The aim of this study was to investigate the possible beneficial role of telmisartan in cerebral edema after traumatic brain injury (TBI) and the potential mechanisms related to the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) pyrin domain-containing 3 (NLRP3) inflammasome activation. TBI model was established by cold-induced brain injury. Male C57BL/6 mice were randomly assigned into 3, 6, 12, 24, 48 and 72 h survival groups to investigate cerebral edema development with time and received 0, 5, 10, 20 and 40 mg/kg telmisartan by oral gavage, 1 h prior to TBI to determine the efficient anti-edemic dose. The therapeutic window was identified by post-treating 30 min, 1 h, 2 h and 4 h after TBI. Blood-brain barrier (BBB) integrity, the neurological function and histological injury were assessed, at the same time, the mRNA and protein expression levels of NLRP3 inflammasome, IL-1β and IL-18 concentrations in peri-contused brain tissue were measured 24 h post TBI. The results showed that the traumatic cerebral edema occurred from 6 h, reached the peak at 24 h and recovered to the baseline 72 h after TBI. A single oral dose of 5, 10 and 20 mg/kg telmisartan could reduce cerebral edema. Post-treatment up to 2 h effectively limited the edema development. Furthermore, prophylactic administration of telmisartan markedly inhibited BBB impairment, NLRP3, apoptotic speck-containing protein (ASC) and Caspase-1 activation, as well as IL-1β and IL-18 maturation, subsequently improved the neurological outcomes. In conclusion, telmisartan can reduce traumatic cerebral edema by inhibiting the NLRP3 inflammasome-regulated IL-1β and IL-18 accumulation.
Animals ; Benzimidazoles ; administration & dosage ; Benzoates ; administration & dosage ; Blood-Brain Barrier ; drug effects ; Brain Edema ; drug therapy ; genetics ; pathology ; Brain Injuries, Traumatic ; drug therapy ; genetics ; pathology ; Caspase 1 ; biosynthesis ; Gene Expression Regulation ; drug effects ; Humans ; Inflammasomes ; adverse effects ; genetics ; Interleukin-18 ; biosynthesis ; Interleukin-1beta ; biosynthesis ; Male ; Mice ; NLR Family, Pyrin Domain-Containing 3 Protein ; biosynthesis ; genetics ; Signal Transduction ; drug effects

The epithelial cytokine response, associated with reactive oxygen species (ROS), is important in Helicobacter pylori (H. pylori)-induced inflammation. H. pylori induces the production of ROS, which may be involved in the activation of mitogen-activated protein kinases (MAPK), janus kinase/signal transducers and activators of transcription (Jak/Stat), and oxidant-sensitive transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB), and thus, expression of interleukin-8 (IL-8) in gastric epithelial cells. alpha-lipoic acid, a naturally occurring thiol compound, is a potential antioxidant. It shows beneficial effects in treatment of oxidant-associated diseases including diabetes. The present study is purposed to investigate whether alpha-lipoic acid inhibits expression of inflammatory cytokine IL-8 by suppressing activation of MAPK, Jak/Stat, and NF-kappaB in H. pylori-infected gastric epithelial cells. Gastric epithelial AGS cells were pretreated with or without alpha-lipoic acid for 2 h and infected with H. pylori in a Korean isolate (HP99) at a ratio of 300:1. IL-8 mRNA expression was analyzed by RT-PCR analysis. IL-8 levels in the medium were determined by enzyme-linked immunosorbent assay. NF-kappaB-DNA binding activity was determined by electrophoretic mobility shift assay. Phospho-specific and total forms of MAPK and Jak/Stat were assessed by Western blot analysis. ROS levels were determined using dichlorofluorescein fluorescence. As a result, H. pylori induced increases in ROS levels, mRNA, and protein levels of IL-8, as well as the activation of MAPK [extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase 1/2 (JNK1/2), p38], Jak/Stat (Jak1/2, Stat3), and NF-kappaB in AGS cells, which was inhibited by alpha-lipoic acid. In conclusion, alpha-lipoic acid may be beneficial for prevention and/or treatment of H. pylori infection-associated gastric inflammation.
Enzyme-Linked Immunosorbent Assay ; Epithelial Cells/metabolism ; Gastric Mucosa/*drug effects/metabolism/microbiology ; Gene Expression Regulation, Bacterial ; Helicobacter Infections/immunology/*metabolism ; Helicobacter pylori/drug effects/*pathogenicity ; Humans ; Interleukin-8/genetics/*metabolism ; JNK Mitogen-Activated Protein Kinases ; Janus Kinase 1 ; Mitogen-Activated Protein Kinases/*biosynthesis ; NF-kappa B/*metabolism ; RNA, Messenger/isolation & purification/metabolism ; Reactive Oxygen Species/metabolism ; STAT3 Transcription Factor ; Stomach/metabolism/*microbiology ; Thioctic Acid/*pharmacology

