Over-expression of caveolin-1 aggravate LPS-induced inflammatory response in AT-1 cells via up-regulation of cPLA2/p38 MAPK

Objective and design

The aim of this study was to study the effect of caveolin-1 on the cytosolic phospholipase A2 (cPLA2), p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor κB (NF-κB) in mouse lung alveolar type-1 cells' (AT-1 cells) inflammatory response induced by LPS.

Materials and methods

Gene clone technique was used to over-express caveolin-1 in AT-1 cells by lentivirus vector. The level of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), cPLA2, p38 MAPK and NF-κB was measured by ELISA, western blotting and EMSA.

Treatment

AT-1 cells were treated with LPS.

Results

Over-expression of caveolin-1 not only increased the production of pro-inflammatory cytokine TNF-α and IL-6, but also enhanced the expression of the cPLA2, p38 MAPK, and NF-κB.

Conclusions

Our data demonstrated that over-expression of caveolin-1 aggravates the AT-1 injury induced by LPS, involving in modulation of the cPLA2 mediated by the cPLA2/p38 MAPK pathway.     

The p38 mitogen-activated protein kinases modulate endothelial cell survival and tissue repair

Objective and design

This study is designed to investigate the role of p38 MAPK in modulating human pulmonary artery endothelial cells (HPAECs) survival and tissue repair functions.

Methods

HPAECs (passage 8?C12) were used for all experiments. Cells were treated with IL-1?? (0.5 or 2?ng/ml) or p38 inhibitor (SB203580 or SB220025, 5???M each). Cells were also transfected with 50?nM siRNAs. Cell length was measured using ImageJ software. Collagen gel contraction and wound close assay were performed to evaluate tissue repair functions.

Results

IL-1?? activated p38 MAPK and induced morphologic change of HPAECs. The p38 inhibitors further augmented IL-1??-induced cell morphologic change, prevented cell death, and augmented collagen gel contraction. Suppression of p38??, ??, or ??, but not p38?? resulted in cell morphologic alteration, and suppressing any one of p38 isoforms by siRNAs increased cell survival. Suppression of p38?? or ?? augmented gel contraction. While p38?? suppression stimulated cell migration, suppressing the rest of three isoforms inhibit cell migration. Nuclear factor p65-siRNA blocked IL-1??-induced cell morphologic change, but did not affect p38 inhibitor-induced change.

Conclusion

These findings suggest that p38 MAPK may negatively modulate tissue repair functions of endothelial cells via p65 independent pathway.     

Discriminators of mouse bladder response to intravesical Bacillus Calmette-Guerin (BCG)

Background

Intravesical Bacillus Calmette-Guerin (BCG) is an effective treatment for bladder superficial carcinoma and it is being tested in interstitial cystitis patients, but its precise mechanism of action remains poorly understood. It is not clear whether BCG induces the release of a unique set of cytokines apart from its pro-inflammatory effects. Therefore, we quantified bladder inflammatory responses and alterations in urinary cytokine protein induced by intravesical BCG and compared the results to non-specific pro-inflammatory stimuli (LPS and TNF-α). We went further to determine whether BCG treatment alters cytokine gene expression in the urinary bladder.

Methods

C57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-α. Morphometric analyses were conducted in bladders isolated from all groups and urine was collected for multiplex analysis of 18 cytokines. In addition, chromatin immune precipitation combined with real-time polymerase chain reaction assay (CHIP/Q-PCR) was used to test whether intravesical BCG would alter bladder cytokine gene expression.

Results

Acute BCG instillation induced edema which was progressively replaced by an inflammatory infiltrate, composed primarily of neutrophils, in response to weekly administrations. Our morphological analysis suggests that these polymorphonuclear neutrophils are of prime importance for the bladder responses to BCG. Overall, the inflammation induced by BCG was higher than LPS or TNF-α treatment but the major difference observed was the unique granuloma formation in response to BCG. Among the cytokines measured, this study highlighted the importance of IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-17, GM-CSF, KC, and Rantes as discriminators between generalized inflammation and BCG-specific inflammatory responses. CHIP/Q-PCR indicates that acute BCG instillation induced an up-regulation of IL-17A, IL-17B, and IL-17RA, whereas chronic BCG induced IL-17B, IL-17RA, and IL-17RB.

