Rapamycin reverses paraquat-induced acute lung injury in a rat model through inhibition of NFκB activation

Objective: To evaluate the role of rapamycin (RAPA) in paraquat (PQ)-induced acute lung injury. Methods: Lung tissues were stained with HE and lung histology was observed. Mortality rate, and neutrophil and leukocyte count in blood and bronchoalveolar lavage fluid (BALF) were recorded. Protein content in BALF was determined by Coomassie blue staining. Malondialdehyde (MDA) content, glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activity in blood were determined by thiobarbituric acid (TBA) assay, pyrogallol autoxidation method, and modified Haefman method, respectively. The NF-κB activity was measured by gel electrophoretic mobility shift assay (EMSA). Carbon dioxide partial pressure (PaCO₂), partial pressure of oxygen (PaO₂) and pH values were measured by automated blood gas analyzer. Results: HE staining results demonstrated RAPA alleviated pathological changes of acute alveolitis in SD rats. Trend of protein content in BALF was PQ group > RAPA treatment group > control group (P < 0.05). Neutrophil and leukocyte count in RAPA treatment group was significantly lower than PQ group at 3, 5, and 7 days after injection (P < 0.05). Trend of MDA content was RAPA treatment group > PQ group > control group (P < 0.05). Trend of GSH-Px and SOD activity was control group > RAPA treatment group > PQ group (P < 0.05). Compared with PQ group, PaO₂ in RAPA treatment group was markedly higher and PaCO₂ was lower (P < 0.05). Conclusion: PQ-induced acute lung injury was effectively reversed with RAPA, through inhibition of NF-κB activation.     

p-Synephrine suppresses lipopolysaccharide-induced acute lung injury by inhibition of the NF-κB signaling pathway

Objective

We investigated whether p-synephrine exerts potent anti-inflammatory effects against acute lung injury (ALI) induced by lipopolysaccharide (LPS) in vivo, and we further investigated the inhibitory mechanism of p-synephrine in LPS-induced ALI.

Methods

Lipopolysaccharide (0.5 mg/kg) was instilled intranasally in phosphate-buffered saline to induce acute lung injury, and 6, 24, and 48 h after LPS was given, bronchoalveolar lavage fluid was obtained to measure pro-inflammatory mediator. We also evaluated the effects of p-synephrine on LPS-induced the severity of pulmonary injury. The phosphorylation of nuclear factor-κB (NF-κB) p65 protein was analyzed by Western blotting.

Results

Our data showed that p-synephrine significantly reduced the amount of inflammatory cells, the lung wet-to-dry weight (W/D) ratio, reactive oxygen species, myeloperoxidase activity and enhanced superoxide dismutase (SOD) in mice with LPS-induced ALI. Tumor necrosis factor α and interleukin (IL)-6 concentrations decreased significantly while the concentration of IL-10 was significantly increased after p-synephrine pretreatment. In addition, p-synephrine suppressed not only the phosphorylation of NF-κB but also the degradation of its inhibitor (IκBα).

Conclusions

These results suggested that the inhibition of NF-κB activation and the regulation of SOD are involved in the mechanism of p-synephrine’s protection against ALI.     

TNF-α对内皮细胞粘附分子NFκB表达的影响

目的 :目前认为动脉粥样硬化 (ＡＳ)是一种炎症性疾病 ,其致病因素多种 ,细胞因子生长因子等在其发病机制中起着重要的作用。因而通过ＴＮＦ α作用于内皮细胞 (ＥＣ)后 ,观察单核细胞对其粘附的变化 ,粘附分子表达以及对调控粘附分子基因的转录因子 (ＮＦκＢ)变化 ,以探讨ＴＮＦ α在ＡＳ发生早期细胞行为中的作用。方法 :人脐静脉内皮细胞和γＴＮＦ α(5ｎｇ/ｍｌ)作用 4ｈ后 ,用细胞计数法测Ｕ937细胞以ＥＣ粘附率 ,用ＥＬＩＳＡ法检测ＥＣ膜上粘附分子ＶＣＡＭ 1、ＩＣＡＭ 1、Ｐ ｓｅｌｅｃｔｉｎ的表达 ,以及采用细胞免疫化学方法测…     

Inhibition of PKR ameliorates lipopolysaccharide-induced acute lung injury by suppressing NF-κB pathway in mice

