Current therapies for ischemia/reperfusion are insufficient because of our poor understanding of the mechanisms of brain injury after ischemic stroke. As a vital component of the innate immune system, NLRP3 inflammasome contributes to ischemic brain injury; however, a detailed understanding of their molecular mechanisms is unknown. This study was designed to investigate the effect of nuclear factor E2-related factor-2 (Nrf2) on NLRP3 inflammasome. BV2 microglial cells were pretreated with tert-butylhydroquinone or Nrf2 CRISPR plasmid before oxygen-glucose deprivation/reoxygenation (OGDR) exposure. Then we observed the effect of Nrf2 on NLRP3 inflammasome. We identified that Nrf2 activation inhibited NLRP3 inflammasome expression and subsequent IL-1 beta generation. Furthermore, the activation of NLRP3 inflammasome was sensitive to the reactive oxygen species (ROS) level and Nrf2 could decrease the production of ROS. Additionally, as a Nrf2-targeted ARE gene, NADPH quinone oxidoreductase 1 was involved in the inhibition of the NLRP3 inflammasome. We elucidated an inhibitory regulation of Nrf2/ARE pathway on ROS-induced NLRP3 inflammasome activation in BV2 microglial cells after OGDR exposure.
ObjectiveThe Shixiangru (Mosla chinensis Maxim) total flavonoids (STF) mainly contain luteolin and apigenin. The study aims to examine the inhibitory effects of STF on anti-H1N1 influenza virus and its related molecular mechanisms in pneumonia mice. Methods The viral pneumonia mice were treated with Ribavirin or various doses of STF. We observed histological changes of lung by immunohistochemistry and measured lung index to value anti-influenza virus effects of STF. The concentrations of inflammatory cytokines and anti-oxidant factors were detected by ELISA. RT-PCR and western blot assays were used to determine the expression level of TLR pathway's key genes and proteins in lung tissues. Results We found that the pathological changes of lung in the viral pneumonia mice obviously alleviated by STF treatments and the STF (288 or 576 mg/kg) could significantly decrease lung indices. Moreover, the up-regulation (IL-6, TNF-alpha, IFN-gamma, and NO) and down-regulation (IL-2, SOD and GSH) of inflammatory cytokines and anti-oxidant factors were associated with higher clearance of virus and reduction of inflammatory lung tissue damage. Meanwhile, the expression levels of TLR3, TLR7, MyD88, TRAF3 and NF-kappa B p65 of the TLR pathway were reduced by STF treatment. Conclusions This study suggested that STF may be a promising candidate for treating H1N1 influenza and subsequent viral pneumonia.
Objective Glucagon-like peptide-1 (GLP-1)-based therapy via G protein-coupled receptor (GPCR) GLP-1R, to attenuate hyperglycemia in critical care has attracted great attention. However, the exaggerated inflammation by GLP-1R agonist, Exendin-4, in a mouse model of burn injury was quite unexpected. Recent studies found that GPCR might elicit proinflammatory effects by switching from G alpha s to G alpha i signaling in the immune system. Thus, we aimed to investigate the possible G alpha s to G alpha i switch in GLP-1R signaling in monocyte following burn injury. Materials and methods Splenic monocytes from sham and burn mice 24 h following burn injury were treated with consecutive doses of Exendin-4 alone or in combination with an inhibitor of G alpha i signaling (pertussis toxin, PTX), or a blocker of protein kinase A (H89). Cell viability was assessed by CCK-8, and the supernatant was collected for cytokine measurement by ELISA. Intracellular cAMP level, phosphorylated PKA activity, and nuclear NF-kappa B p65 were determined by ELISA, ERK1/2 activation was analyzed by Western blot. The expression of GLP-1R downstream molecules, G alpha s, G alpha i and G-protein coupled receptor kinase 2 (GRK2) were examined by immunofluorescence staining and Western blot. Results Exendin-4 could inhibit the viability of monocyte from sham rather than burn mice. Unexpectedly, it could also reduce TNF-alpha secretion from sham monocyte while increase it from burn monocyte. The increased secretion of TNF-alpha by Exendin-4 from burn monocyte could be reversed by pretreatment of PTX or H89. Accordingly, Exendin-4 could stimulates cAMP production dose dependently from sham instead of burn monocyte. However, the blunt cAMP production from burn monocyte was further suppressed by pretreatment of PTX or H89 after 6-h incubation. Nevertheless, phosphorylated PKA activity was significantly increased by low dose of Exendin-4 in sham monocyte, by contrast, it was enhanced by high dose of Exendin-4 in burn monocyte after 1-h incubation. Following Exendin-4 treatment for 2 h ex vivo, total nuclear NF-kappa B and phosphorylated NF-kappa B activity, as well as cytoplasmic pERK1/2 expressions were reduced in sham monocyte, however, only pERK1/2 was increased by Exendin-4 in burn monocytes. Moreover, reduced expressions of GLP-1R, GRK-2 and G alpha s in contrast with increased expression of G alpha i were identified in burn monocyte relative to sham monocyte. Conclusions This study presents an unexpected proinflammatory switch from G alpha s to G alpha i signaling in burn monocyte, which promotes ERK1/2 and NF-kappa B activation and the downstream TNF-alpha secretion. This phenomenon is most probably responsible for proinflammatory response evoked by G alpha s agonist Exendin-4 following burn injury.
