Isovitexin Exerts Anti-Inflammatory and Anti-Oxidant Activities on Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting MAPK and NF-κB and Activating HO-1/Nrf2 Pathways

Oxidative damage and inflammation are closely associated with the pathogenesis of acute lung injury (ALI). Thus, we explored the protective effect of isovitexin (IV), a glycosylflavonoid, in the context of ALI. To accomplish this, we created in vitro and in vivo models by respectively exposing macrophages to lipopolysaccharide (LPS) and using LPS to induce ALI in mice. In vitro, our results showed that IV treatment reduced LPS-induced pro-inflammatory cytokine secretion, iNOS and COX-2 expression and decreased the generation of ROS. Consistent findings were obtained in vivo. Additionally, IV inhibited H₂O₂-induced cytotoxicity and apoptosis. However, these effects were partially reversed following the use of an HO-1 inhibitor in vitro. Further studies revealed that IV significantly inhibited MAPK phosphorylation, reduced NF-κB nuclear translocation, and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) expression in RAW 264.7 cells. In vivo, pretreatment with IV attenuated histopathological changes, infiltration of polymorphonuclear granulocytes and endothelial activation, decreased the expression of ICAM-1 and VCAM-1, reduced the levels of MPO and MDA, and increased the content of GSH and SOD in ALI. Furthermore, IV treatment effectively increased Nrf2 and HO-1 expression in lung tissues. Therefore, IV may offer a protective role against LPS-induced ALI by inhibiting MAPK and NF-κB and activating HO-1/Nrf2 pathways.     

Blocking of tripartite motif 8 protects against lipopolysaccharide (LPS)-induced acute lung injury by regulating AMPKα activity

Acute lung injury (ALI) and its more serious form, respiratory distress syndrome (ARDS), are considered as an acute and severe inflammatory process existing in lungs, and still remain high mortality rates. Tripartite motif 8 (TRIM8) contains an N-terminal RING finger, which is followed by two B-boxes and a coiled-coil domain, belonging to the TRIM/RBCC family and playing significant role in meditating inflammation, oxidative stress and apoptosis. In the study, we investigated the role of TRIM8 in ALI induced by lipopolysaccharide (LPS) and the underlying molecular mechanisms. The in vitro results indicated that LPS time-dependently enhanced TRIM8 expression in lung epithelial cells. Suppressing TRIM8 markedly ameliorated LPS-elicited inflammatory response, as evidenced by the down-regulated mRNA levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) in cells mainly through inactivating nuclear factor-kappa B (NF-κB) signaling pathway; however, over-expressing TRIM8 markedly promoted inflammation in LPS-challenged cells. In addition, LPS-induced oxidative stress was accelerated by TRIM8 over-expression, while being alleviated by TRIM8 knockdown by regulating Nrf2 signaling. Importantly, TRIM8 could negatively meditate AMP-activated protein kinase-α (AMPKα) activation to modulate LPS-triggered inflammatory response and ROS generation in vitro. Additionally, our in vivo findings suggested that TRIM8 knockdown effectively attenuated LPS-induced lung injury nu decrease of lung wet/dry (W/T) ratio, protein concentrations, neutrophil infiltration, myeloperoxidase (MPO) activity, reactive oxygen species (ROS) production and superoxide dismutase (SOD) depletion. Meanwhile, the loss of TRIM8 markedly lessened IL-1β, IL-6 and TNF-α expression in lung tissues of LPS-challenged mice, and reduced NF-κB phosphorylation. Furthermore, TRIM8 knockdown evidently improved nuclear factor-erythroid 2 related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expressions in lung of LPS-treated mice. The anti-inflammation and anti-oxidant role of TRIM8-silence might be associated with AMPKα phosphorylation. Together, our study firstly provided a support that TRIM8 knockdown effectively protected LPS-induced ALI against inflammation and oxidative stress largely dependent on the promotion of AMPKα pathway.     

