Interleukin-6 Protein Expression Research Articles

Significance of cerebrospinal fluid matrix metalloproteinase-9, interleukin-6, and tumor necrosis factor-alpha protein in children with viral encephalitis

Objective To explore the significance of changes of matrix metalloproteinase-9(MMP-9), interleukin-6(IL-6), and tumor necrosis factor-alpha(TNF-α) protein level in the cerebrospinal fluid (CSF) of children with viral encephalitis(VE). Methods The concentration of neuron-specific enolase(NSE), structural proteins 100B (S100B), and MMP-9, IL-6, TNF-α in the CSF of VE children were detected by an enzyme linked immunosorbent assay, and the correlations of them were analyzed. Results NSE, S100B, MMP-9, IL-6, TNF-α protein expression could be found significantly higher than those in the control group, and there were significant differences according to statistics expression trends(all P<0.05). The NSE protein expression was significantly positive related with S100B in the VE group (r=0.467, P=0.009), and the concentration was markedly negative related with the duration of viral encephalitis (r=-0.472, P=0.008). MMP-9, IL-6 protein expression were significantly positive related with NSE, S100B respectively (r=0.698, P=0.00; r=0.559, P=0.00; r=0.812, P=0.00; r=0.664, P=0.00). TNF-α protein expression was positive related with CSF S100B(r=0.363, P=0.049), but there was no correlation between TNF-α and NSE(r=0.245, P=0.193). Conclusions The neurons and the neuroglial cells are damaged in the viral encephalitis children.MMP-9, IL-6, TNF-α protein may participate in the pathological damage process of nerve cells in VE children in different degrees. Key words: Viral encephalitis; Structural proteins 100B; Matrix metalloproteinase-9; Interleukin-6; Tumor necrosis factor-alpha; Child

Estrogen receptor, progesterone receptor, interleukin-6 and interleukin-8 are variable in breast cancer and benign stem/progenitor cell populations.

BackgroundEstrogen receptor positive breast cancers have high recurrence rates despite tamoxifen therapy. Breast cancer stem/progenitor cells (BCSCs) initiate tumors, but expression of estrogen (ER) or progesterone receptors (PR) and response to tamoxifen is unknown. Interleukin-6 (IL-6) and interleukin-8 (IL-8) may influence tumor response to therapy but expression in BCSCs is also unknown.MethodsBCSCs were isolated from breast cancer and benign surgical specimens based on CD49f/CD24 markers. CD44 was measured. Gene and protein expression of ER alpha, ER beta, PR, IL-6 and IL-8 were measured by proximity ligation assay and qRT-PCR.ResultsGene expression was highly variable between patients. On average, BCSCs expressed 10-106 fold less ERα mRNA and 10-103 fold more ERβ than tumors or benign stem/progenitor cells (SC). BCSC lin-CD49f−CD24−cells were the exception and expressed higher ERα mRNA. PR mRNA in BCSCs averaged 10-104 fold less than in tumors or benign tissue, but was similar to benign SCs. ERα and PR protein detection in BCSCs was lower than ER positive and similar to ER negative tumors. IL-8 mRNA was 10-104 higher than tumor and 102 fold higher than benign tissue. IL-6 mRNA levels were equivalent to benign and only higher than tumor in lin-CD49f−CD24−cells. IL-6 and IL-8 proteins showed overlapping levels of expressions among various tissues and cell populations.ConclusionsBCSCs and SCs demonstrate patient-specific variability of gene/protein expression. BCSC gene/protein expression may vary from that of other tumor cells, suggesting a mechanism by which hormone refractory disease may occur.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-733) contains supplementary material, which is available to authorized users.

Endocannabinoids and inflammatory response in periodontal ligament cells.

