ALI Model Research Articles

Overexpression of FoxM1 optimizes the therapeutic effect of bone marrow mesenchymal stem cells on acute respiratory distress syndrome

BackgroundInjury of alveolar epithelial cells and capillary endothelial cells is crucial in the pathogenesis of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Mesenchymal stem cells (MSCs) are a promising cell source for ALI/ARDS treatment. Overexpression of Fork head box protein M1 (FoxM1) facilitates MSC differentiation into alveolar type II (AT II) cells in vitro. Moreover, FoxM1 has been shown to repair the endothelial barrier. Therefore, this study explored whether overexpression of FoxM1 promotes the therapeutic effect of bone marrow-derived MSCs (BMSCs) on ARDS by differentiation of BMSCs into AT II cells or a paracrine mechanism.MethodsA septic ALI model was established in mice by intraperitoneal administration of lipopolysaccharide. The protective effect of BMSCs-FoxM1 on ALI was explored by detecting pathological variations in the lung, total protein concentration in bronchoalveolar lavage fluid (BALF), wet/dry (W/D) lung weight ratio, oxidative stress levels, cytokine levels, and retention of BMSCs in the lung. In addition, we assessed whether FoxM1 overexpression promoted the therapeutic effect of BMSCs on ALI/ARDS by differentiating into AT II cells using SPC−/− mice. Furthermore, the protective effect of BMSCs-FoxM1 on lipopolysaccharide-induced endothelial cell (EC) injury was explored by detecting EC proliferation, apoptosis, scratch wounds, tube formation, permeability, and oxidative stress, and analyzing whether the Wnt/β-catenin pathway contributes to the regulatory mechanism in vitro using a pathway inhibitor.ResultsCompared with BMSCs-Vector, treatment with BMSCs-FoxM1 significantly decreased the W/D lung weight ratio, total BALF protein level, lung injury score, oxidative stress, and cytokine levels. With the detected track of BMSCs-FoxM1, we observed a low residency rate and short duration of residency in the lung. Notably, SPC was not expressed in SPC−/− mice injected with BMSCs-FoxM1. Furthermore, BMSCs-FoxM1 enhanced EC proliferation, migration, and tube formation; inhibited EC apoptosis and inflammation; and maintained vascular integrity through activation of the Wnt/β-catenin pathway, which was partially reversed by XAV-939.ConclusionOverexpression of FoxM1 enhanced the therapeutic effect of BMSCs on ARDS, possibly through a paracrine mechanism rather than by promoting BMSC differentiation into AT II cells in vivo, and prevented LPS-induced EC barrier disruption partially through activating the Wnt/β-catenin signaling pathway in vitro.

Cryptotanshinone attenuates LPS-induced acute lung injury by regulating metabolic reprogramming of macrophage.

Acute lung injury (ALI) is a life-threatening inflammatory disease without effective therapeutic regimen. Macrophage polarization plays a key role in the initiation and resolution of pulmonary inflammation. Therefore, modulating macrophage phenotype is a potentially effective way for acute lung injury. Cryptotanshinone (CTS) is a lipophilic bioactive compound extracted from the root of Salvia miltiorrhiza with a variety of pharmacological effects, especially the anti-inflammatory role. In this study, we investigated the therapeutic and immunomodulatory effects of CTS on ALI. The rat model of ALI was established by intratracheal instillation of LPS (5 mg/kg) to evaluate the lung protective effect of CTS in vivo and to explore the regulation of CTS on the phenotype of lung macrophage polarization. LPS (1 μg/mL) was used to stimulate RAW264.7 macrophages in vitro to further explore the effect of CTS on the polarization and metabolic reprogramming of RAW264.7 macrophages and to clarify the potential mechanism of CTS anti-ALI. CTS significantly improved lung function, reduced pulmonary edema, effectively inhibited pulmonary inflammatory infiltration, and alleviated ALI. Both in vivo and in vitro results revealed that CTS inhibited the differentiation of macrophage into the M1 phenotype and promoted polarization into M2 phenotype during ALI. Further in vitro studies indicated that CTS significantly suppressed LPS-induced metabolic transition from aerobic oxidation to glycolysis in macrophages. Mechanistically, CTS blocked LPS-induced metabolic transformation of macrophages by activating AMPK. These findings demonstrated that CTS regulates macrophage metabolism by activating AMPK, and then induced M1-type macrophages to transform into M2-type macrophages, thereby alleviating the inflammatory response of ALI, suggesting that CTS might be a potential anti-ALI agent.