Helicobacter pylori (H. pylori) induces the activation of nuclear factor-kB (NF-kappaB) and cytokine expression in gastric epithelial cells. The Janus kinase/signal transducers and activators of transcription (Jak/Stat) cascade is the inflammatory signaling in various cells. The purpose of the present study is to determine whether H. pylori-induced activation of NF-kappaB and the expression of interleukin-8 (IL-8) are mediated by the activation of Jak1/Stat3 in gastric epithelial (AGS) cells. Thus, gastric epithelial AGS cells were infected with H. pylori in Korean isolates (HP99) at bacterium/cell ratio of 300:1, and the level of IL-8 in the medium was determined by enzyme-linked immonosorbent assay. Phospho-specific and total forms of Jak1/Stat3 and IkappaBalpha were assessed by Western blot analysis, and NF-kappaB activation was determined by electrophoretic mobility shift assay. The results showed that H. pylori induced the activation of Jak1/Stat3 and IL-8 production, which was inhibited by a Jak/Stat3 specific inhibitor AG490 in AGS cells in a dose-dependent manner. H. pylori-induced activation of NF-kappaB, determined by phosphorylation of IkappaBalpha and NF-kappaB-DNA binding activity, were inhibited by AG490. In conclusion, Jak1/Stat3 activation may mediate the activation of NF-kappaB and the expression of IL-8 in H. pylori-infected AGS cells. Inhibition of Jak1/Stat3 may be beneficial for the treatment of H. pylori-induced gastric inflammation, since the activation of NF-kappaB is inhibited and inflammatory cytokine expression is suppressed.
Blotting, Western ; DNA, Bacterial/analysis/genetics ; Epithelial Cells/metabolism ; Gastric Mucosa/drug effects/*immunology/microbiology ; Gene Expression Regulation/drug effects/*immunology ; Gene Expression Regulation, Bacterial ; Helicobacter Infections/immunology/*metabolism ; Helicobacter pylori/genetics/pathogenicity/*physiology ; Humans ; Interleukin-8/genetics/*metabolism ; Janus Kinase 1 ; NF-kappa B/biosynthesis/*metabolism ; Phosphorylation ; RNA, Messenger/metabolism ; STAT3 Transcription Factor ; Signal Transduction/genetics