Acute lung injury (ALI) is characterized by dramatic lung inflammation and alveolar epithelial cell death. Although protein kinase R (PKR) (double-stranded RNA-activated serine/threonine kinase) has been implicated in inflammatory response to bacterial cell wall components, whether it plays roles in lipopolysaccharide (LPS)-induced ALI remains unclear. This study was aimed to reveal whether and how PKR was involved in LPS-induced ALI pathology and the potential effects of its specific inhibitor, C16 (C₁₃H₈N₄OS). During the experiment, mice received C16 (100 or 500 ug/kg) intraperitoneally 1?h before intratracheal LPS instillation. Then, whole lung lavage was collected for analysis of total protein levels and proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6. The lungs were tested for Western blot, transferase-mediated dUTP nick-end labeling (TUNEL) stain and immunohistochemistry. Results showed that PKR phosphorylation increased significantly after LPS instillation. Furthermore, PKR specific inhibition attenuated LPS-induced lung injury (hematoxylin and eosin stain), reduced lung protein permeability (total protein levels in whole lung lavage) and suppressed proinflammatory cytokines (TNF-α, IL-1β and IL-6) and lung apoptosis (TUNEL stain and caspase3 activation). Moreover, mechanism-study showed that C16 significantly suppressed I kappa B kinase (IKK)/I kappa B alpha (IκBα)/NF-κB signaling pathway after LPS challenge. These findings suggested that PKR inhibition ameliorated LPS-induced lung inflammation and apoptosis in mice by suppressing NF-κB signaling pathway.     

014 TNF-α对内皮细胞粘附分子NFκB表达的影响

目的: 目前认为动脉粥样硬化(AS)是一种炎症性疾病, 其致病因素多种, 细胞因子生长因子等在其发病机制中起着重要的作用. 因而通过TNF-α作用于内皮细胞(EC)后, 观察单核细胞对其粘附的变化, 粘附分子表达以及对调控粘附分子基因的转录因子(NFκB)变化, 以探讨TNF-α在AS发生早期细胞行为中的作用. 方法: 人脐静脉内皮细胞和γTNF-α(5 ng/ml)作用4 h后, 用细胞计数法测U937细胞以EC粘附率, 用ELISA法检测EC膜上粘附分子VCAM-1、 ICAM-1、 P-selectin的表达, 以及采用细胞免疫化学方法测定NFκB核转位百分率. 结果: U937细胞对γTNF-α作用的EC粘附明显增高, 其粘附率为41.30%±1.16%(n=7), 对照组为4.50±1.01%(n=7)(P<0.001), γTNF-α能明显上调EC膜上VCAM-1, ICAM-1及P-selectin表达, 并能明显增加NFκB的核转位其阳性细胞为99.42%±0.84%而对照组为28.93%±14.06%(P<0.001), 当采用银杏提取液事先处理EC而后再用γTNF-α作用, 能抑制U937对EC的粘附百分率, 且有量效依赖关系. 结论: γTNF-α明显促进NFκB核转位, 增强了对粘附分子基因调控, 明显增加EC膜上粘附分子表达粘附分子是细胞粘附分子基础, 增加了单核细胞等对EC粘附, 这为单核细胞迁移到内膜下成为巨噬泡沫细胞提供了前提, 而银杏提取液有明显抑制单核细胞对TNF-α处理的EC粘附作用, 为AS的发病机理和防治提供线索.     

Protectin DX ameliorates inflammation in sepsis-induced acute lung injury through mediating PPARγ/NF-κB pathway

Immunologic Research - Previous reports have demonstrated that the newly identified lipid mediator protectin DX (PDX) could effectively attenuate multiple organ injuries in sepsis. The aim of our...     

Magnesium isoglycyrrhizinate attenuates D-galactosamine/lipopolysaccharides induced acute liver injury of rat via regulation of the p38-MAPK and NF-κB signaling pathways

Context: Acute hepatic failure involves in serious inflammatory responses and leads to a high mortality. Magnesium isoglycyrrhizinate (MgIG), a magnesium salt of 18-α glycyrrhizic acid (GA) stereoisomer, has been shown anti-inflammatory activity previously.

Objective: This study aimed to investigate the protective effects of MgIG, a hepatocyte protective agent, on D-galactosamine and lipopolysaccharide (D-GaIN/LPS)-induced acute liver injury in rats, and meanwhile explore the molecular mechanism.

Materials and methods: Male Sprague–Dawley (SD) rats were injected with D-GaIN/LPS (800?mg/kgBW/10?μg/kgBW) with or without administration of MgIG (225?mg/kg once 6?h after D-GaIN/LPS injection and MgIG 45?mg/kg twice in another 12?h, intraperitoneal injection). Rats were sacrificed 24?h after D-GaIN/LPS injection, the blood and liver samples were collected for future inflammation and hepatotoxicity analyses.