Introduction Graves' orbitopathy (GO) is an autoimmune inflammatory disorder affecting the orbit around the eye. Astragaloside IV (AS-VI) is the main active ingredient of the Chinese herbal medicine-Huangqi (Radix Astragali Mongolici). AS-IV exhibits antioxidant and anti-inflammatory properties, and shows therapeutic potential in a number of ischemic and inflammatory diseases; however, its pharmaceutical activities in GO remain undefined. Materials and methods In this study, we investigated the effects of AS-IV on interleukin (IL)-1 beta-induced orbital fibroblast inflammation in vitro and GO orbital inflammation and ocular histopathological changes in vivo, as well as the underlying mechanisms responsible for these effects. Results and conclusion The results show that IL-1 beta increased mRNA expression of the inflammatory cytokines IL-6, IL-8, TNF-alpha, and MCP-1 in cultured orbital fibroblasts. This IL-1 beta-induced inflammation was accompanied by increased autophagic activity as reflected in increased Beclin-1 and Agt-5 expression, as well as LC3-I to LC3-II conversion. Pretreatment with the autophagy inhibitors 3-MA and bafilomycin A1, or silencing of autophagy-related proteins Beclin-1 and Atg-5, prevented IL-1 beta induced orbital fibroblast inflammation, while pretreatment with the autophagy activator rapamycin had the opposite effects. These data suggested that autophagy was involved in GO orbital inflammation. AS-IV treatment significantly decreased IL-1 beta-induced inflammatory cytokine production in orbital fibroblasts in vitro and attenuated GO orbital inflammation, fat accumulation, collagen deposition, and macrophage infiltration in vivo. These in vitro and in vivo protective effects of AS-IV against GO were accompanied by decreased autophagic activities in orbital fibroblasts and GO orbital tissues, respectively. Collectively, our findings suggested that AS-IV protects against GO through suppression of autophagy. Thus, AS-IV may have preventive benefits for GO.
Recent studies have indicated that piperlongumine (PLM) may exert anti-inflammatory effects. In the present study, we determined the effect of PLM on the proliferation, apoptosis, migration and invasion of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) (referred to herein as RA FLS). We further explored the mechanisms by which the studied compound inhibits the functions of RA FLS. RA FLS viability and apoptosis were tested using MTT and Annexin V/PI assays, respectively. We performed an EDU assay to examine the proliferation of RA FLS. The migration and invasion of these cells were measured using a transwell chamber method and wound closure assay. The MMP-1, MMP-3, and MMP-13 levels in the culture supernatants of RA FLS were detected using a Luminex Assay kit. The intracellular ROS levels were detected using DCFH-DA. The expression levels of signal transduction proteins were measured using western blot. We found that PLM induced apoptosis in RA FLS at concentrations of 15 and 20 mu M. The proliferation of RA FLS was downregulated by PLM at concentrations of 1, 5 and 10 mu M. Migration and invasion of RA FLS were reduced by PLM at concentrations of 1, 5 and 10 mu M. PLM also inhibited cytoskeletal reorganization in migrating RA FLS and decreased TNF-alpha-induced intracellular ROS production. Moreover, we demonstrated the inhibitory effect of PLM on activation of the p38, JNK, NF-kappa B and STAT3 pathways. Our findings suggest that PLM can inhibit proliferation, migration and invasion of RA FLS. Moreover, these data suggests that PLM might have therapeutic potential for the treatment of RA.