Endocannabinoids are associated with multiple regulatory functions in several tissues. The main endocannabinoids, anandamide (AEA) and 2-arachidonylglycerol (2-AG), have been detected in the gingival crevicular fluid of periodontitis patients, but the association between periodontal disease or human periodontal ligament cells (hPdLCs) and endocannabinoids still remain unclear. The aim of the present study was to examine the effects of AEA and 2-AG on the proliferation/viability and cytokine/chemokine production of hPdLCs in the presence/absence of Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS). The proliferation/viability of hPdLCs was measured using 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT)-assay. Interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) levels were examined at gene expression and protein level by real-time PCR and ELISA, respectively. AEA and 2-AG did not reveal any significant effects on proliferation/viability of hPdLCs in the absence of P. gingivalis LPS. However, hPdLCs viability was significantly increased by 10–20 µM AEA in the presence of P. gingivalis LPS (1 µg/ml). In the absence of P. gingivalis LPS, AEA and 2-AG did not exhibit any significant effect on the expression of IL-8 and MCP-1 expression in hPdLCs, whereas IL-6 expression was slightly enhanced by 10 µM 2-AG and not affected by AEA. In P.gingivalis LPS stimulated hPdLCs, 10 µM AEA down-regulated gene-expression and protein production of IL-6, IL-8, and MCP-1. In contrast, 10 µM 2-AG had an opposite effect and induced a significant up-regulation of gene and protein expression of IL-6 and IL-8 (P<0.05) as well as gene-expression of MCP-1 in P. gingivalis LPS stimulated hPdLCs. Our data suggest that AEA appears to have an anti-inflammatory and immune suppressive effect on hPdLCs’ host response to P.gingivalis LPS, whereas 2-AG appears to promote detrimental inflammatory processes. In conclusion, AEA and 2-AG might play an important role in the modulation of periodontal inflammation.

Targeting complement anaphylatoxin C5a receptor in hyperoxic lung injury in mice.

Receptor binding of complement anaphylatoxin C5a results in proinflammatory activation of numerous diseases, but the role of receptor-mediated action during hyperoxic lung injury has, to the best of our knowledge, not yet been investigated. The contribution of the C5a receptor (C5aR) to hyperoxic lung injury in mice was investigated in this study. The effect of C5aR on hyperoxic lung injury in Balb/c mice was examined employing a C5aR antagonist (C5aRA). The mice were ventilated with 100% oxygen for 36 h with or without the administration of C5aRA. C5aR expression levels in non-treated or 100% oxygen-treated mice were assessed by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. The body weight and the relative lung weight of the mice, and the morphological changes in the lung were then determined. The total cell counts and the number of macrophages, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF) were determined using cytocentrifuge slides and a hemocytometer. The levels of interleukin-6 (IL-6), monocyte chemotactic protein (MCP-1) and tumor necrosis factor-α (TNF-α) in BALF and the myeloperoxidase (MPO) activity in the lung tissue were measured by enzyme-linked immunosorbent assay. The relative levels of CD68 and F4/80 messenger ribonucleic acid (mRNA) expression in the lung tissue were detected by RT-PCR. The TNF-α, IL-6 and MCP-1 protein expression levels in the lung tissue were assessed by western blot analysis. The results revealed hyperoxia-induced morphological changes, lung injury and increased expression levels of C5aR in lung tissue. The hyperoxia-induced increases in the total cell count and the number of macrophages, neutrophils and lymphocytes in the BALF were all significantly reduced in the mice receiving C5aRA. Treatment with C5aRA also attenuated the morphological changes and reduced MPO activity, and CD68 and F4/80 mRNA expression levels in the lung tissue, as well as the levels of IL-6, MCP-1 and TNF-α in BALF and lung tissue. In conclusion, C5a-C5aR action accelerated hyperoxia-induced lung injury, but this hyperoxic lung injury was attenuated by treatment with C5aRA.

The association between sex-related interleukin-6 gene polymorphisms and the risk for cerebral palsy.