Downregulated microRNA-27b attenuates lipopolysaccharide-induced acute lung injury via activation of NF-E2-related factor 2 and inhibition of nuclear factor κB signaling pathway

Acute lung injury (ALI) is a life-threatening, diffuse heterogeneous lung injury characterized by acute onset, pulmonary edema, and respiratory failure. Lipopolysaccharide (LPS) is a leading cause for ALI and when administered to a mouse it induces a lung phenotype exhibiting some of the clinical characteristics of human ALI. This study focused on investigating whether microRNA-27b (miR-27b) affects ALI in a mouse model established by LPS-induction and to further explore the underlying mechanism. After model establishment, the mice were treated with miR-27b agomir, miR-27b antagomir, or D-ribofuranosylbenzimidazole (an inhibitor of nuclear factor-E2-related factor 2 [Nrf2]) to determine levels of miR-27b, Nrf2, nuclear factor kappa-light-chain-enhancer of activated B cells nuclear factor κB (NF-κB), p-NF-κB, and heme oxygenase-1 (HO-1). The levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) in bronchoalveolar lavage fluid (BALF) were determined. The results of luciferase activity suggested that Nrf2 was a target gene of miR-27b. It was indicated that the Nrf2 level decreased in lung tissues from ALI mice. The downregulation of miR-27b decreased the levels of IL-1β, IL-6, and TNF-α in BALF of ALI mice. Downregulated miR-27b increased Nrf2 level, thus enhancing HO-1 level along with reduction of NF-κB level as well as the extent of NF-κB phosphorylation in the lung tissues of the transfected mice. Pathological changes were ameliorated in LPS-reduced mice elicited by miR-27b inhibition. The results of this study demonstrate that downregulated miR-27b couldenhance Nrf2 and HO-1 expressions, inhibit NF-κB signaling pathway, which exerts a protective effect on LPS-induced ALI in mice.     

Ghrelin protects against lipopolysaccharide-induced acute respiratory distress syndrome through the PI3K/AKT pathway

Pulmonary endothelial barrier dysfunction is a major pathophysiology observed in acute respiratory distress syndrome (ARDS). Ghrelin, a key regulator of metabolism, has been shown to play protective roles in the respiratory system. However, its effects on lipopolysaccharide (LPS)-induced pulmonary endothelial barrier injury are unknown. In this study, the effects of ghrelin on LPS-induced ARDS and endothelial cell injury were evaluated in vivo and in vitro. In vivo, mice treated with LPS (3 mg/kg intranasal application) were used to establish the ARDS model. Annexin V/propidium iodide apoptosis assay, scratch-wound assay, tube formation assay, transwell permeability assay, and Western blotting experiment were performed to reveal in vitro effects and underlying mechanisms of ghrelin on endothelial barrier function. Our results showed that ghrelin had protective effects on LPS-induced ARDS and endothelial barrier disruption by inhibiting apoptosis, promoting cell migration and tube formation, and activating the PI3K/AKT signaling pathway. Furthermore, ghrelin stabilized LPS-induced endothelial barrier function by decreasing endothelial permeability and increasing the expression of the intercellular junction protein vascular endothelial cadherin. LY294002, a specific inhibitor of the PI3K pathway, reversed the protective effects of ghrelin on the endothelial cell barrier. In conclusion, our findings indicated that ghrelin protected against LPS-induced ARDS by impairing the pulmonary endothelial barrier partly through activating the PI3K/AKT pathway. Thus, ghrelin may be a valuable therapeutic strategy for the prevention or treatment of ARDS.     

Sitagliptin activates the p62–Keap1–Nrf2 signalling pathway to alleviate oxidative stress and excessive autophagy in severe acute pancreatitis-related acute lung injury

