Methane alleviates carbon tetrachloride induced liver injury in mice: anti-inflammatory action demonstrated by increased PI3K/Akt/GSK-3β-mediated IL-10 expression

The inflammatory response plays an important role in carbon tetrachloride (CCl₄)-induced acute liver injury and methane has been shown to exert beneficial effects on inflammation-associated diseases. Thus, we investigated the potential protective effects of methane-rich saline (MS) on CCl₄-induced acute liver injury and explored the underlying mechanism. A CCl₄-induced acute liver injury model was established by injection of CCl₄ (0.6 ml/kg, ip) in mice followed by treatment with MS (16 ml/kg, ip), 24 h later. All groups of mice were sacrificed and blood and liver tissues were collected. Serum aminotransferase, necrotic areas, and inflammatory cell infiltration in liver slices were enhanced after CCl₄ treatment but decreased with MS treatment. IL-6, TNF-α, IL-1β, IFN-γ, ICAM-1, CXCL1, MPO, NF-κB p65, ERK, JNK, and MAPK P38, expression in serum or liver homogenate were greater after CCl₄ treatment but comparatively less after MS treatment. Only IL-10 increased after MS treatment. Anti-IL10 blockade (1.5 mg/kg) restored MS-mediated attenuated phosphorylation of NF-?bB/MAPK and the protective effect of MS was abolished for all indices examined. The PI3K inhibitor, wortmannin had the same effects on MS as anti-IL-10 antibody. MS also induced phosphorylation of GSK-3β and AKT in CCl₄-treated mice. After pre-treatment with wortmannin (0.7 mg/kg), phosphorylation of GSK-3β and AKT proteins were reduced compared to its solvent control group-DMSO-treated animals. Thus, the data provide evidence that MS may activate the PI3K–AKT–GSK-3β pathway to induce IL-10 expression and produce anti-inflammatory effects via the NF-κB and MAPK pathways. The findings provide a new pharmacological strategy for management of inflammatory response after acute liver injury.     

APOE2 promotes the development and progression of subretinal neovascularization in age-related macular degeneration via MAPKs signaling pathway

BackgroundNeovascular age-related macular degeneration (nvAMD) is one of the main pathological features of wet AMD. Apolipoprotein E2 is involved in the formation of nvAMD but the molecular mechanism has not been reported.MethodsThe APOE alleles in AMD patients were detected by genotyping. Mouse models were divided into 4 groups according to transfection different gene segments and laser-induced treatment. APOE2, VEGF, PDGF-BB, b-FGF and inflammatory cytokines (including p-NF-κB, TNF-α, IL-1β and IL-6) were tested by ELISA in mice retinal lysate. The formation of nvAMD in the indicated treatment groups at 3rd, 7th and 14th day after laser-induced damage were detected by FFA. Besides, qRT-PCR was used to determine the mRNA levels of p38, JNK and ERK in ARPE-19 cells. Finally, the inflammatory cytokines and MAPK proteins (including P38, p-P38, JNK, p-JNK, ERK and p-ERK) were detected by western blot.ResultsThe statistics of APOE alleles showed that APOE2 allele carriers were more likely to nvAMD. VEGF, PDGF-BB, b-FGF and related inflammatory cytokines were up-regulated significantly after treatment with APOE2, which were reduced after silencing the MAPK family genes, however. Further, the expression levels of neovascular growth factors and inflammatory cytokines were highly consistent between mouse models and ARPE-19 cells. Besides, the phosphorylation levels of p38, JNK and ERK were affected by APOE2.ConclusionnvAMD was affected directly by the overexpression of VEGF, PDGF-BB and b-FGF, which were regulated by APOE2 through activating MAPKs pathway.     