Results: MgIG significantly inhibited D-GaIN/LPS-induced inflammatory cytokines production and hepatotoxicity as indicated by both diagnostic indicators of liver damage [aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels] and histopathological analysis. Western blot analysis demonstrated that MgIG significantly decreased p38-mitogen activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) activation induced by D-GaIN/LPS.

Conclusion: The results indicated that the protective effects of MgIG on D-GaIN/LPS-induced acute liver injury might be correlated with its capacity to regulate the p38-MAPK and NF-κB signaling pathways.     

Anti-IFN-γ therapy alleviates acute lung injury induced by severe influenza A (H1N1) pdm09 infection in mice

Background/purposeSevere infection with influenza A (H1N1)pdm09 virus is characterized by acute lung injury. The limited efficacy of anti-viral drugs indicates an urgent need for additional therapies. We have previously reported that neutralization of gamma interferon (IFN-γ) could significantly rescue the thymic atrophy induced by severe influenza A (H1N1)pdm09 infection in BALB/c mice. A deeper investigation was conducted into the influence of neutralizing IFN-γ to the BALB/c mice weight, survival rate, and lung injury.MethodsThe BALB/c mice was infected with severe influenza A (H1N1)pdm09. Monoclonal antibodies against IFN-γ were injected into the abdominal cavities of the mice. After neutralization of IFN-γ occurred in mice infected by severe ? influenza A (H1N1)pdm09, observing the influence of neutralizing IFN-γ to the BALB/c mice weight, survival rate, lung injury.ResultOur results here showed that anti-IFN-γ therapy alleviated the acute lung injury in this mouse model. Neutralization of IFN-γ led to a significant reduction in the lung microvascular leak and the cellular infiltrate in the lung tissue, and also improved the outcome in mice mortality. Several pro-inflammatory cytokines, including interleukin (IL)-1α, tumor necrosis factor (TNF)-α and granulocyte-colony stimulating factor (G-CSF) in the bronchoalveolar lavage fluid (BALF), and the chemokines including G-CSF, monocyte chemoattractant protein-1 (MCP-1) in serum samples were found to be significantly reduced after anti-IFN-γ treatment.ConclusionThese results suggested that IFN-γ plays an important role in acute lung injury induced by severe influenza A (H1N1)pdm09 infection, and monoclonal antibodies against IFN-γ could be useful as a potential therapeutic remedy for future influenza pandemics.     

A modified “double-hit” induced acute lung injury model in rats and protective effects of tetramethylpyrazine on the injury via Rho/ROCK pathway

We focused on the production and evaluation of a modified “double-hit” induced acute lung injury (ALI) model, which closely mimics the clinical situation. Further, tetramethylpyrazine (TMP), an alkaloid contained in ligustrazine was evaluated for its potent anti-inflammatory effects in this model. Rats were randomized into 4 groups: G1 (NS control group), G2 (“double-hit” group), G3 (low dosage TMP group) and G4 (high dosage TMP group). The rats in G2, G3 and G4 were intraperitoneally injected with a low dose of LPS followed by intratracheal injection with median dose of LPS to establish the “double-hit” model. The rats in G3, G4 were intraperitoneally injected with low (G3), high (G4) dosage TMP for the protection against ALI. Upon termination of the experiment, TMP attenuated the harmful changes in animal model reaction, breathing frequency, histological examination, lung W/D-weight ratio, BAL fluid PMNs percentage, MPO activity and ROCK2 mRNA expression. We found inhibiting RhoA/ROCK pathway might attribute to TMP-induced protection against ALI.     

Immunomodulatory role of recombinant human erythropoietin in acute kidney injury induced by crush syndrome via inhibition of the TLR4/NF-κB signaling pathway in macrophages

Abstract

Objective: The present study aimed to investigate whether recombinant human erythropoietin (rHuEPO) plays an immunomodulatory function by regulating the TLR4/NF-κB signaling pathway.

Materials and methods: C57BL/6 mice were intraperitoneally injected with rHuEPO and, half an hour later, with 50% glycerol at the dose of 7.5?ml/kg to induce crush syndrome (CS)-acute kidney injury (AKI). The levels of TNF-α, IL-1β, IL-6, serum creatinine (Scr), and creatine kinase (CK) were measured. The kidney tissues were analyzed by HE staining, and macrophage infiltration was detected by immunohistochemistry. Double immunofluorescence staining, RT-qPCR, and western blotting were conducted to analyze TLR4/NF-κB p65 expression. Ferrous myoglobin was co-cultured with RAW264.7 cells to mimic crush injury and the production of proinflammatory cytokines. The expression levels of TLR4 and NF-κB p65 were measured.