Acute lung injury (ALI) is a complication of severe acute pancreatitis (SAP). Sitagliptin (SIT) is a DPP4 inhibitor that exerts anti-inflammatory and antioxidant effects; however, its mechanism of action in SAP-ALI remains unclear. In this study, we investigated the effects of SIT on SAP-ALI and the specific pathways involved in SAP-induced lung inflammation, including oxidative stress, autophagy, and p62–Kelch-like ECH-associated protein 1 (Keap1)–NF-E2-related factor 2 (Nrf2) signalling pathways. Nrf2 knockout (Nrf2^−/−) and wild-type (WT) mice were pre-treated with SIT (100 mg/kg), followed by caerulein and lipopolysaccharide (LPS) administration to induce pancreatic and lung injury. BEAS-2B cells were transfected with siRNA-Nrf2 and treated with LPS, and the changes in inflammation, reactive oxygen species (ROS) levels, and autophagy were measured. SIT reduced histological damage, oedema, and myeloperoxidase activity in the lung, decreased the expression of pro-inflammatory cytokines, and inhibited excessive autophagy and ROS production via the activation of the p62–Keap1–Nrf2 signalling pathway and promotion of the nuclear translocation of Nrf2. In Nrf2-knockout mice, the anti-inflammatory effect of SIT was reduced, resulting in ROS accumulation and excessive autophagy. In BEAS-2B cells, LPS induced ROS production and activated autophagy, further enhanced by Nrf2 knockdown. This study demonstrates that SIT reduces SAP-ALI-associated oxidative stress and excessive autophagy through the p62–Keap1–Nrf2 signalling pathway and nuclear translocation of Nrf2, suggesting its therapeutic potential in SAP-ALI.Subject terms: Macroautophagy, Acute pancreatitis     

Dehydrozingerone ameliorates Lipopolysaccharide induced acute respiratory distress syndrome by inhibiting cytokine storm,oxidative stress via modulating the MAPK/NF-κB pathway

BackgroundInflammation-mediated lung injury is a major cause of health problems in many countries and has been the leading cause of morbidity/mortality in intensive care units. In the current COVID-19 pandemic, the majority of the patients experienced serious pneumonia resulting from inflammation (Acute respiratory distress syndrome/ARDS). Pathogenic infections cause cytokine release syndrome (CRS) by hyperactivation of immune cells, which in turn release excessive cytokines causing ARDS. Currently, there are no standard therapies for viral, bacterial or pathogen-mediated CRS.PurposeThis study aimed to investigate and validate the protective effects of Dehydrozingerone (DHZ) against LPS induced lung cell injury by in-vitro and in-vivo models and to gain insights into the molecular mechanisms that mediate these therapeutic effects.MethodsThe therapeutic activity of DHZ was determined in in-vitro models by pre-treating the cells with DHZ and exposed to LPS to stimulate the inflammatory cascade of events. We analysed the effect of DHZ on LPS induced inflammatory cytokines, chemokines and cell damage markers expression/levels using various cell lines. We performed gene expression, ELISA, and western blot analysis to elucidate the effect of DHZ on inflammation and its modulation of MAPK and NF-κB pathways. Further, the prophylactic and therapeutic effect of DHZ was evaluated against the LPS induced ARDS model in rats.ResultsDHZ significantly (p < 0.01) attenuated the LPS induced ROS, inflammatory cytokine, chemokine gene expression and protein release in macrophages. Similarly, DHZ treatment protected the lung epithelial and endothelial cells by mitigating the LPS induced inflammatory events in a dose-dependent manner. In vivo analysis showed that DHZ treatment significantly (p < 0.001) mitigated the LPS induced ARDS pathophysiology of increase in the inflammatory cells in BALF, inflammatory cytokine and chemokines in lung tissues. LPS stimulated neutrophil-mediated events, apoptosis, alveolar wall thickening and alveolar inflammation were profoundly reduced by DHZ treatment in a rat model.ConclusionThis study demonstrates for the first time that DHZ has the potential to ameliorate LPS induced ARDS by inhibiting cytokine storm and oxidative through modulating the MAPK and NF-κB pathways. This data provides pre-clinical support to develop DHZ as a potential therapeutic agent against ARDS.     

Nuclear erythroid 2 p45-related factor–2 Nrf2 ameliorates cigarette smoking-induced mucus overproduction in airway epithelium and mouse lungs

Background and objectiveNuclear erythroid 2 p45-related factor–2 (Nrf2) is known to play important roles in airway disorders, whereas little has been investigated about its direct role in airway mucus hypersecretion. The aim of this study is to determine whether this factor could protect pulmonary epithelium and mouse airway from cigarette-induced mucus overproduction.MethodsUsing genetic approaches, the role of Nrf2 on cigarette smoking extracts (CSE) induced MUC5AC expression was investigated in lung A549 cells. Nrf2 deficiency mice were smoked for various periods, and the airway inflammation and mucus production was characterized.ResultsAcute smoking exposure induced expression of MUC5AC and Nrf2 in both A549 cells and mouse lungs. Genetic ablation of Nrf2 augmented, whereas overexpression of this molecule ameliorated CSE-induced expression of MUC5AC. Nrf2 knockout mice, after exposure to cigarette smoking, displayed enhanced airway inflammation and mucus production.ConclusionNrf2 negatively regulated smoking-induced mucus production in vitro and in vivo, suggesting therapeutic potentials of this factor in airway diseases with hypersecreted mucus.     