Scoring the Original Soundtrack of an Escape Room

Through the example of Nobody – Vis et ressens (Nice, France, 2021), this article sheds light on the musical creation process involved in the conception of an automatized escape room, where participants experience a multimodal experience (sound, light, scenery, video) driven by an original scenario. As the composer, sound designer, and computer music designer, I propose to study Nobody as a playful game. I show how current ludomusicology insights can be used and adapted to a game that is played in a physical space. After first exposing considerations of both technical and aesthetic aspects, I explain the artistic choices made for creating the soundtrack of six interactive puzzles. Through practice-led research of these different mini music games, I analyze how the influence of electronic dance music (EDM) and related club culture are melded together into the development of the plot and its retro-futuristic theme. I show how constraints forced me to adapt the audio content yet also opened a wide range of musical possibilities. The analysis helps clarify how what I call “game music” differs from “background music”. Moreover, the discussion highlights compositional issues that are similar to video game music-making and shows that escape rooms need to be studied further within the field of ludomusicology. Although some of the analytical tools remain valid because of their technical aspect (transition types, typology of music games, transition speed quantization) or their theoretical aspects (ALI model, branching-layering concept, triple lock of synchronization), traditional concepts such as “immersion” or “game feel” need to be questioned for escape room games, as that type of game takes place in a physical space.

FGF10 ameliorates lipopolysaccharide-induced acute lung injury in mice via the BMP4-autophagy pathway.

Introduction: Damage to alveolar epithelial cells caused by uncontrolled inflammation is considered to be the main pathophysiological change in acute lung injury. FGF10 plays an important role as a fibroblast growth factor in lung development and lung diseases, but its protective effect against acute lung injury is unclear. Therefore, this study aimed to investigate protective effect and mechanism of FGF10 on acute lung injury in mice. Methods: ALI was induced by intratracheal injection of LPS into 57BL/6J mice. Six hours later, lung bronchoalveolar lavage fluid (BALF) was acquired to analyse cells, protein and the determination of pro-inflammatory factor levels, and lung issues were collected for histologic examination and wet/dry (W/D) weight ratio analysis and blot analysis of protein expression. Results: We found that FGF10 can prevent the release of IL-6, TNF-α, and IL-1β, increase the expression of BMP4 and autophagy pathway, promote the regeneration of alveolar epithelial type Ⅱ cells, and improve acute lung injury. BMP4 gene knockdown decreased the protective effect of FGF10 on the lung tissue of mice. However, the activation of autophagy was reduced after BMP4 inhibition by Noggin. Additionally, the inhibition of autophagy by 3-MA also lowered the protective effect of FGF10 on alveolar epithelial cells induced by LPS. Conclusions: These data suggest that the protective effect of FGF10 is related to the activation of autophagy and regeneration of alveolar epithelial cells in an LPS-induced ALI model, and that the activation of autophagy may depend on the increase in BMP4 expression.

Dexmedetomidine alleviates acute lung injury by promoting Tregs differentiation via activation of AMPK/SIRT1 pathway.

To explore the anti-inflammatory effect and the potential mechanism of dexmedetomidine in ARDS/ALI. C57BL/6 mice and EL-4 cells were used in this research. The ALI model was established by CLP. The level of inflammatory cytokines in the lung and blood, the severity of lung injury, the expression of Foxp3, and the proportion of Tregs were detected before and after dexmedetomidine treatment. The expression of the AMPK/SIRT1 after dexmedetomidine treatment was detected in vivo and in vitro. After blocking the AMPK/SIRT1 pathway or depleting Tregs in vivo, the level of the inflammatory response, tissue injury, and Tregs differentiation were detected again to clarify the effect of dexmedetomidine. Dexmedetomidine significantly reduced systemic inflammation and lung injury in CLP mice. Dexmedetomidine enhanced the Foxp3 expression in the lungs and the frequency of Tregs in the spleen. Dexmedetomidine up-regulated the protein expression of p-AMPK and SIRT1 in lungs and EL-4 cells and facilitated the differentiation of naïve CD4+ T cells into Tregs in vitro. Meanwhile, DEX also increased the expression of Helios in Treg cells. DEX could improve ARDS/ALI by facilitating the differentiation of Tregs from naïve CD4+ T cells via activating the AMPK/SIRT1 pathway.