Conclusion

To the best of our knowledge, the present work is the first to report that BCG induces an increase in the IL-17 family genes. In addition, BCG induces a unique type of persisting bladder inflammation different from TNF-α, LPS, and, most likely, other classical pro-inflammatorystimuli.     

Protective effect of gossypol on lipopolysaccharide-induced acute lung injury in mice

Objective

Gossypol has been reported to have anti-inflammatory properties. The purpose of this study was to evaluate the effect of gossypol on acute lung injury (ALI) induced by lipopolysaccharide (LPS) in mice.

Methods

Male BALB/c mice were pretreated with gossypol 1 h before intranasal instillation of LPS. Then, 7 h after LPS administration, the myeloperoxidase in histology of lungs, lung wet/dry ratio and inflammatory cells in the bronchoalveolar lavage fluid (BALF) were determined. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in the BALF were measured by ELISA. The extent of phosphorylation of IκB-α, p65 NF-κB, p46–p54 JNK, p42–p44 ERK, and p38 were detected by western blot.

Results

Gossypol markedly attenuated the LPS-induced histological alterations in the lung and inhibited the production of TNF-α, IL-1β and IL-6. Additionally, gossypol reduced the inflammatory cells in BALF, decreased the wet/dry ratio of lungs and inhibited the phosphorylation of IκB-α, p65 NF-κB, p46–p54 JNK, p42–p44 ERK, and p38 caused by LPS.

Conclusion

The data suggest that anti-inflammatory effects of gossypol against the LPS-induced ALI may be due to its ability of inhibition of the NF-κB and MAPKs signaling pathways. Gossypol may be a promising potential therapeutic reagent for ALI treatment.     

Effect of Toll-like receptor 4 inhibitor on LPS-induced lung injury

Objective and design

Toll-like receptor 4 (TLR4) plays important roles in the recognition of lipopolysaccharide (LPS) and the activation of inflammatory cascade. In this study, we evaluated the effect of TAK-242, a selective TLR4 signal transduction inhibitor, on acute lung injury (ALI).

Materials and methods

C57BL/6J mice were intravenously treated with TAK-242 15 min before the intratracheal administration of LPS or Pam3CSK4, a synthetic lipopeptide. Six hours after the challenge, bronchoalveolar lavage fluid was obtained for a differential cell count and the measurement of cytokine and myeloperoxidase levels. Lung permeability and nuclear factor-κB (NF-κB) DNA binding activity were also evaluated.

Results

TAK-242 effectively attenuated the neutrophil accumulation and activation in the lungs, the increase in lung permeability, production of inflammatory mediators, and NF-κB DNA-binding activity induced by the LPS challenge. In contrast, TAK-242 did not suppress inflammatory changes induced by Pam3CSK4.

Conclusion

TAK-242 may be a promising therapeutic agent for ALI, especially injuries associated with pneumonia caused by Gram-negative bacteria.     

The peritoneal macrophage inflammatory profile in cirrhosis depends on the alcoholic or hepatitis C viral etiology and is related to ERK phosphorylation

Background

Continuous diabetes-associated complications are a major source of immune system exhaustion and an increased incidence of infection. Diabetes can cause poor circulation in the feet, increasing the likelihood of ulcers forming when the skin is damaged and slowing the healing of the ulcers. Whey proteins (WPs) enhance immunity during childhood and have a protective effect on some immune disorders. Therefore, in this study, we investigated the effects of camel WP on the healing and closure of diabetic wounds in a streptozotocin (STZ)-induced type I diabetic mouse model.