Results: In vivo study results revealed that rHuEPO ameliorated renal function, tissue damage, production of proinflammatory cytokines, and macrophage infiltration in the kidneys. The protein and mRNA expression levels of genes involved in the TLR4/NF-κB signaling pathway in CS-induced AKI mice were upregulated (p?<?.05). Meanwhile, the expression levels of TLR4, NF-κB p65, and proinflammatory cytokines in RAW264.7 cells were downregulated in CS-AKI mice injected with rHuEPO (p?<?.05).

Conclusions: Our results demonstrated the immunomodulatory capacity of rHuEPO and confirmed that rHuEPO exerts protective effects against CS-induced AKI by regulating the TLR4/NF-κB signaling pathway in macrophages. Therefore, our findings highlight the therapeutic potential of rHuEPO in improving the prognosis of CS-AKI patients.     

Squamosamide derivative FLZ inhibits TNF-α-induced ICAM-1 expression via down-regulation of the NF-κB signaling pathway in ARPE-19 cells

Dysfunction of the retinal pigment epithelium (RPE) resulting from chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD). It has been reported that tumor necrosis factor-α (TNF-α) could induce intercellular adhesion molecule-1 (ICAM-1) expression in RPE cells. FLZ, a novel synthetic squamosamide derivative from a Chinese herb, Annona glabra, has displayed significant anti-inflammatory activity. However, the effects of FLZ on TNF-α-induced ICAM-1 expression in RPE cells remain unknown. Therefore, in the present study, we evaluated the effects of FLZ on TNF-α-induced ICAM-1 expression in RPE cells. We found that FLZ prevented TNF-α-induced ICAM-1 expression and the ability of monocytes to adhere to ARPE-19 cells induced by TNF-α. Furthermore, FLZ inhibited TNF-α-induced NF-κB p65 expression, as well as phosphorylation of IκBα in ARPE-19 cells. Taken together, these results suggest that FLZ inhibited TNF-α-induced ICAM-1 expression through blocking NF-κB signaling pathway in ARPE-19 cells. Thus, FLZ could be used for designing novel therapeutic agents against AMD.     

MHTP,a synthetic tetratetrahydroisoquinoline alkaloid,attenuates lipopolysaccharide-induced acute lung injury via p38MAPK/p65NF-κB signaling pathway-TLR4 dependent

Introduction

This study investigated the mechanism of action of a synthetic tetrahydroisoquinoline alkaloid, MHTP, in an experimental model of acute lung injury (ALI) in two distinct moments: 72 h and 10 days.

Methodology

To realize this study, 2.5 mg/kg of lipopolysaccharide (LPS) was intranasally administered in BALB/c mice, and nasal instillation of MHTP (1.25; 2.5; 5.0; 10 or 20 mg/kg) was administrated at 1, 24, and 48 h after LPS challenge. The data were statistically analyzed and p < 0.05 was considered statistically significant.

Results

MHTP treatment (2.5, 5.0, 10 or 20 mg/kg) significantly decreased neutrophil migration into the bronchoalveolar lavage fluid (BALF), tissue inflammatory cell infiltration, edema, and hemorrhage as well as collagen fiber deposition on the perialveolar regions at both moments. TNF-α and IL-6 levels were significantly diminished in the MHTP-treated animals at 72 h and maintained them, at a basal level, at 10-day observation. These effects of MHTP are due to downregulating p38MAPkinese/p65NFκB signaling pathway-TLR4 dependent. Also, the MHTP treatment promoted a survival rate at 100% and improved their body weights during the 10-day observation. Unlike, the LPS group (non-treated LPS challenged animals) presented less than 50% of surviving rate at 72 h and the animals that survived did not improve their physiological state at 10-day observation.

Conclusions

These data showed for the first time the beneficial and effective activity of a nasal treatment with a synthetic tetrahydroisoquinoline alkaloid on an experimental model of ALI and pointed out the molecular mechanism related to it.

