Chelerythrine Attenuates the Inflammation of Lipopolysaccharide-Induced Acute Lung Inflammation Through NF-?B Signaling Pathway Mediated by Nrf2.
ABSTRACT: Chelerythrine (CH), is a kind of benzo[c] phenanthridine alkaloid isolated from plants such as Chelidonium, with pharmacological activities as antitumor, antibiosis and anti-inflammation. However, few studies have demonstrated whether CH could protect against lipopolysaccharide (LPS)-induced acute lung injury (ALI), and the underlying mechanism is also uncertain. The purpose of the present study was to investigate the anti-inflammatory effects of CH on LPS-induced ALI in mice and in RAW264.7 cells. In this study, we demonstrated that treatment with CH significantly ameliorated LPS-induced pathological changes in the lung. CH also attenuated LPS-induced W/D ratio, inflammatory cell infiltration. Meanwhile, LPS-induced Tumor necrosis factor-alpha (TNF-?), interleukin 6 (IL-6), and interleukin 1? (IL-1?) production and oxidative stress were markedly suppressed by CH. Furthermore, western blot showed that CH suppressed LPS-stimulated inflammation of RAW264.7 cells through activation of nuclear factor kappa-B (NF-?B) pathway. Knocking down of nuclear factor erythroid 2-related factor 2 (Nrf2) led to the reduction of nuclear translocation of the NF-?B p65, which triggered inflammation. These experimental results provided evidence that CH could be a potential therapeutic candidate for the intervention of ALI caused by LPS.
Project description:Acute lung injury (ALI) is a complex syndrome with sepsis occurring in critical patients, who usually lack effective therapy. Nuciferine is a primary bioactive component extracted from the lotus leaf, and it displays extensive pharmacological functions, including anti-cancer, anti-inflammatory, and antioxidant properties. Nevertheless, the effects of nuciferine on lipopolysaccharide (LPS)-stimulated ALI in mice has not been investigated. ALI of mice stimulated by LPS was used to determine the anti-inflammatory function of nuciferine. The molecular mechanism of nuciferine was performed on RAW264.7 macrophage cells. The results of pathological section, myeloperoxidase activity and lung wet/dry ratio showed that nuciferine alleviated LPS-induced lung injury (p < 0.05). qRT-PCR and ELISA experiments suggested that nuciferine inhibited TNF-?, IL-6, and IL-1? secretion in tissues and RAW264.7 cells but increased IL-10 secretion (p < 0.05). Molecular studies showed that TLR4 expression and nuclear factor (NF)-?B activation were both inhibited by nuciferine treatment (p < 0.05). To further investigate the anti-inflammatory mechanism of nuciferine, TLR4 was knocked down. When TLR4 was silenced, LPS induced the production of IL-1?, and TNF-? was markedly decreased by TLR4-siRNA and nuciferine treatment in LPS-induced RAW264.7 cells (p < 0.05). These results suggested that nuciferine had the ability to protect against LPS-stimulated ALI. Thus, nuciferine may be a potential drug for treating LPS-induced pulmonary inflammation.
Project description:Acute lung injury (ALI), characterized by a severe inflammatory process, is a complex syndrome that can lead to multisystem organ failure. Fatty acid amide hydrolase (FAAH) and N-acylethanolamine acid amidase (NAAA) are two potential therapeutic targets for inflammation-related diseases. Herein, we identified carmofur, a 5-fluorouracil-releasing drug and clinically used as a chemotherapeutic agent, as a dual FAAH and NAAA inhibitor. In Raw264.7 macrophages, carmofur effectively reduced the mRNA expression of pro-inflammatory factors, including IL-1?, IL-6, iNOS, and TNF-?, and down-regulated signaling proteins of the nuclear transcription factor ?B (NF-?B) pathway. Furthermore, carmofur significantly ameliorated the inflammatory responses and promoted resolution of pulmonary injury in lipopolysaccharide (LPS)-induced ALI mice. The pharmacological effects of carmofur were partially blocked by peroxisome proliferator-activated receptor-? (PPAR?) antagonist MK886 and cannabinoid receptor 2 (CB2) antagonist SR144528, indicating that carmofur attenuated LPS-induced ALI in a PPAR?- and CB2-dependent mechanism. Our study suggested that carmofur might be a novel therapeutic agent for ALI, and drug repurposing may provide us effective therapeutic strategies for ALI.