Results

Diabetic mice exhibited delayed wound closure characterized by a significant decrease in an anti-inflammatory cytokine (namely, IL-10) and a prolonged elevation of the levels of inflammatory cytokines (TNF-??, IL-1?? and IL-6) in wound tissue. Moreover, aberrant expression of chemokines that regulate wound healing (MIP-1??, MIP-2, KC and CX3CL1) and growth factors (TGF-??) were observed in the wound tissue of diabetic mice compared with control nondiabetic mice. Interestingly, compared with untreated diabetic mice, supplementation with WP significantly accelerated the closure of diabetic wounds by limiting inflammatory stimuli via the restoration of normal IL-10, TNF-??, IL-1?? and IL-6 levels. Most importantly, the supplementation of diabetic mice with WP significantly modulated the expression of MIP-1??, MIP-2, KC, CX3CL1 and TGF-?? in wound tissue compared with untreated diabetic mice.

Conclusion

Our data demonstrate the benefits of WP supplementation for improving the healing and closure of diabetic wounds and restoring the immune response in diabetic mice.     

Effect of chlorogenic acid on LPS-induced proinflammatory signaling in hepatic stellate cells

Objective and design

This study was aimed at investigating the effect of chlorogenic acid (CGA) on lipopolysaccharide (LPS)-induced proinflammatory signaling in hepatic stellate cells (HSCs).

Methods

An immortalized rat HSC line was cultured in vitro and treated with LPS in the absence or presence of CGA. Reactive oxygen species (ROS) production in the HSCs was monitored by flow cytometer using DCFH-DA. The protein expression levels of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor-κB (NF-κB), and p-IκB-α were determined by Western blot. The mRNA expression levels of TLR4, MyD88, monocyte chemotactic protein 1(MCP-1), and interleukin 6 (IL-6) were detected by RT-PCR. The levels of MCP-1 and IL-6 in the culture supernatant of HSCs were measured by ELISA.

Results

CGA had no effect on expression of TLR4 and MyD88. However, the treatment of CGA can inhibit LPS-induced production of ROS in HSCs. Meanwhile, CGA can inhibit LPS-induced nuclear translocation of NF-κB and IκB-α phosphorylation in HSCs, as well as NAC (a ROS scavenger). The mRNA expression and the levels of MCP-1 and IL-6 in the culture supernatant of the HSCs in this study were elevated by LPS stimulation and inhibited by CGA treatment, as well as NAC and PDTC (a NF-κB inhibitor).

Conclusion

Our results indicate that CGA can efficiently inhibit LPS-induced proinflammatory responses in HSCs and the anti-inflammatory effect may be due to the inhibition of LPS/ROS/NF-κB signaling pathway.     

Urinary trypsin inhibitor reduced neointimal hyperplasia induced by systemic inflammation after balloon injury in rabbits

Objectives

The aims of this study were to evaluate the effect of urinary trypsin inhibitor (UTI) on the regulation of inflammatory cytokines induced by lipopolysaccharide (LPS) and the reduction of neointimal formation in rabbits.

Methods and results

Rabbits subjected to iliac artery balloon injury were randomly divided into three groups: control group (balloon injury), LPS group (LPS + balloon injury) and UTI group (UTI + LPS + balloon injury). Systemic markers of inflammation (serum IL-1β and TNF-α levels measured by ELISA) were increased after LPS administration. Arterial nuclear factor-κB (NF-κB/p65) at 28 days after injury was 31.50 ± 7.08 % of total cells in controls and 73.50 ± 6.90 % in LPS group (P < 0.05). Morphometric analysis of the injured arteries at 28 days revealed significantly increased luminal stenosis (45.81 ± 5.31 vs 27.93 ± 2.85 %, P < 0.05) and neointima-to-media ratio (1.40 ± 0.15 vs 0.68 ± 0.12, P < 0.05) in LPS-treated animals compared with controls. This effect was reduced by UTI administration. Serum IL-1β and TNF-α levels and NF-κB/p65 expression were significantly increased in correlation with the severity of intimal hyperplasia and inhibited by UTI.

Conclusions

Systemic inflammatory response concurrently with arterial vascular injury facilitated neointimal formation. UTI reduced neointimal hyperplasia by regulating inflammatory response and could be considered as a potential anti-restenosis supplement.     

BACE1 inhibitory activity of fungal endophytic extracts from Malaysian medicinal plants

Background

The roots of Sophora flavescens (Leguminosae) have been used in East Asian countries as an herbal medicine and a food ingredient for thousands of years. The aim of the present study was to investigate the effects of S. flavescens fermentation on endotoxin-induced uveitis (EIU) in rats.

