarker of sepsis-induced acute lung injury, and exacerbates lung damage and epithelial cell apoptosis induced by LPS via the IL-17–NFκB–caspase-3 signaling pathway.

PEBP4 deficiency aggravates LPS-induced acute lung injury and alveolar fluid clearance impairment via modulating PI3K/AKT signaling pathway

Acute lung injury (ALI) is a common clinical syndrome, which often results in pulmonary edema and respiratory distress. It has been recently reported that phosphatidylethanolamine binding protein 4 (PEBP4), a basic cytoplasmic protein, has anti-inflammatory and hepatoprotective effects, but its relationship with ALI remains undefined so far. In this study, we generated PEBP4 knockout (KO) mice to investigate the potential function of PEBP4, as well as to evaluate the capacity of alveolar fluid clearance (AFC) and the activity of phosphatidylinositide 3-kinases (PI3K)/serine-theronine protein kinase B (PKB, also known as AKT) signaling pathway in lipopolysaccharide (LPS)-induced ALI mice models. We found that PEBP4 deficiency exacerbated lung pathological damage and edema, and increased the wet/dry weight ratio and total protein concentration of bronchoalveolar lavage fluid (BALF) in LPS-treated mice. Meanwhile, PEBP4 KO promoted an LPS-induced rise in the pulmonary myeloperoxidase (MPO) activity, serum interleuin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α levels, and pulmonary cyclooxygenase-2 (COX-2) expression. Mechanically, PEBP4 deletion further reduced the protein expression of Na+ transport markers, including epithelial sodium channel (ENaC)-α, ENaC-γ, Na,K-ATPase α1, and Na,K-ATPase β1, and strengthened the inhibition of PI3K/AKT signaling in LPS-challenged mice. Furthermore, we demonstrated that selective activation of PI3K/AKT with 740YP or SC79 partially reversed all of the above effects caused by PEBP4 KO in LPS-treated mice. Altogether, our results indicated the PEBP4 deletion has a deterioration effect on LPS-induced ALI by impairing the capacity of AFC, which may be achieved through modulating the PI3K/AKT pathway.

The Protective Effects of Modified Dachaihu Decoction against LPS-induced Acute Lung Injury via Modulating PI3K/Akt Signalling Pathway

Background: Modified Dachaihu decoction (MDD) is a herbal prescription that has shown promising therapeutic benefits in ameliorating pulmonary diseases in clinical practice. However, the detailed mechanisms remain unclear. Objective: This study aimed to elucidate the lung-protective effects of MDD against acute lung injury (ALI) and the involvement of underlying mechanisms. Methods: High-performance liquid chromatography (HPLC) was performed to identify the main active ingredients of MDD. Network pharmacological method was adapted to explore the potential mechanisms. Mice were orally administered MDD (11.25, 22.5, and 45 g/kg) once daily for 7 days. H&E staining was performed to evaluate histological changes in the lungs. Levels of inflammatory cytokines and oxidative stress markers were measured to determine the extent of lung injury. Total protein content in bronchoalveolar lavage fluid (BALF) and lung wet/dry weight ratio were measured to assess the severity of pulmonary edema. TUNEL staining and immunohistochemistry analysis were performed to detect apoptosis. RT-qPCR and western blotting were performed to validate the mechanisms involved. Results: About 10 main active ingredients of MDD were identified. Notably, treatment with MDD resulted in a remarkable reduction in total protein content in BALF and lung W/D weight ratio, as well as substantial mitigation of the inflammatory response and oxidative stress. Mechanistically, the PI3K/Akt signalling pathway was activated. Moreover, MDD pretreatment downregulated p53 and caspase-9 mRNA expression and decreased the Bax/Bcl-2 ratio to ameliorate lung apoptosis. Conclusions: MDD exhibited pronounced therapeutic effects via attenuating inflammatory response, oxidative stress, and apoptosis. These therapeutic effects could be attributed to the synergistic effect of the main active ingredients and are believed to be associated with the activation of the PI3K/Akt pathway.

Fortunellin ameliorates LPS-induced acute lung injury, inflammation, and collagen deposition by restraining the TLR4/NF-κB/NLRP3 pathway.