1141. Phase 3 Trial (in progress) of the SYK Inhibitor Fostamatinib in Patients Hospitalized with COVID-19: Protocol and Study Implementation Updates

Abstract Background Immune dysregulation associated with COVID-19 includes immune cell activation, inflammatory cytokine release, and neutrophil extracellular trap release (NETosis), which are mediated by spleen tyrosine kinase (SYK) (Fig 1). Fostamatinib, an oral spleen tyrosine kinase (SYK) inhibitor, was approved for immune thrombocytopenia (ITP) in 2018, and the Phase 3 trials showed a lower than expected rate of thrombosis.1 Clinical studies showed a reduction in IL-6 in patients with rheumatoid arthritis.2 The active metabolite of fostamatinib (R406) protected against LPS-induced acute lung injury and thrombosis in mice3,4 and reduced MUC1 in a mouse model of ALI.5 Fostamatinib demonstrated abrogation of the hyperimmune response caused by anti-spike IgG,6 including reduction in hyperactivation of platelets7 and NETosis in neutrophils8 in in vitro studies using plasma from patients with severe COVID-19. A phase 2 study (NCT04579393) evaluated fostamatinib vs. placebo (all received standard of care [SOC]) in 59 hospitalized patients with COVID-19 and demonstrated reduction in mortality, ordinal scale scores, and number of days in the intensive care unit (ICU) as well as meeting the primary endpoint of safety.9 A phase 3 clinical study (NCT04629703) of fostamatinib for the treatment of COVID-19 is underway. Figure 1.Mechanism of Disease (COVID-19) and Role of SYK inhibition Methods A Phase 3, randomized, double-blind, placebo-controlled, adaptive design, multi-center study (NCT04629703) is underway to evaluate fostamatinib in 308 adult patients hospitalized with COVID-19 and on oxygen without intubation (Fig 2). Patients will receive fostamatinib 150 mg BID or placebo for 14 days; both arms receive SOC. At baseline, the clinical status score (8-point ordinal scale) had to be 5 or 6. Patients ≥ 65 years had to have ≥ 1 risk factor for severe disease and adults < 65 had to have ≥ 3. The primary outcome is days on oxygen (Day 1 to 29). Other endpoints include change in clinical status score, days in the ICU, time to hospital discharge, all-cause mortality, oxygen-free status and safety. Fostamatinib is investigational for COVID-19. Results Blinded data from this trial in progress are as of 2 December 2021. See Fig 3. Conclusion Final results of this Phase 3 trial are anticipated in 2022. Disclosures Lucy Yan, MD, PhD, Rigel Pharmaceuticals: Employee|Rigel Pharmaceuticals: Stocks/Bonds Ziad Mallat, MD, PhD, Rigel Pharmaceuticals: Advisor/Consultant Suzana Margareth Lobo, MD, PhD, Rigel Pharmaceuticals: Grant/Research Support Anuj Malik, MD, MS, Rigel Pharmaceuticals: Grant/Research Support Wolfgang Dummer, MD, PhD, Rigel Pharmaceuticals: Employee|Rigel Pharmaceuticals: Stocks/Bonds.

Cupping alleviates lung injury through the adenosine/A2BAR pathway

BackgroundAcute lung injury (ALI) is a serious condition. Inflammation plays a crucial role in the pathogenesis of ALI. Cupping, as a part of traditional Chinese medicine, is still a popular complementary and alternative therapy for a variety of ailments including respiratory diseases. However, reliable scientific data about cupping therapy are scarce. Adenosine, a purine nucleoside produced under metabolic stress by the action of extracellular ectonucleotidases (i.e. CD39 and CD73), can attenuate ALI through the A2BAR receptor. The aim of this study was to investigate the protective effect of cupping in a rat model of ALI and the role of adenosine in it. MethodsMale adult rats were subjected to ALI by intratracheal LPS instillation (0.3 mg/kg). Immediately after intratracheal LPS instillation, vacuum pressure was applied to a sanitized plastic bell cup on the back of the rat by suction for 10 min. Pulmonary injury and inflammation were assessed at 4 h after LPS challenge. The role of adenosine and A2BAR in cupping's protection after LPS instillation were evaluated. ResultsCupping alleviated LPS-induced lung injury, reduced inflammation and inhibited NF-kB activation in rats. Cupping upregulated CD39 and CD73 mRNA expression of the skin tissue at the cupping site and increased circulating levels of adenosine. Administration of PSB1115, a specific adenosine A2BAR receptor antagonist, abolished cupping's beneficial effects in LPS-induced ALI. ConclusionsCupping attenuates lung inflammation and injury through the adenosine/A2BAR pathway. The current study provides evidence-based information about cupping therapy in ALI.