Helicobacter pylori (H. pylori) induces the activation of nuclear factor-kB (NF-kappaB) and cytokine expression in gastric epithelial cells. The Janus kinase/signal transducers and activators of transcription (Jak/Stat) cascade is the inflammatory signaling in various cells. The purpose of the present study is to determine whether H. pylori-induced activation of NF-kappaB and the expression of interleukin-8 (IL-8) are mediated by the activation of Jak1/Stat3 in gastric epithelial (AGS) cells. Thus, gastric epithelial AGS cells were infected with H. pylori in Korean isolates (HP99) at bacterium/cell ratio of 300:1, and the level of IL-8 in the medium was determined by enzyme-linked immonosorbent assay. Phospho-specific and total forms of Jak1/Stat3 and IkappaBalpha were assessed by Western blot analysis, and NF-kappaB activation was determined by electrophoretic mobility shift assay. The results showed that H. pylori induced the activation of Jak1/Stat3 and IL-8 production, which was inhibited by a Jak/Stat3 specific inhibitor AG490 in AGS cells in a dose-dependent manner. H. pylori-induced activation of NF-kappaB, determined by phosphorylation of IkappaBalpha and NF-kappaB-DNA binding activity, were inhibited by AG490. In conclusion, Jak1/Stat3 activation may mediate the activation of NF-kappaB and the expression of IL-8 in H. pylori-infected AGS cells. Inhibition of Jak1/Stat3 may be beneficial for the treatment of H. pylori-induced gastric inflammation, since the activation of NF-kappaB is inhibited and inflammatory cytokine expression is suppressed.
Blotting, Western ; DNA, Bacterial/analysis/genetics ; Epithelial Cells/metabolism ; Gastric Mucosa/drug effects/*immunology/microbiology ; Gene Expression Regulation/drug effects/*immunology ; Gene Expression Regulation, Bacterial ; Helicobacter Infections/immunology/*metabolism ; Helicobacter pylori/genetics/pathogenicity/*physiology ; Humans ; Interleukin-8/genetics/*metabolism ; Janus Kinase 1 ; NF-kappa B/biosynthesis/*metabolism ; Phosphorylation ; RNA, Messenger/metabolism ; STAT3 Transcription Factor ; Signal Transduction/genetics

OBJECTIVETo explore the role of HIV-1 tat gene variations in AIDS dementia complex (ADC) pathogenesis.

METHODSHIV-1 tat genes derived from peripheral spleen and central basal ganglia of an AIDS patient with ADC and an AIDS patient without ADC were cloned for sequence analysis. HIV-1 tat gene sequence alignment was performed by using CLUSTAL W and the phylogentic analysis was conducted by using Neighbor-joining with MEGA4 software. All tat genes were used to construct recombinant retroviral expressing vector MSCV-IRES-GFP/tat. The MSCV-IRES-GFP/tat was cotransfected into 293T cells with pCMV-VSV-G and pUMVC vectors to assemble the recombinant retrovirus. After infection of gliomas U87 cells with equal amount of the recombinant retrovirus, TNF-α, and IL-1β concentrations in the supernatant of U87 cells were determined with ELISA.

RESULTSHIV-1 tat genes derived from peripheral spleen and central basal ganglia of the AIDS patient with ADC and the other one without ADC exhibited genetic variations. Tat variations and amino acid mutation sites existed mainly at Tat protein core functional area (38-47aa). All Tat proteins could induce U87 cells to produce TNF-α and IL-1β, but the level of IL-1β production was different among Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen. The level of Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen were obviously higher than that from the non-ADC patient's basal ganglia.

CONCLUSIONTat protein core functional area (38-47aa) may serve as the key area of enhancing the secretion of IL-1β. This may be related with the neurotoxicity of HIV-1 Tat.

AIDS Dementia Complex ; metabolism ; pathology ; virology ; Adult ; Amino Acid Sequence ; Basal Ganglia ; virology ; Cell Line, Tumor ; Gene Expression Regulation, Viral ; Genes, tat ; HIV-1 ; genetics ; pathogenicity ; Humans ; Interleukin-1beta ; biosynthesis ; genetics ; secretion ; Middle Aged ; Molecular Sequence Data ; Neuroglia ; pathology ; secretion ; Spleen ; virology ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics ; secretion ; tat Gene Products, Human Immunodeficiency Virus ; genetics ; physiology