     

Overexpression of integrin-linked kinase (ILK) promotes migration and invasion of colorectal cancer cells by inducing epithelial–mesenchymal transition via NF-κB signaling

Integrin-linked kinase (ILK), a ubiquitously expressed and evolutionally conserved serine/threonine kinase, has been shown to be aberrantly overexpressed and activated in diversified types of human malignancies, including colorectal cancer (CRC). However, the potential role of ILK in cancer cell migration and invasion remains to be elucidated. In this study, we introduced the human ILK gene into a low ILK-expressing human CRC cell line SW480. Cell migration and invasion were evaluated by the wound healing assay and transwell invasion assay, respectively. The epithelial–mesenchymal transition (EMT)-related proteins were detected by Western blot analysis or immunofluorescence. We found that enforced overexpression of ILK in SW480 cells dramatically promoted their migratory and invasive ability in vitro. Furthermore, SW480 cells stably overexpressing ILK underwent EMT, as indicated by mesenchymal morphology, decreased expression of E-cadherin, and increased expression of vimentin, Snail, and Slug. Finally, the nuclear factor (NF)-κB inhibitor BAY 11-7028 or NF-κB p65 small interfering RNA significantly restored the reduced E-cadherin level in ILK-overexpressing cells, suggesting that ILK-mediated down-regulation of E-cadherin is dependent on NF-κB activation. Overall, our study demonstrates a pivotal role of ILK in EMT and metastasis, and suggests novel therapeutic opportunities for the treatment of CRC.     

Time-dependent expression of endothelin-1 in lungs and the effects of TNF-α blocking peptide on acute lung injury in an endotoxemic rat model

Endothelin (ET)-1 is a potent vasoconstrictor that has been implicated in the pathogenesis of a number of diseases, and some studies suggest that circulating ET-1 is elevated in sepsis. The present study investigated whether ET plays a role in sepsis-mediated acute lung injury and whether its expression could be down regulated by blockade of TNF-α in septic lung. Male Wistar rats at 8 weeks of age were administered with either saline or lipopolysaccharide (LPS) at different time points (1, 3, 6 and 10 h) and various tests were then performed. The features of acute lung injury were observed at 1 h after LPS administration, which gradually became severe with time. Systolic and diastolic pressures were reduced just about one hour after LPS administration, whereas pulmonary TNF-α levels were significantly increased at various time points after LPS administration. LPS induced a time-dependent expression of ET-1 and ET(A) receptor in the lungs compared to control, peaking and increasing by 3 fold at 6 h after induction of endotoxemia, whereas levels of ET(B) receptor, which has vasodilating effects, were remarkably down regulated time-dependently. We conclude that time-dependent increase of ET-1 and ET(A) receptor with the down regulation of ET(B) receptor may play a role in the pathogenesis of acute lung injury in endotoxemia. Finally, treatment of LPS-administered rats with TNF-α blocking peptide for three hours significantly suppressed levels of pulmonary ET-1. These data taken together, led us to conclude that differential alteration in ET expression and its receptors may be mediated by TNF-α and may, in part, account for the pathogenesis of acute lung injury in endotoxemia.     

Endothelial cell injury and dysfunction induced by silver nanoparticles through oxidative stress via IKK/NF-κB pathways

Epidemiological studies have indicated that particulate matter (PM) exposure is most likely relevant to atherosclerosis. Endothelial cell injury and dysfunction are considered the early events in the initiation of atherosclerosis. Silver nanoparticles (AgNPs), with a smaller size and higher reactive activity, may induce much higher toxicity to endothelial cells compared with PM. However, few studies have been performed to determine the effect of AgNPs on endothelial cells. In the present study, human umbilical vein endothelial cells (HUVECs) were chosen as model cells to systematically explore the toxicity of AgNPs to endothelial cells. The obtained results indicated that exposure to AgNPs could inhibit proliferation, damage the cell membrane and seriously induce apoptosis. Simultaneously, the inflammatory cytokines, adhesion molecules, and chemokines of HUVECs were clearly up-regulated, which resulted in the adhesion of many monocytes to endothelial cells. More importantly, we found that dysfunctions of endothelial cells could be ascribed to the activation of NF-κB pathways. Furthermore, an oxidation inhibitor, N-acetyl-l-cysteine (NAC), effectively antagonized all of the AgNPs-induced responses, which indicated the key role of ROS production during the exposure of AgNPs in the toxicity of endothelial cells. In summary, our results clearly demonstrated that AgNPs could induce the injury and dysfunction of HUVECs through the activation of IKK/NF-κB, which is associated with oxidative stress, suggesting that exposure to AgNPs may be a potential hazardous factor for early atherosclerosis.