Project description:Acute lung injury (ALI) is a life-threatening inflammatory disease owing to the lack of specific and effective therapies. Oridonin (Ori) is an active diterpenoid isolated from Rabdosiarubescens (R.rubescens) that has been shown to possess a broadspectrum pharmacological properties including anti-inflammatory, antitumour, antioxidative and neuroregulatory effects. However, its potential protective mechanism in ALI is not well characterized. In this study, we demonstrated that Ori reduces the mortality of mice with ALI induced by a high dose of lipopolysaccharide (LPS), which suggests that Ori has a protective effect on LPS induced ALI. Next, our results confirmed that Ori improves LPS-induced localized pulmonary pathology and decreased the concentration of pro-inflammatory cytokines (IL-1?, IL-6, and TNF-?) in the serum. Nuclear factor-kappa B (NF-?B) is capable of regulating the transcription of pro-inflammatory factors. Interestingly, our results showed that Ori inhibits the expression of TLR4/MyD88 and phosphorylation of NF-?B p65 in lung tissues. To confirm this, we further validated the possible regulatory anti-inflammatory mechanisms of Ori in vitro. LPS-induced RAW264.7 cells, which are widely used as an inflammation model to evaluate the potential protective effect of drugs in vitro, were chosen for this study. Similar results were observed, that is, pre-treatment with Ori, markedly inhibited the nuclear translocation and phosphorylation of NF-?B p65 induced by LPS and subsequently decreased the release of pro-inflammatory cytokines that were increased by LPS. Overall, these results demonstrated that Ori exerts a therapeutic effect on ALI by inhibiting the release of pro-inflammatory cytokines, such as IL-1?, IL-6, and TNF-?, through the TLR4/MyD88/NF-?B axis.
Project description:BACKGROUND: Acute respiratory distress syndrome (ARDS) is a severe and life-threatening acute lung injury (ALI) that is caused by noxious stimuli and pathogens. ALI is characterized by marked acute inflammation with elevated alveolar cytokine levels. Mitogen-activated protein kinase (MAPK) pathways are involved in cytokine production, but the mechanisms that regulate these pathways remain poorly characterized. Here, we focused on the role of Sprouty-related EVH1-domain-containing protein (Spred)-2, a negative regulator of the Ras-Raf-extracellular signal-regulated kinase (ERK)-MAPK pathway, in lipopolysaccharide (LPS)-induced acute lung inflammation. METHODS: Wild-type (WT) mice and Spred-2(-/-) mice were exposed to intratracheal LPS (50 µg in 50 µL PBS) to induce pulmonary inflammation. After LPS-injection, the lungs were harvested to assess leukocyte infiltration, cytokine and chemokine production, ERK-MAPK activation and immunopathology. For ex vivo experiments, alveolar macrophages were harvested from untreated WT and Spred-2(-/-) mice and stimulated with LPS. In in vitro experiments, specific knock down of Spred-2 by siRNA or overexpression of Spred-2 by transfection with a plasmid encoding the Spred-2 sense sequence was introduced into murine RAW264.7 macrophage cells or MLE-12 lung epithelial cells. RESULTS: LPS-induced acute lung inflammation was significantly exacerbated in Spred-2(-/-) mice compared with WT mice, as indicated by the numbers of infiltrating leukocytes, levels of alveolar TNF-?, CXCL2 and CCL2 in a later phase, and lung pathology. U0126, a selective MEK/ERK inhibitor, reduced the augmented LPS-induced inflammation in Spred-2(-/-) mice. Specific knock down of Spred-2 augmented LPS-induced cytokine and chemokine responses in RAW264.7 cells and MLE-12 cells, whereas Spred-2 overexpression decreased this response in RAW264.7 cells. CONCLUSIONS: The ERK-MAPK pathway is involved in LPS-induced acute lung inflammation. Spred-2 controls the development of LPS-induced lung inflammation by negatively regulating the ERK-MAPK pathway. Thus, Spred-2 may represent a therapeutic target for the treatment of ALI.