Methods

EIU was induced in rats via a footpad injection of lipopolysaccharide (LPS). Immediately after the LPS inoculation, fermented and non-fermented extracts of S. flavescens (FSE and NFSE, respectively) were administered orally, and the aqueous humor was collected from both eyes 24 hours later. The anti-inflammatory effects of FSE and NFSE were examined in terms of regulation of nuclear factor kappa B (NF-κB) activation and the expression of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), intercellular cell adhesion molecule (ICAM)-1, and cyclooxygenase-2 (COX-2). The regulation of maleic dialdehyde (MDA) levels and polymorphonuclear cell (PMN) infiltration by FSE and NFSE were also examined.

Results

Treatment with FSE significantly inhibited LPS-induced increases in IL-1β and TNF-α production and the expression of iNOS, ICAM-1 and COX-2. Moreover, FSE suppressed LPS-induced NF-κB activation, and reduced both MDA levels and infiltration by PMN.

Conclusion

These results indicate that solid state fermentation may enhance the anti-inflammatory effects of S. flavescens.     

Profiling the proteomic inflammatory state of human astrocytes using DIA mass spectrometry

Background

Astrocytes are the most abundant cells in the central nervous system and are responsible for a wide range of functions critical to normal neuronal development, synapse formation, blood-brain barrier regulation, and brain homeostasis. They are also actively involved in initiating and perpetuating neuroinflammatory responses. However, information about their proteomic phenotypes under inflammation is currently limited.

Method

Data-independent acquisition mass spectrometry was applied to extensively characterize the profile of more than 4000 proteins in immortalized human fetal astrocytes under distinct inflammatory conditions induced by TNF, IL-1β, and LPS, while multiplex immunoassay-based screening was used to quantify a wide range of cytokines released under these inflammatory conditions. Then, immunocytochemistry and western blotting were used to verify the activation of canonical and non-canonical NF-κB upon exposure to the different stimuli. Finally, an in vitro model of the blood-brain barrier consisting of a co-culture of primary human brain microvascular endothelial cells and primary human astrocytes was used to verify the inflammatory response of astrocytes upon LPS exposure in a more complex in vitro system.

Results

We reported on a set of 186 proteins whose levels were significantly modulated by TNF, IL-1β, and LPS. These three stimuli induced proteome perturbations, which led to an increased abundance of key inflammatory proteins involved in antigen presentation and non-canonical NF-κB pathways. TNF and IL-1β, but not LPS, also activated the canonical NF-κB pathway, which in turn led to an extensive inflammatory response and dysregulation of cytoskeletal and adhesion proteins. In addition, TNF and LPS, but not IL-1β, increased the abundance of several interferon-stimulated gene products. Finally, TNF and IL-1β similarly upregulated the secretion of several cytokines and chemokines, whereas LPS only induced a moderate increase in IL-8, IFN-γ, and IL-1β secretion. Upregulation of proteins associated with type I IFN and non-canonical NF-κB signaling was also observed in primary astrocytes co-cultured with primary brain microvascular endothelial cells exposed to LPS.

Conclusions

The present study provides comprehensive information about the proteomic phenotypes of human astrocytes upon exposure to inflammatory stimuli both in monoculture and in co-culture with human brain microvascular endothelial cells.

     

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.     

M3 mAChR-mediated IL-8 expression through PKC/NF-κB signaling pathways

Objective

M3 muscarinic acetylcholine receptor (mAChR) plays an important role in the regulation of cytokine production in inflammatory diseases. In this study, we explored the precise role of M3 mAChR under stimulation with agonist in IL-8 expression and of the signaling pathway involved in this process.

Materials and methods

Recombinant U2OS cells stably expressing M3 mAChR as a model system were stimulated by carbachol to evaluate the role of M3 mAChR in the expression of IL-8.