Ursodeoxycholic acid protects against lung injury induced by fat embolism syndrome

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a life‐threatening disease with a high mortality rate, which was a common complication of fat embolism syndrome (FES). Ursodeoxycholic acid (UDCA) has been reported to exert potent anti‐inflammatory effects under various conditions. In vivo, perinephric fat was injected via tail vein to establish a rat FES model, the anti‐inflammatory effects of UDCA on FES‐induced lung injury were investigated through histological examination, ELISA, qRT‐PCR, Western blot and immunofluorescence. In vitro, human lung microvascular endothelial cells (HPMECs) were employed to understand the protective effects of UDCA. The extent of ALI/ARDS was evaluated and validated by reduced PaO₂/FiO₂ ratios, increased lung wet/dry (W/D) ratios and impaired alveolar‐capillary barrier, up‐regulation of ALI‐related proteins in lung tissues (including myeloperoxidase [MPO], vascular cell adhesion molecule 1 [VCAM‐1], intercellular cell adhesion molecule‐1 [ICAM‐1]), elevated protein concentration and increased proinflammatory cytokines levels (TNF‐α and IL‐1β) in bronchoalveolar lavage fluid (BALF). Pre‐treatment with UDCA remarkably alleviated these pathologic and biochemical changes of FES‐induced ALI/ARDS; our data demonstrated that pre‐treatment with UDCA attenuated the pathologic and biochemical changes of FES‐induced ARDS, which provided a possible preventive therapy for lung injury caused by FES.     

Therapeutic Effect of Intravenous Infusion of Perfluorocarbon Emulsion on LPS-Induced Acute Lung Injury in Rats

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) are the leading causes of death in critical care. Despite extensive efforts in research and clinical medicine, mortality remains high in these diseases. Perfluorocarbon (PFC), a chemical compound known as liquid ventilation medium, is capable of dissolving large amounts of physiologically important gases (mainly oxygen and carbon dioxide). In this study we aimed to investigate the effect of intravenous infusion of PFC emulsion on lipopolysaccharide (LPS) induced ALI in rats and elucidate its mechanism of action. Forty two Wistar rats were randomly divided into three groups: 6 rats were treated with saline solution by intratracheal instillation (control group), 18 rats were treated with LPS by intratracheal instillation (LPS group) and the other 18 rats received PFC through femoral vein prior to LPS instillation (LPS+PFC group). The rats in the control group were sacrificed 6 hours later after saline instillation. At 2, 4 and 6 hours of exposure to LPS, 6 rats in the LPS group and 6 rats in LPS+PFC group were sacrificed at each time point. By analyzing pulmonary pathology, partial pressure of oxygen in the blood (PaO2) and lung wet-dry weight ratio (W/D) of each rat, we found that intravenous infusion of PFC significantly alleviated acute lung injury induced by LPS. Moreover, we showed that the expression of pulmonary myeloperoxidase (MPO), intercellular adhesion molecule-1 (ICAM-1) of endothelial cells and CD11b of polymorphonuclear neutrophils (PMN) induced by LPS were significantly decreased by PFC treatment in vivo. Our results indicate that intravenous infusion of PFC inhibits the infiltration of PMNs into lung tissue, which has been shown as the core pathogenesis of ALI/ARDS. Thus, our study provides a theoretical foundation for using intravenous infusion of PFC to prevent and treat ALI/ARDS in clinical practice.     