5-Methoxyflavone alleviates LPS-mediated lung injury by promoting Nrf2-mediated the suppression of NOX4/TLR4 axis in bronchial epithelial cells and M1 polarization in macrophages

BackgroundAcute lung injury (ALI) arises from sepsis or bacterial infection, which are life-threatening respiratory disorders that cause the leading cause of death worldwide. 5-Methoxyflavone, a methylated flavonoid, is gaining increased attention for its various health benefits. In the current study, we investigated the potential effects of 5-methoxyflavone against LPS-mediated ALI and elucidated the corresponding possible mechanism.MethodsA mouse model with ALI was established by intratracheal instillation of LPS, and lung pathological changes, signaling pathway related proteins and apoptosis in lung tissues were estimated by H&E staining, immunofluorescence and TUNEL assay, respectively. Cell viability was evaluated by MTT assay; protein levels of pro-inflammatory mediators were measured by ELISA assay; levels of ROS and M1 macrophage polarization were assayed by flow cytometry; the expression of Nrf2 signaling, NOX4/TLR4 axis and P-STAT1 were detected by western blotting.ResultsOur results showed that 5-methoxyflavone treatment inhibited LPS-induced expression of NOX4 and TLR4 as well as the activation of downstream signaling (NF-κB and P38 MAPK), which was accompanied by markedly decreased ROS levels and pro-inflammatory cytokines (IL-6, TNF-α, MCP-1, and IL-8) in BEAS-2B cells. Moreover, we revealed that these effects of 5-methoxyflavone were related to its Nrf2 activating property, and blockade of Nrf2 prevented its inhibitory effects on NOX4/TLR4/NF-κB/P38 MAPK signaling, thus abrogating the anti-inflammatory effects of 5-methoxyflavone. Besides, the Nrf2 activating property of 5-methoxyflavone in RAW264.7 cells led to inhibition of LPS/IFN-γ-mediated STAT1 signaling, resulting in suppression of LPS/IFN-γ-induced M1 macrophage polarization and the repolarization of M2 macrophages to M1. In a mouse model of LPS-induced ALI, 5-methoxyflavone administration ameliorated LPS-mediated lung pathological changes, the increased lung index (lung/body weight ratio), and epithelial cell apoptosis. Meanwhile, we found 5-methoxyflavone effectively suppressed the hyperactive signaling pathways and the production of excessive pro-inflammatory mediators. Moreover, 5-methoxyflavone reduced LPS-mediated M1 macrophage polarization associated with elevated P-STAT1 activation in the lung tissues. In addition, 5-methoxyflavone improved the survival of LPS-challenged mice.ConclusionThese results indicated that 5-methoxyflavone might be suitable for the development of a novel drug for ALI therapeutic.

Small extracellular vesicles derived from Nrf2-overexpressing human amniotic mesenchymal stem cells protect against lipopolysaccharide-induced acute lung injury by inhibiting NLRP3

BackgroundAcute lung injury (ALI) is a major cause of respiratory failure in critically ill patients that results in significant morbidity and mortality. Recent studies indicate that cell-based therapies may be beneficial in the treatment of ALI. We recently demonstrated that Nrf2-overexpressing human amniotic mesenchymal stem cells (hAMSCs) reduce lung injury, fibrosis and inflammation in lipopolysaccharide (LPS)-challenged mice. Here we tested whether small extracellular vesicles (sEVs) derived from Nrf2-overexpressing hAMSCs (Nrf2-sEVs) could protect against ALI. sEVs were isolated from hAMSCs that overexpressed (Nrf2-sEVs) or silenced (siNrf2-sEVs) Nrf2. We examined the effects of sEVs treatment on lung inflammation in a mouse model of ALI, where LPS was administered intratracheally to mice, and lung tissues and bronchoalveolar lavage fluid (BALF) were analyzed 24 h later.MethodsHistological analysis, immunofluorescence microscopy, western blotting, RT-PCR and ELISA were used to measure the inflammatory response in the lungs and BALF.ResultsWe found that sEVs from hAMSCs are protective in ALI and that Nrf2 overexpression promotes protection against lung disease. Nrf2-sEVs significantly reduced lung injury in LPS-challenged mice, which was associated with decreased apoptosis, reduced infiltration of neutrophils and macrophages, and inhibition of pro-inflammatory cytokine expression. We further show that Nrf2-sEVs act by inhibiting the activation of the NLRP3 inflammasome and promoting the polarization of M2 macrophages.ConclusionOur data show that overexpression of Nrf2 protects against LPS-induced lung injury, and indicate that a novel therapeutic strategy using Nrf2-sEVs may be beneficial against ALI.