Results

Activation of M3 mAChR with carbachol increased both IL-8 mRNA and protein expression in a concentration-dependent manner. Elevated IL-8 expression was completely antagonized by atropine, 4-DAMP and tiotropium. M3 mAChR-mediated IL-8 expression was almost completely inhibited by the NF-κB inhibitor BAY11-7082 and, to a lesser extent, by U0126, SB203580, and SP600125, which are inhibitors for ERK1/2, p38, and JNK, respectively. Furthermore, M3 mAChR-mediated NF-κB activation and IL-8 expression were simultaneously attenuated by the PKC inhibitor calphostin C, whereas PMA, a PKC activator, mimicked the effects of carbachol, inducing IL-8 expression.

Conclusions

Our findings offer insights into the specific and critical role of M3 mAChR in regulating inflammatory response and indicate M3 mAChR/PKC/NF-κB signaling axis driven by endogenous acetylcholine as a potential therapeutic targets for inflammatory diseases.     

The Antiasthma Effect of Neonatal BCG Vaccination Does Not Depend on the Th17/Th1 but IL-17/IFN-�� Balance in a BALB/c Mouse Asthma Model

Objective

This study aimed to determine whether the protective effects of the Mycobacterium bovis Bacillus Calmette?CGuérin (BCG) vaccination on allergic asthma are associated with the T helper (Th) 17/Th1 balance in a murine asthma model.

Methods

BALB/c neonates were vaccinated with BCG on the first day after birth, sensitized with ovalbumin, and then challenged with allergen. The resulting airway inflammation and responsiveness were measured. The levels of IL-17 and interferon (IFN)-?? in BALF and ratio of Th17/Th1 were investigated.

Results

We found that although BCG neonatal vaccination inhibited airway hyperresponsiveness and inflammation following allergen challenge in a BALB/c mouse asthma model, reduced levels of Th2 cytokines were not observed. However, BCG neonatal vaccination reduced IL-17 production and increased IFN-?? production in both the bronchoalveolar lavage fluid and the lung lymphocytes in asthmatic mice.

Conclusion

The antiasthma effects of neonatal BCG vaccination reversed the IL-17/IFN-?? imbalance in a murine asthma model but did not depend on modifying the Th17/Th1 balance.     

Hepatoprotective activity of <Emphasis Type="Italic">Lepidium sativum</Emphasis> seeds against D-galactosamine/lipopolysaccharide induced hepatotoxicity in animal model

Background

Fulminant hepatic failure (FHF) is clinical syndrome with very poor prognosis and high mortality there is urgent need for the development of safe and non-toxic hepatoprotective agents for the adequate management of hepatitis. Hepatoprotective effect of the Lepidium sativum ethanolic extract (LSEE) was assessed by D-galactosamine-induced/lipopolysaccharide (400 mg/kg and 30 μg/kg) liver damage model in rats.

Methods

Hepatoprotective activity of LSEE (150 and 300 mg/kg) and silymarin on D-GalN/LPS induced FHF in rat was assessed using several liver function enzyme parameters. Antioxidant properties as antioxidant stress enzymes were assessed in hepatic Liver as well as mRNA expression of cytokines genes such as TNF-α, IL-6, and IL-10 and stress related genes iNOS and HO-1 were determined by RT-PCR. Protein expression of apoptotic genes were evaluated through western blot. MPO and NF-κB DNA-binding activity was analyzed by ELISA. The magnitude of hepatic impairment was investigated through histopathological evaluation.

Results

Marked amelioration of hepatic injuries by attenuation of serum and lipid peroxidation has been observed as comparable with silymarin (25 mg/kg p.o). D-GalN/LPS induced significant decrease in oxidative stress markers protein level, and albumin. LSEE significantly down-regulated the D-GalN/LPS induced pro-inflammatory cytokines TNFα and IL-6 mRNA expression in dose dependent fashion about 0.47 and 0.26 fold and up-regulates the IL-10 by 1.9 and 2.8 fold, respectively. While encourages hepatoprotective activity by down-regulating mRNA expression of iNOS and HO-1. MPO activity and NF-κB DNA-binding effect significantly increased and was mitigated by LSEE in a dose-dependent style as paralleled with silymarin.

Conclusion

Our data suggests that pretreatment of LSEE down regulates the caspase 3 and up-regulates the BCl₂ protein expression. The above findings revealed that Lepidium sativum has significant hepatoprotective activity.