Mucin1 relieves acute lung injury by inhibiting inflammation and oxidative stress

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a kind of diffuse inflammatory injury caused by various factors, characterized by respiratory distress and progressive hypoxemia. It is a common clinical critical illness. The aim of this study was to investigate the effect and mechanism of the Mucin1 (MUC1) gene and its recombinant protein on lipopolysaccharide (LPS)-induced ALI/ARDS. We cultured human alveolar epithelial cell line (BEAS-2B) and used MUC1 overexpression lentivirus to detect the effect of MUC1 gene on BEAS-2B cells. In addition, we used LPS to induce ALI/ARDS in C57/BL6 mice and use hematoxylin and eosin (H&E) staining to verify the effect of their modeling. Recombinant MUC1 protein was injected subcutaneously into mice. We examined the effect of MUC1 on ALI/ARDS in mice by detecting the expression of inflammatory factors and oxidative stress molecules in mouse lung tissue, bronchoalveolar lavage fluid (BALF) and serum. Overexpression of MUC1 effectively ameliorated LPS-induced damage to BEAS-2B cells. Results of H&E staining indicate that LPS successfully induced ALI/ARDS in mice and MUC1 attenuated lung injury. MUC1 also reduced the expression of inflammatory factors (IL-1β, TNF-α, IL-6 and IL-8) and oxidative stress levels in mice. In addition, LPS results in an increase in the activity of the TLR4/NF-κB signaling pathway in mice, whereas MUC1 decreased the expression of the TLR4/NF-κB signaling pathway. MUC1 inhibited the activity of TLR4/NF-κB signaling pathway and reduced the level of inflammation and oxidative stress in lung tissue of ALI mice.Key words: Mucin1, acute lung injury, inflammation, oxidative stress, TLR4/NF-κB     

The protective effects of C16 peptide and angiopoietin-1 compound in lipopolysaccharide-induced acute respiratory distress syndrome

C16 peptide and angiopoietin-1 (Ang-1) have been found to have anti-inflammatory activity in various inflammation-related diseases. However, their combined role in acute respiratory distress syndrome (ARDS) has not been investigated yet. The objective of this study was to investigate the effects of C16 peptide and Ang-1 in combination with lipopolysaccharide (LPS)-induced inflammatory insult in vitro and in vivo. Human pulmonary microvascular endothelial cells and human pulmonary alveolar epithelial cells were used as cell culture systems, and an ARDS rodent model was used for in vivo studies. Our results demonstrated that C16 and Ang-1 in combination significantly suppressed inflammatory cell transmigration by 33% in comparison with the vehicle alone, and decreased the lung tissue wet-to-dry lung weight ratio to a maximum of 1.53, compared to 3.55 in the vehicle group in ARDS rats. Moreover, C  +  A treatment reduced the histology injury score to 60% of the vehicle control, enhanced arterial oxygen saturation (SO₂), decreased arterial carbon dioxide partial pressure (PCO₂), and increased oxygen partial pressure (PO₂) in ARDS rats, while also improving the survival rate from 47% (7/15) to 80% (12/15) and diminishing fibrosis, necrosis, and apoptosis in lung tissue. Furthermore, when C  +  A therapy was administered 4 h following LPS injection, the treatment showed significant alleviating effects on pulmonary inflammatory cell infiltration 24 h postinsult. In conclusion, our in vitro and in vivo studies show that C16 and Ang-1 exert protective effects against LPS-induced inflammatory insult. C16 and Ang-1 hold promise as a novel agent against LPS-induced ARDS. Further studies are needed to determine the potential for C16 and Ang-1 in combination in treating inflammatory lung diseases.     

ALI/ARDS患者使用保护性通气策略对于肺部损伤的影响

急性肺损伤(ALI)和急性呼吸窘迫综合征(ARDS)是常见的临床综合征,绝大多数ALI/ARDS患者需机械通气治疗,机械通气在提供可接受的肺部气体交换的同时治疗基础疾病,但机械通气本身也会引起肺部损伤,即机械通气性肺损伤(VILI)。而通过调整机械通气参数的设置,使用保护性通气策略可显著减低ALI/ARDS患者机械通气性肺损伤程度,从而减少肺部感染,缩短机械通气时间和住院时间,降低28天死亡率,明显改善ALI/ARDS患者的生存质量,起到最大程度地肺保护作用。本文从气道平台压,通气容积,呼气末正压等几个不同通气参数方面分别进行综述,讨论ALI/ARDS患者机械通气时使用保护性通气策略对于肺部损伤的影响。