BackgroundThe relationship between genetic factors and the development of cerebral palsy (CP) has recently attracted much attention. Polymorphisms in the genes encoding proinflammatory cytokines have been shown to be associated with susceptibility to perinatal brain injury and development of CP. Interleukin-6 (IL-6) is a proinflammatory cytokine that plays a pivotal role in neonatal brain injury, but conflicting results have been reported regarding the association between IL-6 single nucleotide polymorphisms (SNPs) and CP. The purpose of this study was to analyze IL-6 gene polymorphisms and protein expression and to explore the role of IL-6 in the Chinese CP population.MethodsA total of 753 healthy controls and 713 CP patients were studied to detect the presence of five SNPs (rs1800796, rs2069837, rs2066992, rs2069840, and rs10242595) in the IL-6 locus. Of these, 77 healthy controls and 87 CP patients were selected for measurement of plasma IL-6 by Luminex assay. The SHEsis program was used to analyze the genotyping data. For all comparisons; multiple testing on each individual SNP was corrected by the SNPSpD program.ResultsThere were no differences in allele or genotype frequencies between the overall CP patients and controls among the five genetic polymorphisms. However, subgroup analysis found significant sex-related differences in allele and genotype frequencies. Differences were found between spastic CP and controls in males for rs2069837; between CP with periventricular leukomalacia and controls in males for rs1800796 and rs2066992; and between term CP and controls in males for rs2069837. Plasma IL-6 levels were higher in CP patients than in the controls, and this difference was more robust in full-term male spastic CP patients. Furthermore, the genotype has an effect on IL-6 synthesis.ConclusionsThe influence of IL-6 gene polymorphisms on IL-6 synthesis and the susceptibility to CP is related to sex and gestational age.

Lipopolysaccharide-regulated production of bone sialoprotein and interleukin-8 in human periodontal ligament fibroblasts: the role of toll-like receptors 2 and 4 and the MAPK pathway.

Lipopolysaccharide (LPS) on the cell wall of periodontal pathogens is a major mediator of the inflammatory response and can enhance alveolar bone resorption in periodontitis. Bone sialoprotein is an early marker of osteoblast differentiation. The proinflammatory cytokine, interleukin-8 (IL-8), induces osteoclast differentiation, maturation and maintenance of bone resorption activity. However, the effects of LPS from periodontal pathogens on the expression of bone sialoprotein and IL-8 in human osteoblasts and the mechanism of periodontal bone metabolism regulation are rather unclear. The objectives of this study were to determine the effects of Porphyromonas gingivalis LPS on the production of bone sialoprotein and IL-8 in human periodontal ligament fibroblasts (hPDLFs), and to investigate whether toll-like receptor (TLR) 2, TLR4 and MAPKs pathways are involved in the regulation of production of bone sialoprotein and IL-8 by P.gingivalis LPS. The third-generation of hPDLFs were cultured with mineralization-inducing culture medium. After hPDLFs were treated with P.gingivalis LPS, bone sialoprotein and IL-8 mRNA expression were detected using Real time PCR. Then hPDLFs were transiently transfected with siTLR2 or siTLR4 (20nm) or inhibited by MAPK signaling pathways inhibitors, and then bone sialoprotein and IL-8 mRNA and protein expression were also detected using Real time PCR and western blotting. Treatments with 0.01 and 0.1mg/L of P.gingivalis LPS for 8h up-regulated bone sialoprotein mRNA expression, whereas 10 and 100mg/L of P.gingivalis LPS induced a significant decrease in the expression of bone sialoprotein mRNA. In contrast, IL8 mRNA levels were increased significantly by 10mg/L of P.gingivalis LPS. Interestingly, small interfering RNA (siRNA) knock down of the TLR2 and ERK1/2 inhibitor, PD98059, abolished the effects of P.gingivalis LPS on the bone sialoprotein mRNA level, whereas siRNA knock down of the TLR2 and p38 MAPK inhibitor, SB203580, blocked the effect of P.gingivalis LPS on IL-8 in hPDLFs. This study suggests that in hPDLFs, P.gingivalis LPS suppresses bone sialoprotein and enhances IL-8 gene and protein expression via TLR2 and ERK1/2 or the p38 MAPK signaling pathway, respectively.

Proteasomal degradation of O-GlcNAc transferase elevates hypoxia-induced vascular endothelial inflammatory response†.