Acute lung injury (ALI) is the prevalent respiratory disease of acute inflammation with high morbidity and mortality. Fortunellin has anti-inflammation property, but its role in ALI remains elusive. Thus, this study clarified the function of fortunellin on ALI pathogenesis. The ALI mouse model was established by lipopolysaccharide (LPS) induction, and lung tissue damage was evaluated utilizing hematoxylin-eosin (HE) staining. The edema of lung tissue was measured by the lung wet/dry (W/D) ratio. The lung capillary permeability was reflected by the protein content in bronchoalveolar lavage fluid (BALF). Inflammatory cell infiltration was measured by the evaluation of the content of myeloperoxidase (MPO), neutrophils, and leukocytes in BALF. Cell apoptosis was measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The secretions of inflammatory cytokines were quantified using enzyme-linked immunosorbent assay (ELISA) assays. Lung tissue collagen deposition was evaluated by Masson staining. Fortunellin attenuated LPS-induced lung tissue damage and reduced the W/D ratio, the content of MPO in lung tissue, the total protein contents in BALF, and the neutrophils and leukocytes number. Besides, fortunellin alleviated LPS-stimulated lung tissue apoptosis, inflammatory response, and collagen deposition. Furthermore, Fortunellin repressed the activity of the Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB)/NLR Family Pyrin Domain Containing 3 (NLRP3) pathway in the LPS-stimulated ALI model and LPS-induced RAW264.7 cells. Moreover, fortunellin attenuated LPS-stimulated tissue injury, apoptosis, inflammation, and collagen deposition of the lung via restraining the TLR4/NF-κB/NLRP3 pathway. Fortunellin attenuated LPS-stimulated ALI through repressing the TLR4/NF-κB/NLRP3 pathway. Fortunellin may be a valuable drug for ALI therapy.

Blockage of mechanosensitive Piezo1 channel alleviates the severity of experimental malaria-associated acute lung injury

BackgroundMalaria-associated acute lung injury (MA-ALI) is a well-recognized clinical complication of severe, complicated malaria that is partly driven by sequestrations of infected red blood cells (iRBCs) on lung postcapillary induced impaired blood flow. In earlier studies the mechanosensitive Piezo1 channel emerged as a regulator of mechanical stimuli, but the function and underlying mechanism of Piezo1 impacting MA-ALI severity via sensing the impaired pulmonary blood flow are still not fully elucidated. Thus, the present study aimed to explore the role of Piezo1 in the severity of murine MA-ALI.MethodsHere, we utilized a widely accepted murine model of MA-ALI using C57BL/6 mice with Plasmodium berghei ANKA infection and then added a Piezo1 inhibitor (GsMTx4) to the model. The iRBC-stimulated Raw264.7 macrophages in vitro were also targeted with GsMTx4 to further explore the potential mechanism.ResultsOur data showed an elevation in the expression of Piezo1 and number of Piezo1+-CD68+ macrophages in lung tissues of the experimental MA-ALI mice. Compared to the infected control mice, the blockage of Piezo1 with GsMTx4 dramatically improved the survival rate but decreased body weight loss, peripheral blood parasitemia/lung parasite burden, experimental cerebral malaria incidence, total protein concentrations in bronchoalveolar lavage fluid, lung wet/dry weight ratio, vascular leakage, pathological damage, apoptosis and number of CD68+ and CD86+ macrophages in lung tissues. This was accompanied by a dramatic increase in the number of CD206+ macrophages (M2-like subtype), upregulation of anti-inflammatory cytokines (e.g. IL-4 and IL-10) and downregulation of pro-inflammatory cytokines (e.g. TNF-α and IL-1β). In addition, GsMTx4 treatment remarkably decreased pulmonary intracellular iron accumulation, protein level of 4-HNE (an activator of ferroptosis) and the number of CD68+-Piezo1+ and CD68+-4-HNE+ macrophages but significantly increased protein levels of GPX4 (an inhibitor of ferroptosis) in experimental MA-ALI mice. Similarly, in vitro study showed that the administration of GsMTx4 led to a remarkable elevation in the mRNA levels of CD206, IL-4, IL-10 and GPX-4 but to a substantial decline in CD86, TNF-α, IL-1β and 4-HNE in the iRBC-stimulated Raw264.7 cells.ConclusionsOur findings indicated that blockage of Piezo1 with GsMTx4 alleviated the severity of experimental MA-ALI in mice partly by triggering pulmonary macrophage M2 polarization and subsequent anti-inflammatory responses but inhibited apoptosis and ferroptosis in lung tissue. Our data suggested that targeting Piezo1 in macrophages could be a promising therapeutic strategy for treating MA-ALI.Graphical