Inhibition of Rho kinase by fasudil hydrochloride attenuates lung injury induced by intestinal ischemia and reperfusion

AimThe aim of this study is to evaluate the role of Rho-kinase in the pathogenesis of lung injury induced by intestinal ischemia/reperfusion (I/R) and the preconditioning effects of fasudil hydrochloride. The novel therapeutic approach of using Rho-kinase inhibitors in the treatment of intestinal I/R is introduced.MethodsSprague–Dawley (SD) rats were divided into 4 groups: intestinal I/R group, two fasudil pretreatment groups (7.5 mg/kg and 15 mg/kg), and controls. Intestinal and lung histopathology was evaluated; myeloperoxidase (MPO) and superoxide dismutase (SOD) levels in lung parenchyma were determined. Serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured. eNOS and P-ERM expression were measured by Western Blot.ResultsLung and intestinal injury were induced by intestinal I/R, characterized by histological damage and a significant increase in BALF protein. Compared to controls, serum TNF-α, IL-6, and lung MPO activity increased significantly in the I/R group, while SOD activity decreased. A strongly positive P-ERM expression was observed, while eNOS expression was weak. After fasudil administration, injury was ameliorated. Serum TNF-α, IL-6, lung MPO and P-ERM expression decreased significantly as compared to the I/R group, while SOD activity and eNOS expression increased significantly.SignificanceRho-kinase plays a key role in the pathogenesis of lung injury induced by intestinal I/R. The inhibition of the Rho-kinase pathway by fasudil hydrochloride may prevent lung injury.     

Casuarinin suppresses TNF-α-induced ICAM-1 expression via blockade of NF-κB activation in HaCaT cells

Hippophae rhamnoides has been extensively used in oriental traditional medicines for treatment of asthma, skin diseases, gastric ulcers, and lung disorders. In this study, we isolated casuarinin from the leaves of H.rhamnoides and examined the effect of casuarinin on the TNF-α-induced ICAM-1 expression in a human keratinocytes cell line HaCaT. Pretreatment with casuarinin inhibited TNF-α-induced protein and mRNA expression of ICAM-1 and subsequent monocyte adhesiveness in HaCaT cells. Casuarinin significantly inhibited TNF-α-induced NF-κB activation. In addition, casuarinin inhibited activation of ERK and p38 MAPK in a dose-dependent manner. Furthermore, pretreatment with casuarinin decreased TNF-α-induced pro-inflammatory mediators, such as IL-1β, IL-6, IL-8, and MCP-1. These results demonstrated that casuarinin exerts its anti-inflammatory activity by suppressing TNF-α-induced expression of ICAM-1 and pro-inflammatory cytokines/chemokines via blockage of activation of NF-κB and ERK/p38 MAPK and can be used as a therapeutic agent against inflammatory skin diseases.     

Cytokine effects on cell survival and death of A549 lung carcinoma cells

PurposeIL-13, TNF-α and IL-1β have various effects on lung cancer growth and death, but the signaling pathways mediating these effects have not been extensively analyzed. Therefore, the effects of IL-13, TNF-α and IL-1β alone, and in combination with Fas, on cell viability and death as well as major signaling pathways involved in these effects were investigated in A549 lung carcinoma cells.ResultsUsing MTT and flow cytometry, IL-13, TNF-α and IL-1β pretreatment decreased Fas-induced cell death. These anti-cell death effects were attenuated by pretreatment with inhibitors of Nuclear factor-κB [NF-κB], Phoshatidylinositole-3 kinase [PI3-K], JNK, p38 and ERK1/2 pathways.Using Western blot, IL-13, TNF-α and IL-1β treated cells showed time-dependent expression of p-ERK1/2, p-p38, p-JNK, p-Akt and p-IκBα proteins, decreased IκBα protein expression, no cleavage of Caspase-3 and PARP1 proteins and no notable alterations of Fas protein. IL-13 and TNF-α treated cells showed time-dependent increase of FLIP_L expression.ConclusionIL-13, TNF-α and IL-1β attenuate the pro-cell death effects of Fas on A549 cells, at least partially, by pathways involving the NF-κB, PI3-K and MAP kinases, but not by alterations of Fas protein expression. The IL-13 and TNF-α cell survival effects may also be due to increased expression of FLIP_L protein.     