Hypoxia induces vascular inflammation by a mechanism not fully understood. Emerging evidence implicates O-GlcNAc transferase (OGT) in inflammation. This study explored the role of OGT in hypoxia-induced vascular endothelial inflammatory response. Hypoxia was either induced (1% O2 chamber) or mimicked by exposure to hypoxia-mimetic agents in cultured endothelial cells. Hypoxia increased hypoxia-inducible factor (HIF-1α) and inflammatory response (gene and protein expression of interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, and E-selectin) but, surprisingly, reduced OGT protein (not mRNA) levels. Hypoxia-mimetic CoCl2 failed to reduce OGT when proteasome inhibitors were present, suggesting proteasome involvement. Indeed, CoCl2 enhanced 26S proteasome functionality evidenced by diminished reporter (Ub(G76V)-GFP) proteins in proteasome reporter cells, likely due to increased chymotrypsin-like activities. Mechanistically, β-TrCP1 mediated OGT degradation, since siRNA ablation of this E3 ubiquitin ligase stabilized OGT. Administration of the oxidative stress inhibitors reversed both proteasome activation and OGT degradation. Furthermore, up-regulation of OGT by stabilization, overexpression, or activation mitigated CoCl2-elicited inflammatory response. These observations were recapitulated in a mouse (C57BL/6J) model mimicking hypoxia, in which lung tissues presented higher levels of HIF-1α, proteasome activity, and inflammatory response, but lower levels of OGT (n = 5/group, hypoxia vs. normoxia, P < 0.05). However, administration of an activator of OGT (glucosamine: 1 mg/g/day, vehicle: saline, ip, 5 days) abolished the up-regulation of proteasome activity and inflammatory response (n = 5/group, the treated vs. untreated hypoxia groups, P < 0.05). 26S proteasome-mediated OGT reduction contributed to hypoxia-induced vascular endothelial inflammatory response. Modulation of OGT may represent a new approach to treat diseases characterized by hypoxic inflammation.

猪圆环病毒2型&amp;lt;italic&amp;gt;Cap&amp;lt;/italic&amp;gt;基因单核苷酸缺失突变体的构建与生物学特性

Porcine circovirus type 2 (PCV2) is a causative agent for PCV-associated disease. To analyze the role of the genomic nucleotide deletion in PCV2 replication, four mutant infectious clones of single nucleotide deletion within ORF2 Δ1376 PCV2, Δ1377 PCV2, Δ1378 PCV2, and Δ1379 PCV2, were constructed by the Dpn I site-directed mutagenesis. These mutants were rescued and verified by indirect immunoinfluscent assay (IFA) on the transfected PK15 cells. The data showed that only infectious clone Δ1376 PCV2 can successfully generate infectious virions, indicating that the nucleotide 1376G within ORF2 is not essential for PCV2 replication. The ability of virus multiplication revealed that virus titer of Δ1376 PCV2 was approximately 10 folds more than that of its parent virus. The proinflammatory factors analysis demonstrated that splenic transcripts and serum concentration of Interleukin-6 (IL-6) in Δ1376 PCV2-infected mice are higher than those in its parent PCV2-infected mice. Our data suggested that the replication ability of PCV2 with the nucleotide 1376G deleted within ORF2 was improved and the mRNA transcription and protein expression of IL-6 were strengthened.

Inhibition of Interleukin-6 Expression by Curcumin in Rat Vascular Smooth Muscle Explants &lt;I&gt;In Vitro&lt;/I&gt;

In atherosclerotic plaques, infectious agents from microbes may release Lipopolysaccharide (LPS) and heat shock proteins that can stimulate the production of mediators, i.e., pro-inflammatory cytokines, by vascular endothelial cells and Smooth Muscle Cells (SMCs). The elevated level of Interleukin-6 (IL-6) is strongly associated with the development of atherosclerosis. Here, we investigated whether curcumin isolated from the rhizome of Curcuma longa affected the expression of IL-6 at protein and gene levels in rat smooth muscle cells treated with LPS in vitro by conducting ELISA and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assays. LPS at 2 µg mL-1 activated the expression of IL-6 protein and mRNA in SMCs. Curcumin (1-15 µM) caused the decreased levels of IL-6 protein and mRNA in the dose-dependent manner in LPS-induced SMCs, indicating its potential antiatherosclerotic effect for cardiovascular risk management. In addition, curcumin also partially blocked the activation of LPS-induced phosphorylation of MAP kinases, i.e., ERK1/2, p38 and JNK, in SMCs, suggesting it may inhibit IL-6 expression via attenuating MAP kinase signaling pathways in LPS-induced SMCs. These data may in part explain the molecular action of antiatherosclerotic effects of curcumin.