Project description:Inflammation plays an important role in cases of acute lung injury (ALI), and the Toll-like receptor 4/nuclear factor-?B (TLR4/NF-?B) pathway, which can be regulated by Polygonatum sibiricum polysaccharides (PSPs), is closely related to the dynamics of lipopolysaccharide (LPS)-induced inflammation. Thus, we sought to evaluate whether or not PSPs prevent LPS-induced ALI by way of inhibiting inflammation via the TLR4/NF-?B pathway in rats. We established an ALI rat model by tracheal instillation of LPS, and by pre-injection of PSPs into rats to examine PSPs in the ALI rat model. We found that PSPs attenuated LPS-induced lung pathological changes in ALI rats, decreased LPS-induced myeloperoxidase (MOP) activity, and elevated malondialdehyde (MDA) levels in lung tissue. However, PSPs also decreased the LPS-induced increase in the neutrophil ratio, and decreased inflammatory factor levels in bronchoalveolar lavage fluid (BALF). Moreover, PSPs decreased LPS-induced increases in inflammatory factors measured by mRNA expression, and altered the levels of expression of TLR4, medullary differentiation protein 88 (Myd88), p-IKB-?/IKB-? and p-p65/p65 proteins in lung tissue. In vitro, PSPs also reduced apoptosis induced by LPS in BEAS-2B cells by suppressing inflammation through its effect of inhibiting the TLR4/NF-?B pathway. In conclusion, our results suggest that PSPs may be a potential drug for effective treatment of LPS-induced ALI, due to the ability to inhibit inflammation through effects exerted on the TLR4/Myd88/NF-?B pathway.
Project description:The cellular and molecular mechanisms by which indole-3-acetic acid (IAA), a tryptophan-derived metabolite from gut microbiota, attenuates inflammation and oxidative stress has not been fully elucidated. The present study was to unearth the protective effect and underlying mechanism of IAA against lipopolysaccharide (LPS)-induced inflammatory response and free radical generation in RAW264.7 macrophages. IAA significantly ameliorated LPS-induced expression of interleukin-1? (IL-1?), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) as well as generation of reactive oxidative species (ROS) and nitric oxide (NO). LPS-triggered nuclear translocation of nuclear factor kappa B (NF-?B) p65 was mitigated by IAA treatment. Further, an up-regulation of heme oxygenase-1 (HO-1) was observed in IAA-treated cells in dose-dependent manner under both normal and LPS-stimulated condition. Interference of HO-1 activity by tin protoporphyrin IX (SnPP) impeded the alleviative effects of IAA on expression of IL-1? and IL-6 induced by LPS, whereas demonstrated no effect on its suppression of ROS and NO production. This result suggests a HO-1-dependent anti-inflammatory effect of IAA and its direct scavenging action on free radicals. Treatment with CH-223191, a specific antagonist of aryl hydrocarbon receptor (AhR), showed no significant effects on the beneficial role of IAA against inflammation and free radical generation. In summary, our findings indicate that IAA alleviates LPS-elicited inflammatory response and free radical generation in RAW264.7 macrophages by induction of HO-1 and direct neutralization of free radicals, a mechanism independent of AhR.
Project description:Acute lung injury (ALI) is a highly lethal pulmonary disease that causes edema, hypoxemia and respiratory failure. Recent evidence indicates that nuclear factor-kappa B (NF-?B) plays a crucial role in ALI development. However, the regulatory mechanism of NF-?B on ALI remains enigmatic. In this study, we investigated potential molecular mechanism of NF-?B on ALI induced by lipopolysaccharide (LPS). BALB/c mice were subjected to intratracheal spraying of LPS to generate an ALI mode, with the activity of NF-?B in mice tissues being detected by enzyme linked immunosorbent assay (ELISA), and the number of inflammatory cells in bronchoalveolar lavage fluid being counted. Then, the macrophage cell line RAW264.7 exposed to LPS were treated with ammonium pyrrolidinedithiocarbamate (PDTC) (inhibitor of NF-?B), miR-194 mimic, or oe-chemokine receptor type 4 (CXCR4) separately or in combination. After that, ELISA and reverse transcription quantitative polymerase chain reaction (RT-qPCR) were used to detect the expression level of IL-1?, IL-6, TNF-?, miR-194 and CXCR4, respectively. In addition, the targeting relationship between miR-194 and CXCR4 was verified by dual-luciferase reporter gene assay. The dry/wet ratio of lung and the MPO activity were also measured to assess the inflammatory response in mice. Activation of NF-?B down-regulated the miR-194 expression in LPS-induced ALI. Overexpression of miR-194 alleviated LPS-induced ALI and reduced the expression of inflammatory factors IL-1?, IL-6 and TNF-? via targeting CXCR4. In LPS-induced ALI, NF-?B mediates the CXCR4 expression by inhibiting the expression of miR-194, thus promoting the inflammatory injury of lung.