Unilateral acute lung injury in pig: a promising animal model

BackgroundAcute lung injury (ALI) occurs in 23% unilateral. Models of unilateral ALI were developed and used previously without clearly demonstrating the strictly unilateral nature and severity of lung injury by the key parameters characterizing ALI as defined by the American Thoracic Society (ATS). Thus, the use of unilateral ALI remained rare despite the innovative approach. Therefore, we developed a unilateral model of ALI and focused on the crucial parameters characterizing ALI. This model can serve for direct comparisons between the injured and intact lungs within single animals, thus, reducing the number of animals required for valid experimental conclusions.MethodsWe established the model in nine pigs, followed by an evaluation of key parameters in six pigs (main study). Pigs were ventilated using an adapted left double-lumen tube for lung separation and two ventilators. ALI was induced in the left lung with cyclic rinsing (NaCl 0.9% + Triton® X-100), after which pigs were ventilated for different time spans to test for the timing of ALI onset. Ventilatory and metabolic parameters were evaluated, and bronchoalveolar lavage (BAL) was performed for measurements of inflammatory mediators. Finally, histopathological specimens were collected and examined in respect of characteristics defining the lung injury score (LIS) as suggested by the ATS.ResultsAfter adjustments of the model (n = 9) we were able to induce strictly left unilateral ALI in all six pigs of the evaluation study. The median lung injury score was 0.72 (IQR 0.62–0.79) in the left lung vs 0.14 (IQR 0.14–0.16; p < 0.05) in the right lung, confirming unilateral ALI. A significant and sustained drop in pulmonary compliance (Cdyn) of the left lung occurred immediately, whereas Cdyn of the right lung remained unchanged (p < 0.05). BAL fluid concentrations of interleukin-6 and -8 were increased in both lungs.ConclusionsWe established a model of unilateral ALI in pigs, confirmed by histopathology, and typical changes in respiratory mechanics and an inflammatory response. This thoroughly evaluated model could serve as a basis for future studies and for comparing pathophysiological and pharmacological changes in the uninjured and injured lung within the same animal.

Inhalation of L-arginine-modified liposomes targeting M1 macrophages to enhance curcumin therapeutic efficacy in ALI

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS), characterized by uncontrolled lung inflammation, is one of the most devastating diseases with high morbidity and mortality. As the first line of defense system, macrophages play a crucial role in the pathogenesis of ALI/ARDS. Therefore, it has great potential to selectively target M1 macrophages to improve the therapeutic effect of anti-inflammatory drugs. l-arginine plays a key role in regulating the immune function of macrophages. The receptors mediating l-arginine uptake are highly expressed on the surface of M1-type macrophages. In this study, we designed an l-arginine-modified liposome for aerosol inhalation to target M1 macrophages in the lung, and the anti-inflammatory drug curcumin was encapsulated in liposomes as model drug. Compared with unmodified curcumin liposome (Cur-Lip), l-arginine functionalized Cur-Lip (Arg-Cur-Lip) exhibited higher uptake by M1 macrophages in vitro and higher accumulation in inflamed lungs in vivo. Furthermore, Arg-Cur-Lip showed more potent therapeutic effects in LPS-induced RAW 264.7 cells and the rat model of ALI. Overall, these findings indicate that l-arginine-modified liposomes have great potential to enhance curcumin treatment of ALI/ARDS by targeting M1 macrophages, which may provide an option for the treatment of acute lung inflammatory diseases such as coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome and middle east respiratory syndrome.

Glycyrrhizin mitigates acute lung injury by inhibiting the NLRP3 inflammasome in vitro and in vivo

Ethnopharmacological relevanceGlycyrrhiza glabra L. is a widely used traditional Chinese medicine with antipyretic, detoxification, antibacterial and therapeutic effects against various diseases, including liver diseases. Glycyrrhizin (GL), the most significant active ingredient of Glycyrrhiza glabra L., exerts anti-inflammatory activity. However, the anti-inflammatory effect of GL remains to be determined. Aim of this studyConsequently, this research was carried out to discover the effects and mechanism of action of GL on ALI. Materials and methodsCell experiments established an in vitro model of LPS-induced RAW 264.7 macrophages to verify the mechanism. The levels of NO, PEG2, and inflammatory cytokines were estimated by ELISA. The expression levels of proteins related to the NF-κB signalling pathway and NLRP3 inflammasome were determined by Western blotting. The nuclear translocation of NF-κB p65 and ASC was tested through immunofluorescence analysis. The inhibitory effect of NLRP3 inhibitor MCC950 on macrophage was evaluated. Male BALB/C mice were selected to establish the ALI model. The experiment was randomly divided into five groups: control, ALI, GLL, GLH, and DEX. Pathological alterations were explored by H&E staining. The weight ratios of lung W/D, MPO, and inflammatory cytokines were evaluated by ELISA. The expression levels of proteins related to the NF-κB signalling pathway or NLRP3 inflammasome were analysed by Western blotting. ResultsHere, we demonstrate that GL attenuates inflammation, nitric oxide, IL-18, IL-1β, TNF-α, IL-6, and PGE2 levels and alveolar epithelial barrier permeability in macrophages and mice challenged with LPS. In addition, GL inhibits NLRP3 inflammasome initiation and activation and NF-κB signalling pathway activation. ConclusionThis research demonstrates that GL may alleviate ALI inflammation by interfering with the NF-κB/NLRP3 inflammasome signalling pathway.