Inhibition of the p38 pathway upregulates macrophage JNK and ERK activities,and the ERK,JNK, and p38 MAP kinase pathways are reprogrammed during differentiation of the murine myeloid M1 cell line

Mitogen-activated protein (MAP) kinases have been implicated as important mediators of the inflammatory response. Here we report that c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAP kinase activities are reprogrammed during the IL-6 induced macrophage-like differentiation of the murine myeloid M1 cell line. Moreover, p38 inhibition upregulates JNK and ERK activity in M1 cells and in thioglycollate-elicited peritoneal exudate macrophages. IL-6-induced M1 differentiation also induces expression of the anti-inflammatory cytokine IL-10, and p38 inhibition potentiates this increase in IL-10 expression in an ERK-dependent manner. Thus, we speculate that during inflammatory conditions in vivo macrophage p38 may regulate JNK and ERK activity and inhibit IL-10 expression. These data highlight the importance of p38 in the molecular mechanisms of macrophage function.     

LL-37 and its analog FF/CAP18 attenuate neutrophil migration in sepsis-induced acute lung injury

Introduction: Sepsis can result in acute lung injury. LL-37 is a small cationic host defense peptide involved in anti-inflammatory. In the current study, it was hypothesized that antimicrobial peptide LL-37 could play a protective role in attenuating the progression of sepsis-induced acute lung injury. Methods: Forty male C57BL/6 mice were induced into sepsis using cecal ligation and puncture, and subsequently administered with recombinant mouse osteopontin. Peptides LL-37, the LL-37 analog (FF/CAP18, called sLL-37), or normal saline was intravenously administered into septic mice for 20 hours. Then, proinflammatory cytokines (IL-6 and IL-1β), acute lung injury markers (alanine aminotransferase [ALT], aspartate aminotransferase [AST], and lactate dehydrogenase [LDH]), the neutrophil infiltration marker (myeloperoxidase [MPO]), and neutrophil infiltration were detected. Furthermore, the neutrophil migration and expression of migration-related factors (focal adhesion kinase [FAK], ERK, and P38) in differentiated HL-60 cells were detected. Results: Septic mice had upregulated IL-6, IL-1β, ALT, AST, LDH, MPO, p-FAK, p-ERK, and p-P38, infiltrated neutrophils, and migrated neutrophil-like HL-60 cells. In contrast, the administration of peptide LL-37 and sLL-37 inhibited all these changes. Compared with septic mice, it was found that proinflammatory cytokines, lung injury markers, MPO, and infiltrated neutrophils decreased in mice treated with LL-37 and sLL-37. In addition, the migrated neutrophil-like HL-60 cells and activated p-FAK, p-ERK, and p-P38 proteins were suppressed by LL-37 and sLL-37 treatments. Conclusions: Peptide LL-37 and its analog sLL-37 attenuated the progression of sepsis-induced acute lung injury by inhibiting neutrophil infiltration and migration through the FAK, ERK, and P38 pathways.     

Polysaccharide-rich fraction of Termitomyces eurhizus accelerate healing of indomethacin induced gastric ulcer in mice

The current study aims to determine the healing activity of water soluble polysaccharide-rich fraction of a wild mushroom, Termitomyces eurhizus (TEps) against the indomethacin induced gastric ulceration in mice model. Gastric tissue histology, myeloperoxidase (MPO) activity, cyclooxygenases (COX) 1 and 2 expression, prostaglandin E₂ (PGE₂) synthesis, and modulation of pro/anti inflammatory cytokines expression were studied for this purpose. Histological study shows that TEps (20 mg/kg) effectively healed the gastric ulceration. Based on biochemical results, the healing capacities of TEps could be attributed to reduction of MPO activity and protection of mucosal mucin content. Enhanced synthesis of PGE₂ by modulation of COX-1 and COX-2 expression and a prominent shift of cytokines expression from pro (TNF-α, IL-1ß) to anti inflammatory (IL-10) side are also held responsible for ulcer healing. The preliminary study highlights the anti-ulcerogenic property of polysaccharide-rich fraction of Termitomyces eurhizus and opens an alternative cure for NSAID induced gastroduodenal diseases.     