Regulatory effect of miR-149 on interleukin-6 expression in silica-induced pulmonary fibrosis

To investigate the regulatory effect of miR-149 on interleukin-6 (IL-6) expression in silica-induced pulmonary fibrosis. A mouse model of pulmonary fibrosis was established using silica dust; the level of miR-149 in the lung tissues of mice with silica-induced pulmonary fibrosis was measured by quantitative real-time polymerase chain reaction (qRT-PCR), while the protein expression of IL-6 was measured by immunohistochemistry and Western blot. Type II alveolar epithelial cells (A549) and bronchial epithelial cells (HBE) were exposed to silica dust to establish a model; the level of miR-149 was measured by qRT-PCR, while the protein expression of IL-6 was measured by Western blot. A549 cells were transfected with miR-149 mimics and inhibitor in vitro, and the cellular expression of IL-6 was measured by Western blot. Serum samples from patients with coal workers' pneumoconiosis were examined by double-antibody sandwich ELISA to measure the protein expression of IL-6. At three time points after silica treatment, the miR-149 expression in lung tissues was significantly down-regulated while an evident increase in IL-6 expression was observed in lung tissues (P < 0.01). Silica-stimulated epithelial cell (A549 and HBE) had up-regulated IL-6 expression and down-regulated miR-149 expression (P < 0.01). Increased levels of miR-149 attenuated IL-6 expression, whereas adverse results were found when miR-149 was inhibited. Compared with that in control group, serum level of IL-6 was significantly increased in patients with stage II and III coal workers' pneumoconiosis (P < 0.01). Down-regulation of miR-149 and up-regulation of IL-6 might be involved in the progression of silica-induced pulmonary fibrosis; miR-149 could negatively regulate IL-6 expression.

Interleukin-8 promotes canine hemangiosarcoma growth by regulating the tumor microenvironment

Interleukin-8 (IL-8) gene expression is highly up-regulated in canine hemangiosarcoma (HSA); however, its role in the pathogenesis of this disease is unknown. We investigated the expression of IL-8 in canine HSA tissues and cell lines, as well and the effects of IL-8 on canine HSA in vitro, and in vivo using a mouse xenograft model for the latter. Constitutive expression of IL-8 mRNA, IL-8 protein, and IL-8 receptor were variable among different tumor samples and cell lines, but they showed stable steady states in each cell line. Upon the addition of IL-8, HSA cells showed transient intracellular calcium fluxes, suggesting that their IL-8 receptors are functional and that IL-8 binding activates relevant signaling pathways. Yet, neither addition of exogenous IL-8 nor blockade of endogenous IL-8 by neutralizing anti-IL-8 antibody (α-IL-8 Ab) affected HSA cell proliferation or survival in vitro. To assess potential effects of IL-8 in other tumor constituents, we stratified HSA cell lines and whole tumor samples into “IL-8 high” and “IL-8 low” groups. Genome-wide gene expression profiling showed that samples in the “IL-8 high” tumor group were enriched for genes associated with a “reactive microenvironment,” including activation of coagulation, inflammation, and fibrosis networks. Based on these findings, we hypothesized that the effects of IL-8 on these tumors were mostly indirect, regulating interactions with the microenvironment. This hypothesis was supported by in vivo xenograft experiments where survival and engraftment of tumor cells was inhibited by administration of neutralizing α-IL-8 Ab. Together, our results suggest that IL-8 contributes to establishing a permissive microenvironment during the early stages of tumorigenesis in HSA.

Transcriptional and posttranscriptional regulation and endocytosis were involved in zinc oxide nanoparticle-induced interleukin-8 overexpression in human bronchial epithelial cells