This study aimed to investigate the anti-inflammatory effects of a novel spirocyclopiperazinium salt compound LXM-15, and explore the underlying mechanisms. Xylene-induced mouse ear oedema and carrageenan-induced rat paw oedema tests were used to evaluate the anti-inflammatory effects of LXM-15. The protein levels of TNF-alpha, IL-6, phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) were detected by ELISA or Western blot analysis. Additionally, receptor blocking test was performed to explore the possible target. Intragastric administration with LXM-15 (2, 1, 0.5 mg/kg in mice, and 6, 3, 1.5 mg/kg in rats) produced distinct anti-inflammatory effects in vivo, the highest inhibition percentage was 60 and 52%, respectively (P < 0.01). Following treatment with LXM-15 (6 mg/kg, i.g.), the levels of TNF-alpha and IL-6 in the rats paws were attenuated by 40 and 41%; and the phosphorylation of JAK2 and STAT3 was restrained by 35 and 45%, respectively (P < 0.01). All effects of LXM-15 were blocked by pretreatment with methyllycaconitine citrate or tropicamide. This study provides the first report that the spirocyclopiperazinium salt compound LXM-15 displays considerable anti-inflammatory effects. The underlying mechanism may be through activating the peripheral alpha 7 nicotinic acetylcholine receptor and M4 muscarinic acetylcholine receptor, leading to the inhibition of the JAK2/STAT3 signalling pathway, eventually resulting in the reduction of TNF-alpha and IL-6.
ObjectiveTetraspanin family plays an important role in the pathogenesis of cancer, but its role in lung fibrosis is unknown. To determine whether tetraspanin 1 (TSPAN1), a member of the family, may be involved in the pathogenesis of pulmonary fibrosis.MethodsTNF -stimulated human alveolar epithelial (A549) and alveolar epithelial type II cell (AT2) were treated in vitro. Murine pulmonary fibrosis model was generated by injection of bleomycin (BLM). The expression of TSPAN1 was examined in vivo using the bleomycin-induced lung fibrosis model and tissue sample of IPF patients. Then we transfected the cells with TSPAN1 siRNA or plasmid and detected the expression changes of related proteins and cell apoptosis.ResultsIn our study, we found that TSPAN1 was markedly down-regulated in lung tissue of patients with idiopathic pulmonary fibrosis (IPF) and in bleomycin-induced pulmonary fibrosis in mice. We also found that TSPAN1 was significantly down-regulated in A549 and primary (AT2) cells following exposure to TNF. Meanwhile, TSPAN1 inhibited p-IB, which attenuated nuclear NF-B translocation and activation and inhibited apoptosis. We demonstrated that TSPAN1 reduced Bax translocation and caspase-3 activation, inhibited the apoptosis by regulating the NF-B pathway in response to TNF.ConclusionsWe conclude that TSPAN1 mediated apoptosis resistance of alveolar epithelial cells by regulating the NF-B pathway. TSPAN1 may be a potential therapeutic target for pulmonary fibrosis or acute lung injury.
Aim and objectiveS100B has been found abundantly expressed in microglia during cerebral ischemia. However, S100B effects on phenotype changes and migration of microglia are unclear.MethodsReal-time PCR of S100B, M1 and M2 markers were tested to characterize phenotypic changes in microglia in mice middle cerebral artery occlusion (MCAO) model. Migration assay and additional mechanism studies were performed to elucidate the role of NF-B in S100B-mediated microglia M1/M2 phenotype change and migration. Finally, S100B treatment on MCAO models was performed to show the in vivo evidence.ResultsS100B was identified as an induced gene with its pattern in accordance with M1 markers in mice MCAO model. That S100B was promoted by M1 stimuli whereas inhibited by M2 stimuli further confirmed S100B a M1 marker. Moreover, S100B promotes microglia M1 polarization with enhanced migration ability and inhibits M2 polarization. Additionally, NF-B is essential in S100B control in microglia M1/M2 polarization and migration. Furthermore, S100B aggravated cerebral ischemia in murine MCAO model and exacerbated the microglia M1 polarization and migration.ConclusionsOur findings demonstrate that S100B promotes microglia M1 polarization to aggravate cerebral ischemia, and provide a better understanding on the therapeutic effects of S100B and/or its antagonist/neutralization antibody in stroke.