Ebosin is a novel exopolysaccharide produced by Streptomyces sp.139 with remarkable activity against rheumatic arthritis in vivo. In this paper, we reported effects of Ebosin on the inflammatory cytokines including interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) in THP-1 cells. With the special fluorogenic peptide as substrates, the enzymatic activities of interleukin-1beta converting enzyme (ICE) and TNFalpha-converting enzyme (TACE) were inhibited by Ebosin separately. Using the real-time reverse transcription polymerase chain reaction (real-time PCR), the mRNA synthesis of the three cytokines were identified decline separately by Ebosin. The secretion quantum of three cytokines in THP-1 cells with Ebosin was lower than that of normal THP-1 cells determined by ELISA assay and Western blotting. All of these results showed that Ebosin has remarkably suppressed synthesis of the three cytokines in THP-1 cells through different pathways. The primary study of Ebosin on anti-inflammation mechanism was promoted developing the new drugs treating rheumatic arthritis.
ADAM Proteins ; metabolism ; ADAM17 Protein ; Anti-Inflammatory Agents ; pharmacology ; Caspase 1 ; metabolism ; Cell Line, Tumor ; Humans ; Interleukin-1beta ; genetics ; metabolism ; Interleukin-6 ; genetics ; metabolism ; Leukemia, Monocytic, Acute ; metabolism ; pathology ; Polysaccharides, Bacterial ; biosynthesis ; pharmacology ; RNA, Messenger ; metabolism ; Streptomyces ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

The fungus Trichophyton schoenleinii (T. schoenleinii) is the causative agent of Trichophytosis and Tinea favosa of the scalp in certain regions of Eurasia and Africa. Human innate immune system plays an important role in combating with various pathogens including fungi. The inflammasome is one of the most critical arms of host innate immunity, which is a protein complex controlling maturation of IL-1β. To clarify whether T. schoenleinii is able to activate the inflammasome, we analyzed human monocytic cell line THP-1 for IL-1β production upon infection with T. schoenleinii strain isolated from Tinea favosa patients, and rapid IL-1β secretion from THP-1 cells was observed. Moreover, applying competitive inhibitors and gene specific silencing with shRNA, we found that T. schoenleinii induced IL-1β secretion, ASC pyroptosome formation as well as caspase-1 activation were all dependent on NLRP3. Cathepsin B activity, ROS production and K⁺ efflux were required for the inflammasome activation by T. schoenleinii. Our data thus reveal that the NLRP3 inflammasome plays an important role in host defense against T. schoenleinii, and suggest that manipulating NLRP3 signaling can be a novel approach for control of diseases caused by T. schoenleinii infection.
Animals ; Bone Marrow Cells ; cytology ; Carrier Proteins ; metabolism ; Caspase 1 ; metabolism ; Cell Line ; Dendritic Cells ; cytology ; metabolism ; microbiology ; Enzyme Activation ; Hot Temperature ; Humans ; Inflammasomes ; metabolism ; Interleukin-1beta ; biosynthesis ; metabolism ; Lysosomes ; metabolism ; Mice ; Monocytes ; cytology ; metabolism ; microbiology ; NLR Family, Pyrin Domain-Containing 3 Protein ; Potassium ; metabolism ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; Trichophyton ; physiology

The paracaspase MALT1 is essential for the activation of NF-κB in response to T cell receptor (TCR) stimulation. It recruits downstream TRAF6 and activates the E3 ligase activity of TRAF6 to polyubiquitinate several targets, which ultimately leads to NF-κB activation. Here we identified ubiquitin-specific protease 2a (USP2a) as a MALT1-associated protein by biochemical affinity purification. Endogenous USP2a constitutively interacted with TRAF6, but dynamically interacted with MALT1 and CARMA1 in a stimulation-dependent manner. RNA interference (RNAi)-mediated silencing of USP2a attenuated TCR-induced NF-κB activation and production of interleukin-2 (IL-2). In addition, the ubiquitination of MALT1 and TRAF6 were both suppressed by USP2a knockdown. By knockdown and reconstitution assays, we found that USP2a mediated the interaction between MALT1 and TRAF6 in a catalytic activity-dependent manner. Furthermore, USP2a deSUMOylated TRAF6. Our findings implicate that USP2a plays an important role in TCR signaling by deSUMOylating TRAF6 and mediating TRAF6-MALT1 interaction.
Caspases ; metabolism ; Endopeptidases ; deficiency ; genetics ; metabolism ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Interleukin-2 ; biosynthesis ; Jurkat Cells ; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein ; NF-kappa B ; metabolism ; Neoplasm Proteins ; metabolism ; Receptors, Antigen, T-Cell ; metabolism ; Signal Transduction ; Sumoylation ; TNF Receptor-Associated Factor 6 ; metabolism