Inhaled zinc oxide nanoparticles (ZnO-NPs) can induce lung inflammation through released inflammatory mediators, such as interleukin 8 (IL-8), from airways. However, the mechanisms underlying ZnO-NP-induced IL-8 gene expression have not been fully characterized. The transcription inhibitor actinomycin D (Act D) and the BEAS-2B cells stably overexpressing wild-type or mutated IL-8 promoter at the NFκB or C/EBPβ binding site were used to determine the involvement of transcriptional mechanisms. The effect of ZnO-NPs on IL-8 mRNA stability was examined using mRNA decay assay. The phagocytosis inhibitor cytochalasin B (CB) was utilized to define the role of endocytosis in ZnO-NP-induced IL-8 expression. In addition, the solubility of ZnO-NPs in culture medium was assessed using atomic absorption spectroscopy. Exposure to ZnO-NPs significantly increased the expression of IL-8 mRNA and protein in a dose-dependent manner. Pretreatment with Act D blocked ZnO-NP-induced IL-8 expression. Both NFκB and C/EBPβ transcription factors were required for ZnO-NP-induced IL-8 transcription. mRNA decay assay showed that ZnO-NP stimulation delayed IL-8 mRNA degradation in BEAS-2B cells. Pretreatment of BEAS-2B cells with CB blocked ZnO-NP-induced IL-8 expression by 30%. Exposure to ZnO-NPs induced IL-8 gene expression through transcriptional activation and mRNA stabilization. Internalization of nanoparticles was partially involved in ZnO-NP-induced IL-8 expression.

Intramuscular fat and inflammation differ in older adults: the impact of frailty and inactivity.

Intramuscular adipose tissue (IMAT) is recognized as a negative predictor of both muscle and mobility function in older adults, however the mechanism by which IMAT may negatively influence muscle and mobility function is currently unknown. The release of pro-inflammatory cytokines from IMAT provides a potential reason for these negative associations. To explore this hypothesis we compared IMAT and muscular inflammation in age-and BMI-matched older non-obese frail and non-frail adults. We also sought to examine the relationship between IMAT and inflammation, and muscle and mobility function in this group of older adults. A case-control sampling was used for this study. Age-and BMI-matched non-obese frail and non-frail individuals (<65 years) were recruited. MRI was used to quantify thigh IMAT and lean tissue. Unilateral muscle biopsies were used to quantify muscular inflammation as represented by interleukin-6 (IL-6) and tumor-necrosis factor alpha (TNF-α). Muscle and mobility function was also measured using a maximal voluntary isometric contraction, six-minute walk, and self-selected gait speed. 26 older (80.7 +/- 5.4 years) individuals (8 frail and 18 non-frail) were enrolled. The frail-group had increased IMAT (p<0.01) and decreased lean tissue (p<0.01), and elevated IL-6 muscle mRNA (p=0.02) and IL-6 protein content (p=0.02) compared to the non-frail group. IMAT was significantly associated with IL-6 mRNA (r=0.43, p=.04) and protein expression within the muscle (r=0.41, p= 0.045). IL-6 mRNA was significantly associated with six-minute walk (r=-0.63, p<0.01), and gait speed (r=-0.60, p <0.01) and IL-6 protein was significantly associated with muscle force (r=-0.54, p=0.01), six-minute walk (r=-0.66, p<0.01), and gait speed (r=-0.76, p<0.01). No significant relationships were found for any variables with TNF-a. Non-obese, older, frail individuals have increased IMAT and muscular inflammation when compared to their non-frail, age- and BMI-matched peers. A significant relationship exists between IMAT and muscle IL-6 expression as well as between IL-6 and muscle and mobility function of these older adults. This IMAT-inflammatory pathway provides a potential link between IMATs and decreased muscle and mobility function.

Stress-related hormone norepinephrine induces interleukin-6 expression in GES-1 cells

In the current literature, there is evidence that psychological factors can affect the incidence and progression of some cancers. Interleukin 6 (IL-6) is known to be elevated in individuals experiencing chronic stress and is also involved in oncogenesis and cancer progression. However, the precise mechanism of IL-6 induction by the stress-related hormone norepinephrine (NE) is not clear, and, furthermore, there are no reports about the effect of NE on IL-6 expression in gastric epithelial cells. In this study, we examined the effect of NE on IL-6 expression in immortalized human gastric epithelial cells (GES-1 cells). Using real-time PCR and enzyme-linked immunoassay, we demonstrated that NE can induce IL-6 mRNA and protein expression in GES-1 cells. The induction is through the β-adrenergic receptor-cAMP-protein kinase A pathway and mainly at the transcriptional level. Progressive 5′-deletions and site-directed mutagenesis of the parental construct show that, although activating-protein-1 (AP-1), cAMP-responsive element binding protein (CREB), CCAAT-enhancer binding protein-β (C/EBP-β), and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) binding sites are all required in the basal transcription of IL-6, only AP-1 and CREB binding sites in the IL-6 promoter are required in NE-induced IL-6 expression. The results suggest that chronic stress may increase IL-6 secretion of human gastric epithelial cells, at least in part, by the stress-associated hormone norepinephrine, and provides basic data on stress and gastric cancer progression.