Objective and designWe investigated the expressions of lncRNA MEG3 and PTEN in ovarian cancer tissues and their effects on cell proliferation, cycle and apoptosis of ovarian cancer.MethodsExpression levels of MEG3 in ovarian cancer cell lines and normal ovarian cell lines were detected by qRT-PCR. Cell viability was detected by MTT assay. Cell apoptosis and cell cycle distribution were measured by flow cytometry. Cell invasion capability was tested by transwell assay. Cell migration capacity was tested by wound healing. The xenograft model was constructed to explore the effect of lncRNA MEG3 on ovarian cancer in vivo.ResultCompared with normal ovarian cells, expression levels of MEG3 and PTEN were relatively lower in ovarian cancer cells. There was a positive correlation between the expression of PTEN and the expression of MEG3. Enhanced expression level of PTEN suppressed SKOV3 cell proliferation, increased cell apoptosis rate, and decreased cell invasion and migration.ConclusionLncRNA MEG3 and PTEN were down-regulated in ovarian cancer cells. LncRNA MEG3 regulated the downstream gene PTEN in ovarian cancer cells to prohibit cell proliferation, promote apoptosis and block cell cycle progression.
ObjectiveTo determine whether the long noncoding RNA MALAT1 may be involved in the inflammatory effect of Amadori-glycated albumin (AGA) in retinal microglia via a microRNA-124 (miR-124)-dependent mechanism.MethodsDiabetes mellitus was induced by streptozotocin (STZ) injection. The expression of monocyte chemotactic protein-1 (MCP-1) in the retinas of rats was determined using quantitative reverse transcription-PCR (qRT-PCR) analyses and enzyme-linked immunosorbent assay (ELISA). Both qRT-PCR and ELISA were used to detect the levels of MCP-1 mRNA and soluble MCP-1 protein in the primary rat retinal microglia treated with AGA. The regulation of a putative target of miR-124 was validated by luciferase reporter assays.ResultsMALAT1 knockdown ameliorated diabetic retinopathy (DR) and inhibited MCP-1 release in the retinas of STZ-induced diabetic rats. The cultured retinal microglial cells treated with AGA-released MCP-1 in a dose- and time-dependent manner. In addition, AGA consistently induced MALAT1 expression in the retinal microglial cells. Next, we demonstrated that the expression of MCP-1 is controlled by miR-124, which binds to the 3-UTR of MCP-1 in microglial cells. Luciferase reporter assays and RNA-binding protein immunoprecipitation assays showed that MALAT1 targets miR-124. Finally, we demonstrated that MALAT1 acts as a competing endogenous RNA by directly binding to miR-124 to regulate AGA-induced MCP-1 expression in microglial cells.ConclusionsMALAT1-miR-124-MCP-1 signaling pathway may be involved in AGA-induced MCP-1 expression in microglial cells, which may provide a new approach for the treatment of DR.
BackgroundNuciferine, a major bioactive component from the lotus leaf, has been reported to have notable anti-inflammatory activities such as renal inflammation and acute lung injury in previous studies. Mastitis is one of the most prevalent diseases in the dairy cattle, which causes large economic losses for the dairy industry. However, the effects of nuciferine on lipopolysaccharide (LPS)-induced mastitis have not been reported.Methods and resultsHere, we investigated the anti-inflammatory effects of nuciferine on LPS-induced mastitis in mice and illuminated its potential mechanism on the TLR4-mediated signaling pathway in mouse mammary epithelial cells (mMECs). Histopathological changes and myeloperoxidase (MPO) activity assay showed that nuciferine treatment significantly alleviated the LPS-induced injury of mammary gland flocculus, inflammatory cells infiltration. qPCR and ELISA assays indicated that nuciferine dose-dependently reduced the levels of TNF- and IL-1, which indicated that nuciferine might have therapeutic effects on mastitis. Furthermore, nuciferine treatment significantly decreased the expression of TLR4 in a dose-dependent manner. Besides, nuciferine was also found to suppress LPS-induced NF-B activation.ConclusionThese findings indicate that nuciferine potently ameliorates LPS-induced mastitis by inhibition of the TLR4-NF-B signaling pathway.