Acid-sensitive ion channel 1a regulates TNF-α expression in LPS-induced acute lung injury via ERS-CHOP-C/EBPα signaling pathway

BackgroundAcute lung injury (ALI) is the local inflammatory response of the lungs involved in a variety of inflammatory cells. Macrophages are immune cells and inflammatory cells widely distributed in the body. Acid-sensitive ion channel 1a (ASIC1a) is involved in the occurrence of ALI, but the mechanism is still unclear. MethodsKunming mouse were stimulated by Lipopolysaccharides (LPS) to establish ALI model in vivo, and RAW264.7 cells were stimulated by LPS to establish inflammatory model in vitro. Amiloride was used as a blocker of ASIC1a to treat mice, and dexamethasone was used as a positive drug for ALI. After blockers and RNAi blocked or silenced the expression of ASIC1a, the expressions of ASIC1a, endoplasmic reticulum-related proteins GRP78, CHOP, C/EBPα and TNF-α were detected. The Ca2+ concentration was measured by a laser confocal microscope. The interaction between CHOP and C/EBPα and the effect of C/EBPα on the activity of TNF-α promoter were detected by immunoprecipitation and luciferase reporter. ResultsThe expressions of ASIC1a and TNF-α were increased significantly in LPS group. After the blocker and RNAi blocked or silenced ASIC1a, the expressions of TNF-α, GRP78, CHOP were reduced, and the intracellular Ca2+ influx was weakened. The results of immunoprecipitation showed that CHOP and C/EBPα interacted in the macrophages. After silencing CHOP, C/EBPα expression was increased, and TNF-α expression was decreased. The results of the luciferase reporter indicated that C/EBPα directly binds to TNF-α. ConclusionASIC1a regulates the expression of TNF-α in LPS-induced acute lung injury via ERS-CHOP-C/EBPα signaling pathway.

Olfactory mucosa tissue-derived mesenchymal stem cells lysate ameliorates LPS-induced acute liver injury in mice

BackgroundAcute liver injury (ALI) induced by sepsis seriously endangers the health of human beings every year. Mesenchymal stem cells (MSCs) lysate containing various regulators had a positive effect on anti-inflammation, hoping to provide a promising strategy in ALI.MethodsOlfactory mucosa-derived mesenchymal stem cells (OM-MSCs) were extracted and identified. The collected OM-MSCs were prepared after repeated freeze–thaw in phosphate buffer solution (PBS). Then, OM-MSCs lysate was filtered for future experiments. To understand the composes of OM-MSCs clearly, we detected the components of OM-MSCs lysate by western blotting. In vitro, OM-MSCs lysate was applied to evaluate the effects on normal human liver cells (LO-2) under stimulation of LPS. Lipopolysaccharide (LPS) was also injected intraperitoneally to build ALI model in mice. We further assessed the anti-inflammatory capacity of OM-MSCs lysate on ALI in vivo by aminotransferase determination, pathology observation, and immunohistochemical staining. Moreover, the immunoblot technique was performed to recognize the changes in inflammatory factors and related proteins.ResultsIn this study, we found that OM-MSCs lysate could protect structure effectively, improve the plasma aminotransferases, diminish inflammation by releasing interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β). A significant decrease in tumor necrosis factor-α (TNF-α) also occurred under the treatment of OM-MSCs lysate. In addition, trophic factors originating from OM-MSCs lysate provided a supportive micro-environment for liver recovery. Especially, up-expression of vascular endothelial growth factor (VEGF) in vivo revealed that OM-MSCs might have a great potential for healing.ConclusionsOur results demonstrated that OM-MSCs lysate could alleviate LPS-induced ALI via decreasing inflammatory cytokines and promoting recovery.

The transcription factor KLF14 attenuates LPS-induced acute lung injury by ameliorating apoptosis of alveolar epithelial cells in mice

Lipopolysaccharide (LPS)-induced lung injury (ALI) is characterized by severe lung inflammation with high mortality. Pulmonary inflammation and apoptosis of alveolar epithelial cells cause the disruption of alveolar epithelial cell integrity, resulting in the failure of gas exchange, which is correlated with poor prognosis. However, the underlying regulatory mechanism remains unclear. In this study, we found that the Krüppel-like transcription factor KLF14 can suppress the apoptosis of alveolar epithelial cells in a LPS-mediated murine acute lung injury model by western blot and TUNEL assays. First, we found that the expression of KLF14 in alveolar epithelial cells of a murine ALI model was upregulated, and KLF14 deletion exacerbated lung injury, increased mortality, and promoted the apoptosis of alveolar epithelial cells. Furthermore, we identified that KLF14 protects lung tissue from LPS-mediated damage and suppresses the apoptosis of alveolar epithelial cells by inhibiting the NF-κB pathway. Our study provides further research insight and helps to explore the protective mechanism of KLF14 in inflammation and cell death. Our results suggested that the transcription factor KLF14 may act as a new target for the prevention and treatment of acute lung injury.