Interaction of bone marrow stromal cells with Kupffer cells induces immune tolerance

Objective To investigate the phenomenon of bone marrow stromal cells (BMSCs) regulating the function of Kupffer cells (KCs) under direct contact co-culture in vitro.Methods In co-culture experiments,pure rat BMSCs were directly added to the KCs plates with the ratio of 1∶ 1.KCs were separately cultured in a well as control group.After stimulation with lipopolysaccharide (LPS,1 mg/L) for 24 h,the gene and protein expression of interleukin (IL)-10 was detected by using real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting,respectively.The abundance of IL-10,prostaglandin E2 (PGE2) and IL-6 was detected by using enzyme linked immunosorbent assay (ELISA) kits.Flow cytometry was used to detect the expression of major histocompatibility complex (MHC) Ⅱ,CD40,CD80 and CD86.Results As compared with control group,the gene and protein expression of IL-10 in co-culture system was significantly increased (P ＜ 0.05).After co-culture with BMSCs,the levels of IL-10 [KCs:(1000.12±49.06) ng/L; KCs + BMSCs:(2307.44 ±308.97) ng/L] and PGE2[KCs:(244.48 ±28.15) ng/L; KCs + BMSCs:(424.48 ± 47.86) ng/L] were significantly increased (P ＜ 0.01),while the abundance of IL-6 [KCs:(283.18 ± 20.50) ng/L; KCs + BMSCs:(78.14 ± 10.58) ng/L] was significantly down-regulated (P ＜ 0.01).In co-culture system,the expression of MHC Ⅱ,CD40,CD80 and CD86 was lower than in control group.Conclusion BMSCs may reprogram the expression patterns of KCs,regulate the function of KCs,and then induce immune tolerance. Key words: Bone marrow stromal cells; Kupffer cells; Co-culture; Reprogram

Anti-inflammatory mechanism of polyunsaturated fatty acids in Helicobacter pylori-infected gastric epithelial cells.

Helicobacter pylori is an important risk factor for gastric inflammation, which is mediated by multiple signaling pathways. The aim of this study was to investigate the effects of polyunsaturated fatty acids (PUFAs), such as linoleic acid (LA), alpha-linolenic acid (ALA), and docosahexaenoic acid (DHA), on the expression of the proinflammatory chemokine interleukin-8 (IL-8) in H. pylori-infected gastric epithelial AGS cells. To investigate whether PUFAs modulate H. pylori-induced inflammatory signaling, we determined the activation of epidermal growth factor receptor (EGFR), protein kinase C-δ (PKCδ), mitogen-activated protein kinases (MAPKs), nuclear factor-kappa B (NF-κB), and activator protein-1 (AP-1) as well as IL-8 expression in H. pylori-infected gastric epithelial cells that had been treated with or without PUFAs. We found that PUFAs inhibited IL-8 mRNA and protein expression in H. pylori-infected cells. ω-3 fatty acids (ALA, and DHA) suppressed the activation of EGFR, PKCδ, MAPK, NF-κB, and AP-1 in these infected cells. LA did not prevent EGFR transactivation and exhibited a less potent inhibitory effect on IL-8 expression than did ALA and DHA. In conclusion, PUFAs may be beneficial for prevention of H. pylori-associated gastric inflammation by inhibiting proinflammatory IL-8 expression.