Polysaccharide isolated from seeds of Plantago asiatica L. induces maturation of dendritic cells through MAPK and NF-κB pathway

Plantago species are used as traditional medicine in Asian and Europe. Polysaccharide isolated from the seeds of Plantago asiatica L. could stimulate maturation transformation of bone-marrow derived dendritic cells (DCs). We found that blocking p38, ERK1/2 and JNK MAPK signal transduction could significantly decreased the PLP-2 induced expression of MHC II, CD86 surface molecules on DCs. Blocking p38 and JNK signal also significantly inhibited the cytokine secretion of TNF-α and IL-12p70 as well, while blocking ERK1/2 signal only decreased the secretion of TNF-α. Meanwhile, DCs in the three MAPK signal-blocking groups showed dramatically attenuated effects on stimulating proliferation of T lymphocytes. Similarly, blocking signal transduction of NF-κB pathway also significantly impaired the phenotypic and functional maturation development of DCs induced by PLP-2. These data suggest that MAPK and NF-κB pathway mediates the PLP-induced maturation on DCs. Especially, among the three MAPK pathways, activation of JNK signal transduction is the most important for DCs development after PLP-2 incubation. And PLP-2 may activate the MAPK and NF-κB pathway by triggering toll-like receptor 4 on DCs.     

A PPAR-γ ligand, 15-deoxy-Δ12,14-prostaglandin J2, inhibited gastric mucosal injury induced by ischemia–reperfusion in rats

Abstract

Introduction: Recent studies have demonstrated the anti-inflammatory action of 15-deoxy-Δ12,14-prostaglandin J₂ (15d-PGJ₂), a derivative of the PGD₂ metabolic pathway. Acute inflammation, including neutrophil activation, plays a critical role in the pathogenesis of ischemia–reperfusion (I/R). The aim of the present study was to determine the effect of 15d-PGJ₂ on I/R-induced gastric mucosal injury in rats.

Methods: Gastric mucosal damage was induced in male Wistar rats by clamping the celiac artery for 30 min followed by reperfusion. 15d-PGJ₂ (0.01–1.0 mg/kg) was given to the rats intraperitoneally 1 h before the vascular clamping. The area of gastric mucosal erosions (erosion index) was measured. Thiobarbituric acid reactive substances (TBARS) and tissue-associated myeloperoxidase (MPO) activity were measured in the gastric mucosa as indices of lipid peroxidation and neutrophil infiltration. The expression of tumor necrosis factor-α (TNF-α) in gastric mucosa was measured by ELISA. In addition, to elucidate whether the protective effects of 15d-PGJ₂ are related to the activation of the PPAR-γ receptor, we also investigated the effects of a PPAR-γ antagonist, GW9662.

Results: After 60 min of reperfusion, the area of gastric erosion index had significantly increased from the mean basal levels. The increase in the erosion index was significantly inhibited by pretreatment with 15d-PGJ₂ in a dose-dependent manner. On the other hand, GW9662 reversed the protective effect of 15d-PGJ₂. The concentration of TBARS and MPO activity in the gastric mucosa were both significantly increased after I/R, and pretreatment with 15d-PGJ₂ significantly reduced these increases. The TNF-α content was significantly higher in the I/R group than in the sham-operated group. However, the increase in TNF-α was significantly inhibited by pretreatment with 15d-PGJ₂.

Conclusions: 15d-PGJ₂ significantly inhibited the severity of acute gastric mucosal injury induced by I/R in rats through PPAR-γ-dependent mechanisms. This effect may be due, in part, to a reduction in the infiltration of neutrophils into the gastric mucosa, possibly via the inhibition of inflammatory cytokine.     