Xuanfei Baidu Formula attenuates LPS-induced acute lung injury by inhibiting the NF-κB signaling pathway

Ethnopharmacological relevanceAcute lung injury (ALI) is a common manifestation of COVID-19. Xuanfei Baidu Formula(XFBD) is used in China to treat mild or common damp-toxin obstructive pulmonary syndrome in COVID-19 patients. However, the active ingredients of XFBD have not been extensively studied, and its mechanism of action in the treatment of ALI is not well understood. Aim of the studyThe purpose of this study was to investigate the mechanism of action of XFBD in treating ALI in rats, by evaluating its active components. Materials and methodsFirstly, the chemical composition of XFBD was identified using ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry. The potential targets of XFBD for ALI treatment were predicted using network pharmacological analysis. Finally, the molecular mechanism of XFBD was validated using a RAW264.7 cell inflammation model and a mouse ALI model. ResultsA total of 113 compounds were identified in XFBD. Network pharmacology revealed 34 hub targets between the 113 compounds and ALI. The results of Kyoto Encyclopedia of Genes and Genomes and gene ontology analyses indicated that the NF-κB signaling pathway was the main pathway for XFBD in the treatment of ALI. We found that XFBD reduced proinflammatory factor levels in LPS-induced cellular models. By examining the lung wet/dry weight ratio and pathological sections in vivo, XFBD was found that XFBD could alleviate ALI. Immunohistochemistry results showed that XFBD inhibited ALI-induced increases in p-IKK, p–NF–κB p65, and iNOS proteins. In vitro experiments demonstrated that XFBD inhibited LPS-induced activation of the NF-κB pathway. ConclusionThis study identified the potential practical components of XFBD, combined with network pharmacology and experimental validation to demonstrate that XFBD can alleviate lung injury caused by ALI by inhibiting the NF-κB signaling pathway.

Schisandrin B protects against LPS-induced inflammatory lung injury by targeting MyD88

BackgroundAcute lung injury (ALI) is a challenging clinical syndrome that manifests as an acute inflammatory response. Schisandrin B (Sch B), a bioactive lignan from Schisandra genus plants, has been shown to suppress inflammatory responses and oxidative stress. However, the underlying molecular mechanisms have remained elusive. Hypothesis/purposeThis study performed an in-depth investigation of the anti-inflammatory mechanism of Sch B in macrophages and in an animal model of ALI. MethodsqPCR array was used to probe the differential effects and potential target of Sch B. ALI was induced by intratracheal administration of LPS in experimental mice with or without Sch B treatment. ResultsOur studies show that Sch B differentially modulates inflammatory factor induction by LPS in macrophages by directly binding myeloid differentiation response factor-88 (MyD88), an essential adaptor protein in the toll-like receptor-4 (TLR4) pathway. Sch B spares non-MyD88-pathways downstream of TLR4. Such inhibition suppressed key signaling mediators such as TAK1, MAPKs, and NF-κB, and pro-inflammatory factor induction. Pull down assay using biotinylated-Sch B validate the direct interaction between Sch B and MyD88 in macrophages. Treatment of mice with Sch B prior to LPS challenge reduced inflammatory cell infiltration in lungs, induction of MyD88-pathway signaling proteins, and prevented inflammatory cytokine induction. ConclusionIn summary, our studies have identified MyD88 as a direct target of Sch B for its anti-inflammatory activity, and suggest that Sch B may have therapeutic value for acute lung injury and other MyD88-dependent inflammatory diseases.

Inhibiting P2Y12 receptor relieves LPS-induced inflammation and endothelial dysfunction.