Electroacupuncture alleviates ventilator-induced lung injury in mice by inhibiting the TLR4/NF-κB signaling pathway

ObjectiveThis study was aimed to explore the protective effect of electroacupuncture (EA) pretreatment at Zusanli point (ST36) on ventilation-induced lung injury (VILI) and its potential anti-inflammatory mechanism.MethodsHigh tidal volume ventilation was used to induce the VILI in mice, and EA pretreatment at ST36 was given for 7 consecutive days. The wet/dry ratio and pathological injury score of lung tissue, and total protein content of pulmonary alveolar lavage fluid (BALF) were detected after 4 h of mechanical ventilation (MV). Meanwhile, the expressions of TLR4 and NF- κB in lung tissue were evaluated by Western Blot, and the inflammatory factors in lung tissue were detected by ELISA.ResultsAfter four hours of mechanical ventilation, mice with ventilator-induced lung injury showed significant increases in lung wet/dry ratio, tissue damage scores, and protein content in bronchoalveolar lavage fluid. Pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) and TLR4/NF-κB expression levels in the lung were also markedly elevated (P < 0.05). Conversely, ST36 acupuncture point pre-treatment significantly reduced these parameters (P < 0.05).ConclusionEA pretreatment at ST36 could alleviate the inflammatory response for VILI via inhibiting TLR4/NF- κB pathway.

QiShenYiQi pills preserve endothelial barrier integrity to mitigate sepsis-induced acute lung injury by inhibiting ferroptosis

Ethnopharmacological relevanceThe QiShengYiQi pill (QSYQ) is a traditional Chinese medicinal formulation. The effectiveness and safety of QSYQ in treating respiratory system disorders have been confirmed. Its pharmacological actions include anti-inflammation, antioxidative stress, and improving energy metabolism. However, the mechanism of QSYQ in treating sepsis-induced acute lung injury (si-ALI) remains unclear. Aim of the studySi-ALI presents a clinical challenge with high incidence and mortality rates. This study aims to confirm the efficacy of QSYQ in si-ALI and to explore the potential mechanisms, providing a scientific foundation for its application and insights for optimizing treatment strategies and identifying potential active components. Materials and methodsThe impact of QSYQ on si-ALI was evaluated using the cecal ligation and puncture (CLP) experimental sepsis animal model. The effects of QSYQ on endothelial cells were observed through coculturing with LPS-stimulated macrophage-conditioned medium. Inflammatory cytokine levels, HE staining, Evans blue staining, lung wet/dry ratio, and cell count and protein content in bronchoalveolar lavage fluid were used to assess the degree of lung injury. Network pharmacology was utilized to investigate the potential mechanisms of QSYQ in treating si-ALI. Western blot and immunofluorescence analyses were used to evaluate barrier integrity and validate mechanistically relevant proteins. ResultsQSYQ reduced the inflammation and alleviated pulmonary vascular barrier damage in CLP mice (all P < 0.05). A total of 127 potential targets through which QSYQ regulates si-ALI were identified, predominantly enriched in the RAGE pathway. The results of protein-protein interaction analysis suggest that COX2, a well-established critical marker of ferroptosis, is among the key targets. In vitro and in vivo studies demonstrated that QSYQ mitigated ferroptosis and vascular barrier damage in sepsis (all P < 0.05), accompanied by a reduction in oxidative stress and the inhibition of the COX2 and RAGE (all P < 0.05). ConclusionsThis study demonstrated that QSYQ maintains pulmonary vascular barrier integrity by inhibiting ferroptosis in CLP mice. These findings partially elucidate the mechanism of QSYQ in si-ALI and further clarify the active components of QSYQ, thereby providing a scientific theoretical basis for treating si-ALI with QSYQ.