Project description:Long non-coding RNAs (lncRNAs) are involved in various biological processes as well as many respiratory diseases, while the role of lncRNAs in acute lung injury (ALI) remains unclear. The present study aimed to profile the expression of lung lncRNAs and mRNAs in lipopolysaccharide (LPS)-induced ALI mouse model. C57BL/6 mice were exposed to LPS or phosphate-buffered saline for 24 h, and lncRNAs and mRNAs were profiled by Arraystar mouse LncRNA Array V3.0. Bioinformatics analysis gene ontology including (GO) and pathway analysis and cell study in vitro was used to investigate potential mechanisms. Based on the microarray results, 2632 lncRNAs and 2352 mRNAs were differentially expressed between ALI and control mice. The microarray results were confirmed by the quantitative real-time PCR (qRT-PCR) results of ten randomized selected lncRNAs. GO analysis showed that the altered mRNAs were mainly related to the processes of immune system, immune response and defense response. Pathway analysis suggests that tumor necrosis factor (TNF) signaling pathway, NOD-like receptor pathway, and cytokine-cytokine receptor interaction may be involved in ALI. LncRNA-mRNA co-expression network analysis indicated that one individual lncRNA may interact with several mRNAs, and one individual mRNA may also interact with several lncRNAs. Small interfering RNA (siRNA) for ENSMUST00000170214.1, - ENSMUST00000016031.13 significantly inhibited LPS-induced TNF-? and interleukin (IL)-1? production in murine RAW264.7 macrophages. Our results found significant changes of lncRNAs and mRNAs in the lungs of LPS-induced ALI mouse model, and intervention targeting lncRNAs may attenuate LPS-induced inflammation, which may help to elucidate the role of lncRNAs in the pathogenesis and treatment of ALI.
Project description:Oregano is an aromatic plant widely distributed throughout the Mediterranean area and in Asia. Recent studies have revealed that the anti-inflammatory effect of essential oil in this plant. However, the mechanisms underlying the therapeutic potential have not been well elucidated. This study determined whether oregano essential oil (OEO) exerts an anti-inflammatory effect on lipopolysaccharide (LPS)-treated murine macrophage cells (RAW264.7 cells) in vitro and elucidated the possible underlying molecular mechanisms. The results showed that OEO (2.5?10 ?g/mL) inhibited the expression and secretion of interleukin-1 beta (IL-1?), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-?) in RAW264.7 cells treated with LPS (1 ?g/mL). Consistent with the pro-inflammatory gene expression, the OEO treatment efficiently reduced the LPS-induced activation of mitogen-activated protein kinase, protein kinase B, and nuclear factor ?B in RAW264.7 cells. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibition in Nox2 protein-silenced cells attenuated the mRNA expression of IL-1?, IL-6, and TNF-? in the LPS-induced RAW264.7 cells. The OEO inhibited the LPS-induced elevation of NADPH oxidase and oxidative stress. This result suggests that LPS induces RAW264.7 cell inflammation through the NADPH oxidase-mediated production of reactive oxygen species (ROS). In conclusion, OEO protects against the LPS-induced RAW264.7 cell inflammatory response through the NADPH oxidase/ROS pathway.
Project description:Simvastatin inhibits inflammatory responses in vitro and in murine models of lung inflammation in vivo. As simvastatin modulates a number of the underlying processes described in acute lung injury (ALI), it may be a potential therapeutic option.To investigate in vivo if simvastatin modulates mechanisms important in the development of ALI in a model of acute lung inflammation induced by inhalation of lipopolysaccharide (LPS) in healthy human volunteers.Thirty healthy subjects were enrolled in a double-blind, placebo-controlled study. Subjects were randomized to receive 40 mg or 80 mg of simvastatin or placebo (n = 10/group) for 4 days before inhalation of 50 microg LPS. Measurements were performed in bronchoalveolar lavage fluid (BALF) obtained at 6 hours and plasma obtained at 24 hours after LPS challenge. Nuclear translocation of nuclear factor-kappaB (NF-kappaB) was measured in monocyte-derived macrophages.Pretreatment with simvastatin reduced LPS-induced BALF neutrophilia, myeloperoxidase, tumor necrosis factor-alpha, matrix metalloproteinases 7, 8, and 9, and C-reactive protein (CRP) as well as plasma CRP (all P < 0.05 vs. placebo). There was no significant difference between simvastatin 40 mg and 80 mg. BALF from subjects post-LPS inhalation induced a threefold up-regulation in nuclear NF-kappaB in monocyte-derived macrophages (P < 0.001); pretreatment with simvastatin reduced this by 35% (P < 0.001).Simvastatin has antiinflammatory effects in the pulmonary and systemic compartment in humans exposed to inhaled LPS.