12(S)-Hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) is an enzymatic product of prostaglandin H2 (PGH2) derived from cyclooxygenase (COX)-mediated arachidonic acid metabolism. Despite the high level of 12-HHT present in tissues and bodily fluids, its precise function remains largely unknown. In this study, we found that 12-HHT treatment in HaCaT cells remarkably down-regulated the ultraviolet B (UVB) irradiation-induced synthesis of interleukin-6 (IL-6), a pro-inflammatory cytokine associated with cutaneous inflammation. In an approach to identify the down-stream signaling mechanism by which 12-HHT down-regulates UVB-induced IL-6 synthesis in keratinocytes, we observed that 12-HHT inhibits the UVB-stimulated activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kappaB). In addition, we found that 12-HHT markedly up-regulates MAPK phosphatase-1 (MKP-1), a critical negative regulator of p38 MAPK. When MKP-1 was suppressed by siRNA knock-down, the 12-HHT-mediated inhibitory effects on the UVB-stimulated activation of p38 MAPK and NF-kappaB, as well as the production of IL-6, were attenuated in HaCaT cells. Taken together, our results suggest that 12-HHT exerts anti-inflammatory effect via up-regulation of MKP-1, which negatively regulates p38 MAPK and NF-kappaB, thus attenuating IL-6 production in UVB-irradiated HaCaT cells. Considering the critical role of IL-6 in cutaneous inflammation, our findings provide the basis for the application of 12-HHT as a potential anti-inflammatory therapeutic agent in UV-induced skin diseases.
Anti-Inflammatory Agents, Non-Steroidal/pharmacology ; Cell Line ; Dual Specificity Phosphatase 1/biosynthesis/genetics ; Enzyme Activation ; Fatty Acids, Unsaturated/*pharmacology ; Humans ; Interleukin-6/*biosynthesis ; Keratinocytes/*metabolism/radiation effects ; NF-kappa B/metabolism ; RNA Interference ; RNA, Small Interfering ; Receptors, Leukotriene B4/genetics ; Signal Transduction/drug effects ; Skin Diseases/drug therapy ; *Ultraviolet Rays ; Up-Regulation ; p38 Mitogen-Activated Protein Kinases/metabolism

OBJECTIVETo construct pPICZalphaA-soluble interleukin-1 receptor type I (sIL-1RI) recombinant expression vector containing the gene fragment encoding the extracellular domain of sIL-1RI for its expression in Pichia pastoris.

METHODSsIL-1RI gene was amplified by RT-PCR and inserted into the yeast expression vector pPICZalphaA by digestion ligation. The recombinant plasmid pPICZalphaA-sIL1RI was transformed into E.coli Stb13, and the positive clones were analyzed by PCR and DNA sequencing. The pPICZalphaA-sIL1RI recombinant plasmid was electroporated into GS115 cells and the transformants were analyzed by PCR. After phenotype identification, the recombinant strains were induced by methanol to express the target protein, which was analyzed by Western blotting of the cell extract and supernatant.

RESULTSThe recombinant plasmid pPICZalphaA-sIL-1RI was constructed successfully, and the results of Western blotting showed that yeast induced by methanol expressed a protein of about 39 kD.

CONCLUSIONsIL-1RI protein has been successfully expressed in P.pastoris expression system, which provides the basis for further study of sIL-1RI.

Escherichia coli ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Pichia ; metabolism ; Plasmids ; Receptors, Interleukin-1 Type I ; biosynthesis ; genetics