Veronica linariifolia subsp. dilatata ameliorates LPS-induced acute lung injury by attenuating endothelial cell barrier dysfunction via EGFR/Akt/ZO-1 pathway

Ethnopharmacological relevanceThe dried aerial parts of Veronica linariifolia subsp. dilatata (Nakai & Kitag.) D.Y.Hong named Shui Man Jing (SMJ) is a traditional Chinese medicine with a long history of clinical use in the treatment of chronic bronchitis and coughing up blood, however, its role on acute lung injury (ALI) has not been revealed yet. Aim of the studyTo assess the efficiency of SMJ on ALI and to investigate whether it inhibited endothelial barrier dysfunction by regulating the EGFR/Akt/ZO-1 pathway to alleviate ALI in vivo and in vitro based on the result of network pharmacology. Materials and methodsAn in vivo model of ALI was established using inhalation of atomized lipopolysaccharide (LPS), and the effects of SMJ on ALI were evaluated through histopathological examination and inflammatory cytokines, lung histology and edema, vascular and alveolar barrier disruption. Network pharmacology was applied to predict the mechanism of SMJ in the treatment of ALI. The crucial targets were validated by RT-PCR, Western Blotting, molecular docking, immunohistochemistry and immunofluorescence methods in vivo and in virto. ResultsAdministration of SMJ protected mice against LPS-induced ALI, including ameliorating the histological alterations in the lung tissues, and decreasing lung edema, protein content of bronchoalveolar lavage fluid, infiltration of inflammatory cell and secretion of cytokines. SMJ exerted protective effects in ALI by inhibiting endothelial barrier dysfunction in mice and bEnd.3 cell. SMJ relieved endothelial barrier dysfunction induced by LPS through upregulating the EGFR expression. SMJ also increased the phosphorylation of Akt, and ZO-1 expression both in vivo and in vitro. ConclusionSMJ attenuates vascular endothelial barrier dysfunction for LPS-induced ALI via EGFR/Akt/ZO-1 pathway, and is a promising novel therapeutic candidate for ALI.

Sodium butyrate ameliorates sepsis-associated lung injury by enhancing gut and lung barrier function in combination with modulation of CD4+Foxp3+ regulatory T cells

Sepsis-associated lung injury often coexists with intestinal dysfunction. Butyrate, an essential gut microbiota metabolite, participates in gut-lung crosstalk and has immunoregulatory effects. This study aims to investigate the effect and mechanism of sodium butyrate (NaB) on lung injury. Sepsis-associated lung injury was established in mice by cecal ligation and puncture (CLP). Mice in treatment groups received NaB gavage after surgery. The survival rate, the oxygenation index and the lung wet-to-dry weight (W/D) ratio were calculated respectively. Pulmonary and intestinal histologic changes were observed. The total protein concentration in bronchoalveolar lavage fluid (BALF) was measured, and inflammatory factors in serum and BALF were examined. Diamine oxidase (DAO), lipopolysaccharide (LPS), and surfactant-associated protein D (SP-D) levels in serum and amphiregulin in lung tissue were assessed. Intercellular junction protein expression in the lung and intestinal tissues were examined. Changes in immune cells were analyzed. NaB treatment improved the survival rate, the oxygenation index and the histologic changes. NaB decreased the W/D ratio, total protein concentration, and the levels of proinflammatory cytokines, as well as SP-D, DAO and LPS, while increased the levels of anti-inflammatory cytokines and amphiregulin. The intercellular junction protein expression were improved by NaB. Furthermore, the CD4+/CD8+ T-cell ratio and the proportion of CD4+Foxp3+ regulatory T cells (Tregs) were increased by NaB. Our data suggested that NaB gavage effectively improved the survival rate and mitigated lung injury in CLP mice. The possible mechanism was that NaB augmented CD4+Foxp3+ Tregs and enhanced the barrier function of the gut and the lung.

MiR-2113 overexpression attenuates sepsis-induced acute pulmonary dysfunction, inflammation and fibrosis by inhibition of HMGB1