α2A-AR antagonism by BRL-44408 maleate attenuates acute lung injury in rats with downregulation of ERK1/2, p38MAPK,and p65 pathway

Acute respiratory distress syndrome (ARDS), characterized by acute hypoxic respiratory dysfunction or failure, is a manifestation of multiple organ failure in the lung, and the most common risk factor is sepsis. We previously showed that blocking α₂-adrenoceptor (α₂-AR) could attenuate lung injury induced by endotoxin in rats. α_2A-adrenoceptor (α_2A-AR), a subtype of α₂-AR plays a key role in inflammatory diseases, but the mechanism remains unknown. Here, we explored the effect of BRL-44408 maleate (BRL), a specific α_2A-AR antagonist, on cecal ligation puncture (CLP)-induced ARDS in rats and the underlying mechanism. Preadministration of BRL-44408 maleate significantly alleviated CLP-induced histological injury, macrophage infiltration, inflammatory response, and wet/dry ratio in lung tissue. However, there was no statistical difference in survival rate between the CLP and CLP+BRL groups. Extracellular regulated protein kinase (ERK1/2), p38MAPK, and p65 were activated in the CLP group, and BRL-44408 maleate inhibited the activation of these signal molecules, c-Jun N-terminal kinase (JNK) and protein kinase A (PKA) showed no changes in activation between these two groups. BRL-44408 maleate decreased lipopolysaccharide (LPS)-induced expression of cytokines in NR8383 rat alveolar macrophages and reduced phosphorylation of ERK1/2, p38MAPK, and p65. JNK and PKA were not influenced by LPS. Together, these findings suggest that antagonism of α_2A-AR improves CLP-induced acute lung injury and involves the downregulation of ERK1/2, p38MAPK, and p65 pathway independent of the activation of JNK and PKA.     

Ferulic acid suppresses expression of tryptophan metabolic key enzyme indoleamine 2, 3-dioxygenase via NFκB and p38 MAPK in lipopolysaccharide-stimulated microglial cells

Ferulic acid (FA) is a phenol compound found in plants that has anti-inflammatory properties. Indoleamine 2, 3-dioxygenase (IDO) is a tryptophan catabolic enzyme induced in immune cells, including glial cells, during inflammation. Enhanced IDO expression leads to reduced tryptophan levels and increased levels of toxic metabolites, including quinolinic acid. Therefore, inhibition of IDO expression may be effective in suppressing progression of neurodegenerative diseases. In this study, we examined the effect of FA in microglial cells on IDO expression levels and related inflammatory signal molecules. FA suppressed LPS-induced IDO mRNA expression and also suppressed nuclear translocation of NF-κB and phosphorylation of p38 MAPK. However, FA did not affect the production of LPS-induced inflammatory mediators and phosphorylation of JNK. Our results indicate that FA suppresses LPS-induced IDO mRNA expression, which may be mediated by inhibition of the NF-κB and p38 MAPK pathways in microglial cells.     

Stabilization of Nrf2 by tBHQ prevents LPS-induced apoptosis in differentiated PC12 cells

The inflammatory reaction plays an important role in the pathogenesis of the neurodegenerative disorders. tert-butylhydroquinone (tBHQ) exhibits a wide range of pharmacological activities including anti-oxidative and anti-inflammatory action. In this study, we tried to elucidate possible effects of tBHQ on lipopolysaccharide (LPS)-induced inflammatory reaction and its underlying mechanism in neuron-like PC12 cells. tBHQ inhibited LPS-induced generation of reactive oxygen species (ROS) and elevation of intracellular calcium level. It also inhibited LPS-induced cyclooxygenase 2 (COX-2), TNF-α, nuclear factor KappaB (NF-kB), and caspase-3 expression in a dose-dependent manner while stabilizing nuclear factor-erythroid 2 p45-related factor 2. Moreover, the phosphorylations of p38, ERK1/2, and JNK were suppressed by tBHQ. These results suggest that the anti-inflammatory properties of tBHQ might result from inhibition of COX-2 and TNF-α expression, inhibition of NF-kB nuclear translocation along with suppression of MAP kinases (p38, ERK1/2, and JNK) phosphorylation in PC12 cells, so may be a useful agent for prevention of inflammatory diseases.     