BackgroundAcute lung injury (ALI) is characterized by abnormal inflammatory response without effective therapies. P2Y12 receptor (P2Y12R) plays a vital role in inflammatory response. This study intends to explore whether P2Y12R antagonists can inhibit LPS‐induced inflammatory injury of human pulmonary microvascular endothelial cells (HPMVECs) and endothelial cell dysfunction.MethodsUsing a cell model of ALI, the role of P2Y12R was investigated in LPS‐induced HPMVECs. The expression of P2Y12R was detected by RT‐qPCR and Western blot analysis assay and TNF‐α, IL‐1β, and IL‐6 levels were analyzed by RT‐qPCR. NO levels were also analyzed through NO kit. The levels of NF‐κB p65, P‐IκB‐α, and IκB‐α, as well as p‐AKT and eNOS levels were detected by Western blot analysis assay. Wound healing assay was performed to evaluate HPMVECs migration. FITC‐dextran was used to evaluate endothelial cell permeability, and the analysis of adherens junction protein VE‐cadherin and endothelial cell tight junction proteins ZO‐1, Claudin 5 and Occludin expression was performed by RT‐qPCR and Western blot analysis assay.ResultsIn vitro, LPS increased the expression levels of P2Y12R and pro‐inflammatory mediators (TNF‐α, IL‐1β, and IL‐6), followed by a decrease in HPMVECs migration. In addition, LPS led to an increase in endothelial cell permeability. P2Y12R antagonists Ticagrelor or clopidogrel treatment significantly reversed these effects of LPS.ConclusionThe inhibitor of P2Y12R was able to decrease inflammatory response, promote migration and improve endothelial cell function and permeability, suggesting a key role of P2Y12R in ALI.

Aurantiamide Acetate Ameliorates Lung Inflammation in Lipopolysaccharide-Induced Acute Lung Injury in Mice.

Purpose Aurantiamide acetate (AA) is a dipeptide derivative with complex pharmacological activities and remarkable effects on preventing and treating various diseases. In the current study, we aimed to investigate whether AA can exert protective effects in a mouse model of ALI induced by LPS. Materials and Methods In this model, mice were given intranasal LPS for 3 days prior to receiving AA (2.5, 5, and 10 mg/kg) via oral gavage. An assessment of histopathological changes was performed by hematoxylin and eosin (HE). Proinflammatory cytokines were detected in bronchoalveolar lavage fluids (BALFs) by enzyme-linked immunosorbent assays (ELISAs). The effects of AA on protein expression of NF-κB and PI3K/AKT signaling pathways were determined by Western blot. In addition, lung wet/dry (W/D) weight ratio, myeloperoxidase (MPO) activity, cell counts, and protein content were also measured. Results According to results, AA pretreatment significantly reduced lung pathological changes, W/D ratio, MPO activity, and protein content. Additionally, AA resulted in a significant reduction in the number of total cells, neutrophils, and proinflammatory cytokines in the BALF after LPS stimulation. The subsequent study revealed that pretreatment with AA dose dependently suppressed LPS-induced activation of NF-κB as well as PI3K/AKT phosphorylation. Conclusion The results indicated that the AA had a protective effect on LPS-induced ALI in mice and could be a potential drug for ALI.

Total terpenoids of Inula japonica activated the Nrf2 receptor to alleviate the inflammation and oxidative stress in LPS-induced acute lung injury

BackgroundAcute lung injury (ALI) is a life-threatening lung disease and characterized by pulmonary edema and atelectasis. Inula japonica Thunb. is a commonly used traditional Chinese medicine for the treatment of lung diseases. However, the potential effect and mechanism of total terpenoids of I. japonica (TTIJ) on ALI remain obscure. PurposeThis study focused on the protective effect of TTIJ on lipopolysaccharide (LPS)-induced ALI in mice and its potential mechanism. Study design and methodsA mouse model of ALI was established by intratracheal instillation of LPS to investigate the protective effect of TTIJ. RNA-seq and bioinformatics were then performed to reveal the underlying mechanism. Finally, western blot and real-time qPCR were used to verify the effects of TTIJ on the inflammation and oxidative stress. ResultsTTIJ notably attenuated LPS-induced histopathological changes of lung. The RNA-seq result suggested that the protective effect of TTIJ on LPS-induced ALI were associated with the Toll-like receptor 4 (TLR4) and nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling pathways. Pretreatment with TTIJ significantly reduced the inflammation and oxidative stress via regulating levels of pro-inflammatory and anti-oxidative cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), superoxide dismutase (SOD), and glutathione (GSH), in LPS-induced ALI mice. TTIJ treatment could suppress the cyclooxygenase-2 (COX-2) expression level and the phosphorylation of p65, p38, ERK, and JNK through the inactivation of the MAPK/NF-κB signaling pathway in a TLR4-independent manner. Meanwhile, TTIJ treatment upregulated expression levels of proteins involved in the Nrf2 signaling pathway, such as heme oxygenase-1 (HO-1), NAD(P)H: quinoneoxidoreductase-1 (NQO-1), glutamate-cysteine ligase catalytic subunit (GCLC), and glutamate-cysteine ligase modifier subunit (GCLM), via activating the Nrf2 receptor, which was confirmed by the luciferase assay. ConclusionTTIJ could activate the Nrf2 receptor to alleviate the inflammatory response and oxidative stress in LPS-induced ALI mice, which suggested that TTIJ could serve as the potential agent in the treatment of ALI.