PurposeSepsis-induced acute lung injury is related to high mortality. MiR-2113 possesses important functions in human diseases. This research aimed to clarify the role and mechanism of miR-2113 in sepsis-induced acute lung injury. MethodsThe expression of miR-2113 in lipopolysaccharide (LPS)-induced MLE-12 cells, serum of sepsis patients, and cecal ligation and puncture mouse models was examined using quantitative real-time PCR. The functions of miR-2113 in LPS-treated MLE-12 cells were estimated by Cell Counting Kit-8 assay, flow cytometry, enzyme-linked immunosorbent assay, Western blot, and immunofluorescence. The influences of miR-2113 in cecal ligation and puncture-induced acute lung injury in mice were assessed by hematoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay, acute pulmonary dysfunction analysis, lactate dehydrogenase levels and total protein concentrations in bronchoalveolar lavage fluid, and Masson staining. Also, the mechanism of miR-2113 was examined using a dual-luciferase reporter assay. ResultsMiR-2113 expression was decreased in LPS-induced MLE-12 cells, serum of sepsis patients, and cecal ligation and puncture mouse models. miR-2113 overexpression restored LPS-reduced MLE-12 cell proliferation, but alleviated LPS-induced apoptosis and markers of inflammation and fibrosis in MLE-12 cells. Moreover, we found that miR-2113 mimic reduced LPS-induced MLE-12 cell injury by negatively regulating high-mobility group box 1. In vivo data further confirmed that miR-2113 overexpression alleviated acute pulmonary dysfunction, inflammation and fibrosis in cecal ligation and puncture-induced sepsis mice. ConclusionMiR-2113 relieved sepsis-induced acute pulmonary dysfunction, inflammation and fibrosis through decreasing high-mobility group box 1.

Ginkgo biloba Extract 50 (GBE50) Exerts Antifibrotic and Antioxidant Effects on Pulmonary Fibrosis in Mice by Regulating Nrf2 and TGF-β1/Smad Pathways.

Pulmonary fibrosis (PF) is a progressive lung disorder with a poor prognosis. GBE50 is a new standardized Ginkgo biloba extract that has been widely used in cardiovascular diseases. However, the protective mechanism of GBE50 against PF remains to be elucidated. C57BL/6J mice were treated with bleomycin (Bleo) to induce PF in the presence or absence of GBE50. Protein content in bronchoalveolar lavage fluid (BALF) and wet weight/dry weight ratio were examined for analysis of pulmonary edema. Hematoxylin-eosin staining and Masson trichrome staining were used for histopathological observation of murine lung tissues. Ashcroft score was used for semi-quantitation of lung fibrosis degree. RT-qPCR was utilized for assessing mRNA levels of pro-fibrotic mediators in lung tissues. TUNEL staining was implemented for cell apoptosis assessment. The levels of oxidative stress- and inflammation-related markers were evaluated by corresponding commercial assay kits. Western blotting was used to evaluate levels of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling- and transforming growth factor (TGF)-β1/SMAD signaling-related proteins. GBE50 alleviated lung injury and severity of fibrosis, reduced collagen deposition and cell apoptosis in lung tissues, and suppressed inflammatory response and oxidative stress injury in Bleo-stimulated PF mice. GBE50 activated Nrf2 signaling pathway and inactivated TGF-β1/SMAD signaling pathway in the lungs of Bleo-induced PF mice. Inhibition of Nrf2 signaling reversed GBE50-mediated inactivation of TGF-β1/SMAD signaling and attenuation of inflammation and oxidative stress in Bleo-induced PF mice. GBE50 protects against Bleo-induced PF in mice by mitigating fibrosis, inflammation and oxidative stress via Nrf2 and TGF-β1/SMAD signaling pathways.

Pleural inflammatory response, mesothelin content and DNA damage in mice at one-year after intra-pleural carbon nanotube administration

Many in vitro and in vivo studies have shown that exposure to carbon nanotubes (CNTs) is associated with inflammation, oxidative stress and genotoxicity, although there is a paucity of studies on these effects in the pleural cavity. In the present study, we investigated adverse outcomes of pleural exposure to multi-walled CNTs (MWCNT-7, NM-401 and NM-403) and single-walled CNTs (NM-411). Female C57BL/6 mice were exposed to 0.2 or 5 µg of CNTs by intra-pleural injection and sacrificed one-year post-exposure. Exposure to long and straight types of MWCNTs (i.e. MWCNT-7 and NM-401) was associated with decreased number of macrophages and increased number of neutrophils and eosinophils in pleural lavage fluid. Increased protein content in the pleural lavage fluid was also observed in mice exposed to MWCNT-7 and NM-401. The concentration of mesothelin was increased in mice exposed to MWCNT-7 and NM-411. Levels of DNA strand breaks and DNA oxidation damage, measured by the comet assay, were unaltered in cells from pleural scrape. Extra-pleural effects were seen in CNT exposed mice, including enlarged and pigmented mediastinal lymph nodes (all four types of CNTs), pericardial plaques (MWCNT-7 and NM-401), macroscopic abnormalities on the liver (MWCNT-7) and ovaries/uterus (NM-411). In conclusion, the results demonstrate that intra-pleural exposure to long and straight MWCNTs is associated with adverse outcomes. Certain observations such as increased content of mesothelin in pleural lavage fluid and ovarian/uterine abnormalities in mice exposed to NM-411 suggests that exposure to SWCNTs may also be associated with some adverse outcomes.