The peptide lycosin-I has anti-bacterial and anti-cancer capacities. However, the anti-inflammatory activity of lycosin-I remains unknown. We investigated whether lycosin-I could attenuate inflammation. Human umbilical vein endothelial cells (HUVECs) were treated with lycosin-I before exposure to tumor necrosis factor-alpha (TNF-alpha). The expression of intercellular cell adhesion molecule-1 (ICAM-1), nuclear transcription factor-kappa B (NF-kappa B) p65 and inhibitory subunit of NF-kappa B alpha (I kappa B alpha) was evaluated by western blot. The expression of interleukin-6 (IL-6) and interleukin-8 (IL-8) was detected by quantitative RT-PCR or ELISA. Immunofluorescence analysis was used to determine the impact of lycosin-I on NF-kappa B pathway. C57BL/6 mice were pretreated with lycosin-I before exposure with lipopolysaccharide (LPS). Lycosin-I significantly reduced the TNF-alpha-enhanced expression of IL-6, IL-8 and ICAM-1. Lycosin-I also inhibited the human monocyte cells adhesion to HUVECs. We further demonstrated that lycosin-I could effectively suppress the reaction of endothelial cells to TNF-alpha by inhibiting I kappa B alpha degradation. Subsequently, the phosphorylation and translocation of NF-kappa B p65 could also be attenuated. Furthermore, lycosin-I exhibited a significant protection of C57BL/6 mice against LPS-induced death. Our results suggested that the anti-inflammatory activity of lycosin-I was associated with NF-kappa B activation and lycosin-I had potential to be a novel therapeutic candidate for inflammatory diseases.
The objective of this study is to investigate the role of cathepsin H (CatH), a lysosomal cysteine protease, in the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. EAE was induced in CatH-deficient mice (CatH (-/-)) and wild-type littermates (+/+) using myelin oligodendrocyte glycoprotein (MOG) 35-55. The effects of CatH deficiency were determined by clinical scoring, mRNA expression levels of Tbx21, Rorc and FoxP3, protein levels of poly(I:C)-induced toll-like receptor 3 (TLR3) and phosphorylation of IRF3, and secretion of interferon-beta (IFN-beta) by splenocytes. CatH (-/-) showed a significantly earlier disease onset of EAE and increased Th1 cell differentiation in splenocytes. Splenocytes prepared from immunized CatH (-/-) showed a significant decrease in poly(I:C)-induced increased TLR3 expression, interferon regulatory factor 3 (IRF3) phospholylation and IFN-beta secretion. Therefore, CatH deficiency impaired TLR3-mediated activation of IRF3 and consequent secretion of IFN-beta from dendritic cells, leading to the enhancement of Th1 cell differentiation and consequent early disease onset of EAE.
Background Chronic obstructive pulmonary disease (COPD) is a chronic and progressive lung disease characterized by a mixture of small airway disease and lung tissue parenchymal destruction. Abnormal inflammatory responses to cigarette smoking and other noxious particles are generally thought to be responsible for causing of COPD. Since airway inflammation is a key factor in COPD progress, it is crucial to unravel its underlying molecular mechanisms. Unbiased analysis of genome-wide gene expression profiles in lung small airway epithelial cells provides a powerful tool to investigate this. Methods Gene expression data of GSE611906, GSE20257, GSE8545 were downloaded from GEO database. All 288 lung small airway samples in these cohorts, including donors with (n = 61) and without (n = 227) COPD, were chosen for differential gene expression analysis. The gene ontology (GO) function, Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses, gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) network analysis were performed. Subsequently, the analyses of IL1B expression level, the Pearson correlation between IL1B and several COPD biomarkers were performed using other cohorts to validate our main findings. Results With a change >= twofold and P value < 0.05 cutoff, we found 38 genes were up-regulated and 114 genes were down-regulated in patients with COPD compared with health controls, while using cutoff fold change 1.5 and P value < 0.05, there were 318 genes up-regulated and 333 genes down-regulated. Among the most up-regulated genes were IL1B, CCL2, CCL23, and CXCL14, all implicated in inflammation triggering. GO, KEGG and WGCNA analysis all disclosed IL1B was highly correlated to COPD disease trait. The expression profile of IL1B was further validated using independent cohorts from COPD airway epithelium, lung tissue, sputum, and blood. We demonstrated higher IL1B gene expression in COPD small airway epithelial cells, but not in COPD lung tissue, sputum, and blood. Strong co-expression of IL1B with COPD biomarkers, such as DUOX2, MMP12, CCL2, and CXCL14, were validated in silico analysis. Finally, PPI network analysis using enriched data showed IL1B, CCL2, CCL7 and BMP7 were in the same hub node with high degrees. Conclusions We identified IL1B was significantly up-regulated in COPD small airway epithelial cells and propose IL1B as a novel player in airway inflammation in COPD.