Ohioensin F suppresses TNF-α-induced adhesion molecule expression by inactivation of the MAPK, Akt and NF-κB pathways in vascular smooth muscle cells

AimsThe expression of cell adhesion molecules on vascular smooth muscle cells is central to leukocyte recruitment and progression of atherosclerotic disease. Ohioensin F, a chemical compound of the Antarctic moss Polyerichastrum alpinum, exhibited inhibitory activity against protein tyrosine phosphatase 1B and antioxidant activity. However, published scientific information regarding other biological activities and pharmacological function of ohioensin F is scarce. In the present study, we aimed to examine the in vitro effects of ohioensin F on the ability to suppress TNF-α-induced adhesion molecule expression in vascular smooth muscle cells (VSMCs).Main methodsThe inhibitory effect of ohioensin F on TNF-α-induced upregulation in expression of adhesion molecules was investigated by enzyme-linked immunosorbent assay, cell adhesion assay, RT-PCR, western blot analysis, immunofluorescence, and transfection and reporter assay, respectively.Key findingsPretreatment of VSMCs with ohioensin F at nontoxic concentrations of 0.1–10 μg/ml dose-dependently inhibited TNF-α-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). In addition, ohioensin F suppressed adhesion of THP-1 monocytes to TNF-α-stimulated VSMCs. Ohioensin F reduced TNF-α-induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38, ERK, JNK and Akt. Finally, ohioensin F inhibited TNF-α-induced CAM mRNA expression and NK-κB translocation.SignificanceThese results suggest a new mechanism of ohioensin F's anti-inflammatory action, owing to the negative regulation of TNF-α-induced adhesion molecule expression, monocyte adhesion and ROS production in vascular smooth muscle cells. Our finding also supports ohioensin F as a potential pharmacological, anti-inflammatory molecule that has a protective effect on the atherosclerotic lesion.     

Differential activation of mitogen-activated protein kinases in AGS gastric epithelial cells by cag+ and cag- Helicobacter pylori.

The aim of this study was to determine whether Helicobacter pylori activates mitogen-activated protein (MAP) kinases in gastric epithelial cells. Infection of AGS cells with an H. pylori cag+ strain rapidly (5 min) induced a dose-dependent activation of extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK) MAP kinases, as determined by Western blot analysis and in vitro kinase assay. Compared with cag+ strains, cag- clinical isolates were less potent in inducing MAP kinase, particularly JNK and p38, activation. Isogenic inactivation of the picB region of the cag pathogenicity island resulted in a similar loss of JNK and p38 MAP kinase activation. The specific MAP kinase inhibitors, PD98059 (25 microM; MAP kinase kinase (MEK-1) inhibitor) and SB203580 (10 microM; p38 inhibitor), reduced H. pylori-induced IL-8 production in AGS cells by 78 and 82%, respectively (p < 0.01 for each). Both inhibitors together completely blocked IL-8 production (p < 0.001). However, the MAP kinase inhibitors did not prevent H. pylori-induced IkappaBalpha degradation or NF-kappaB activation. Thus, H. pylori rapidly activates ERK, p38, and JNK MAP kinases in gastric epithelial cells; cag+ isolates are more potent than cag- strains in inducing MAP kinase phosphorylation and gene products of the cag pathogenicity island are required for maximal MAP kinase activation. p38 and MEK-1 activity are required for H. pylori-induced IL-8 production, but do not appear to be essential for H. pylori-induced NF-kappaB activation. Since MAP kinases regulate cell proliferation, differentiation, programmed death, stress, and inflammatory responses, activation of gastric epithelial cell MAP kinases by H. pylori cag+ strains may be instrumental in inducing gastroduodenal inflammation, ulceration, and neoplasia.