Comparison on anti-inflammatory activity of Gynostemma pentaphyllum processed with different methods

The aim of this study is to investigate the effects of Gynostemma pentaphyllum dried with two different methods(air drying and heating) on inflammation in acute lung injury(ALI) mice in vivo and in vitro. Lipopolysaccharide(LPS) was sprayed into the airway of wild type C57BL/6J male mice to establish the model, and the drug was injected into the tail vein 24 h after modeling. Lung function, lung tissue wet/dry weight(W/D) ratio, the total protein concentration, interleukin 6(IL-6), IL-1β, and tumor necrosis factor-α(TNF-α) in the bronchoalveolar lavage fluid(BALF), and pathological changes of the lung tissue were used to evaluate the effects of different gypenosides on ALI mice. The results showed that total gypenosides(YGGPs) and the gypenosides substituted with one or two glycosyl(GPs_(1-2)) in the air-dried sample improved the lung function, significantly lowered the levels of IL-1β and TNF-α in BALF, and alleviated the lung inflammation of ALI mice. Moreover, GPs_(1-2) had a more significant effect on inhibiting NO release in RAW264.7 cells. This study showed that different drying methods affected the anti-inflammatory activity of G. pentaphyllum, and the rare saponins in the air-dried sample without heating had better anti-inflammatory activity.

Systemic antibiotics cause deterioration of emphysema associated with exaggerated inflammation and autophagy

The interaction between the microbial environment and the host is important for immune homeostasis. Recent research suggests that microbiota dysbiosis can be involved in respiratory diseases. Emphysema is a chronic inflammatory disease, but it is unclear whether dysbiosis caused by antibiotics can affect disease progression. Here, we tried to elucidate the effect of systemic antibiotics on smoking-exposed emphysema models. In this study, the antibiotic mixture caused more alveolar destruction and airspace expansion in the smoking group than in the smoking only or control groups. This emphysema aggravation as a result of antibiotic exposure was associated with increased levels of inflammatory cells, IL-6, IFNγ and protein concentrations in bronchoalveolar lavage fluid. Proteomics analysis indicated that autophagy could be involved in antibiotic-associated emphysema aggravation, and increased protein levels of LC3B, atg3, and atg7 were identified by Western blotting. In microbiome and metabolome analyses, the composition of the gut microbiota was different with smoking and antibiotic exposure, and the levels of short-chain fatty acids (SCFAs), including acetate and propionate, were reduced by antibiotic exposure. SCFA administration restored emphysema development with reduced inflammatory cells, IL-6, and IFNγ and decreased LC3B, atg3, and atg7 levels. In conclusion, antibiotics can aggravate emphysema, and inflammation and autophagy may be associated with this aggravation. This study provides important insight into the systemic impact of microbial dysbiosis and the therapeutic potential of utilizing the gut microbiota in emphysema.

Notoginsenoside R1 protects against hypobaric hypoxia-induced high-altitude pulmonary edema by inhibiting apoptosis via ERK1/2-P90rsk-BAD ignaling pathway

High-altitude pulmonary edema (HAPE) is a potentially fatal disease. Notoginsenoside R1 is a novel phytoestrogen with anti-inflammatory, antioxidant and anti-apoptosis properties. However, its effects and underlying mechanisms in the protection of hypobaric hypoxia-induced HAPE rats remains unclear. This study aimed to explore the protective effects and underlying mechanisms of Notoginsenoside R1 in hypobaric hypoxia-induced HAPE. We found that Notoginsenoside R1 alleviated the lung tissue injury, decreased lung wet/dry ratio, and reduced inflammation and oxidative stress. Additionally, Notoginsenoside R1 ameliorated the changes in arterial blood gas, decreased the total protein concentration in bronchoalveolar lavage fluid, and inhibited the occurrence of apoptosis caused by HAPE. In the process of further exploration of the mechanism, it was found that Notoginsenoside R1 could promote the activation of ERK1/2-P90rsk-BAD signaling pathway, and the effect of Notoginsenoside R1 was attenuated after the use of ERK1/2 inhibitor U0126. Our study indicated that the protective effects of Notoginsenoside R1 against HAPE were mainly related to the inhibition of inflammation, oxidative stress, and apoptosis. Notoginsenoside R1 may be a potential candidate for preventing HAPE.

Tamibarotene targets heparin-binding protein for attenuating lung injury in sepsis.

Excessively active pulmonary inflammation is a hallmark of sepsis-induced lung damage. A synthetic retinoid drug called tamibarotene reduces inflammation in a variety of conditions, including acute promyelocytic leukemia (APL), renal fibrosis, and neuroinflammation. Its effect on sepsis-related lung injury, however, has not been explained. The purpose of the study was to investigate how tamibarotene affected lung damage induced by cecal ligation and puncture (CLP) procedure. A CLP sepsis mouse model was developed, and tamibarotene was pretreated to determine whether it improved lung injury and survival. The degree of lung injury was evaluated using the Hematoxylin and eosin staining and lung injury score. In order to determine pulmonary vascular permeability, measurements were taken for total protein and cell content of bronchoalveolar lavage fluid (BALF), wet/dry ratio of the lung, and Evans blue stain. The BALF inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β, and IL-17A were discovered by enzyme-linked immunosorbent serologic assay (ELISA). Then, the levels of heparin-binding protein (HBP), and phospho-nuclear factor kappa-B (p-NF-κB) P65, and NF-κB P65 were determined using ELISA and Western blot analysis, respectively. Tamibarotene considerably increases survival and lessens lung damage stimulated by sepsis. Specifically, tamibarotene significantly relieves pulmonary vascular permeability and inhibits inflammation response in sepsis. Moreover, we further confirmed that these ameliorating effects of tamibarotene on sepsis may be exerted by targeting HBP and regulating the activation of NF-κB signaling pathway. These findings demonstrated that tamibarotene lessens sepsis-induced lung injury, and the effect could be exerted by targeting HBP and thereby deregulating the NF-κB signaling pathway.

A study of the role of myeloid-derived growth factor on acute lung injury in vitro and In vivo

Objectives: Bone marrow-derived mesenchymal stem cells (BMSCs) are considered to have potential clinical application value in the treatment of acute lung injury (ALI). Myeloid-derived growth factor (MYDGF) can promote the proliferation of stem cell. We hypothesized that MYDGF may play a role in reducing lung injury in vitro and in vivo through bone marrow mesenchymal stem cells. Methods: An in vitro model of lipopolysaccharide (LPS)(MLE-12) was established, which was divided into five groups: A: MLE-12; B: MLE-12+LPS; C: MLE-12+LPS + BMSCs; D: MLE-12+LPS + MYDGF; and E: MLE-12+LPS + BMSCs + MYDGF. A Cell Counting Kit-8 was used to detect the OD value. And an ALI model was constructed by inducing mice with a lipopolysaccharide. Forty male Balb/c mice were randomly divided into five groups: A control group; B: model group; C: LPS + BMSCs; D: LPS + MYDGF; E: LPS +BMSCs +MYDGF. Specimens were collected after 24 h. Hematoxylin-eosin (HE)-staining was performed on the tissue sections. The protein concentration in the alveolar lavage fluid was measure by bicinchoninic acid (BCA). The NF-κB, p-Akt, Bax, and Bcl-2 protein expression was detected through Western blotting, and Enzyme linked immunosorbent assay (ELISA) was used to measure the expression of serum interleukin-6, interleukin-10, and TNF-α. Results: Compared with the model group, BMSCs and MYDGF can alleviate the ALI induced by lipopolysaccharide in vitro and vivo ( p &lt; .05). Conclusion: We found that the combined treatment effect of MYDGF and BMSCs was better than using MYDGF or BMSCs alone. We speculate that a pretreatment with MYDGF after ALI in mice may improve the survival and growth of transplanted MSCs, thereby improving the curative effect of cell transplantation.