Mitogen-activated protein kinase-activated protein kinase 2 regulates tumor necrosis factor-induced interleukin-6 expression via human antigen R

Background Human antigen R (HuR) is a ubiquitously expressed member of the ELAV family, and has relatively high cytoplasmic abundance in lung tissue regenerating after injury. In this study, we investigated whether mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) and HuR participate in the tumor necrosis factor (TNF)-induced expression of interleukin-6 (IL-6). Methods Human pulmonary microvascular endothelial cells were treated with TNF following short interfering RNA-mediated knockdown of MK2 or HuR. Cell supernatants were collected to detect the mRNA and protein expression of IL-6 at different time points. The expression and half-life of IL-6 mRNA were then determined in cells that had been treated with actinomycin D. Finally, after knockdown of MK2, the cytoplasmic expression of HuR protein was analyzed using Western blotting. Results MK2 or HuR knockdown decreased both the mRNA and protein expression of IL-6 in TNF-stimulated cells. In MK2 knockdown cells, the half-life of IL-6 mRNA was reduced to 36 minutes, compared with 67 minutes in the control group. In HuR knockdown cells, the half-life of IL-6 mRNA decreased from 62 minutes to 24 minutes. Further analysis revealed that knockdown of MK2 resulted in reduced HuR protein expression in the cytoplasm. Conclusions MK2 regulates the TNF-induced expression of IL-6 by influencing the cytoplasmic levels of HuR.

Serotonin acts as a novel regulator of interleukin-6 secretion in osteocytes through the activation of the 5-HT2B receptor and the ERK1/2 signalling pathway

Interleukin-6 (IL-6) is a potent stimulator of osteoclastic bone resorption. Osteocyte secretion of IL-6 plays an important role in bone metabolism. Serotonin (5-HT) has recently been reported to regulate bone metabolism. The aim of this study was to evaluate the effect of serotonin on osteocyte expression of IL-6. The requirement for the 5-HT receptor(s) and the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) in serotonin-induced IL-6 synthesis were examined. In this study, real-time PCR and ELISA were used to analyse IL-6 gene and protein expression in serotonin-stimulated MLO-Y4 cells. ERK1/2 pathway activation was determined by Western blot. We found that serotonin significantly activated the ERK1/2 pathway and induced IL-6 mRNA expression and protein synthesis in cultured MLO-Y4 cells. However, these effects were abolished by pre-treatment of MLO-Y4 cells with a 5-HT2B receptor antagonist, RS127445 or the ERK1/2 inhibitor, PD98059. Our results indicate that serotonin stimulates osteocyte secretion of IL-6 and that this effect is associated with activation of 5-HT2B receptor and the ERK1/2 pathway. These findings provide support for a role of serotonin in bone metabolism by indicating serotonin regulates bone remodelling by mediating an inflammatory cytokine.

Impairment of the Interleukin System in Equine Endometrium During the Course of Endometrosis1

The aim of the study was to characterize endometrial mRNA transcription, immunolocalization, and protein expression of interleukin (IL) 1alpha, IL1beta, IL6, and IL1RI, IL1RII, and IL6Ralpha/beta in the course of endometrosis during the estrous cycle. Additionally, the influence of IL1alpha, IL1beta, and IL6 on prostaglandin (PG) secretion and PG synthase mRNA transcription in endometrial tissue during endometrosis was investigated. The endometrial samples were obtained at the early (n = 12), mid- (n = 12), and late (n = 12) luteal phases and at the follicular (n = 12) phase of the estrous cycle. Within each of these phases, there were four samples within each category I, II, and III of endometrium, according to the Kenney classification. In experiment 1, transcription of IL1alpha, IL1beta, IL6, and their receptor's (IL1RI, IL1RII, and IL6Ralpha/beta) mRNAs and their immunolocalization and protein expression were determined using real-time PCR and immunohistochemistry, respectively. In Experiment 2, endometrial samples (n = 5 samples within categories I, II, and III) were obtained for tissue culture in the midluteal phase of the estrous cycle. The endometrial tissues were stimulated with IL1alpha (10 ng/ml), IL1beta (10 ng/ml), IL6 (10 ng/ml), and oxytocin (positive control; 10⁻⁷ M) for 24 h. The PG concentration was determined using ELISA. In addition, transcription of PTGS-2, PGES, and PGFS mRNAs was determined using real-time PCR. ILs were found to regulate PG secretion via modulation of PG synthases in equine endometrium. The alterations in IL and the expression of their receptors, and in endometrial secretory functions, were observed during the course of endometrosis, and suggest serious changes in the endometrial microenvironment. The described disturbances may be closely related to impaired endometrial processes responsible for the subfertility or the infertility in endometrosis.

Effects of resveratrol on obesity-related inflammation markers in adipose tissue of genetically obese rats

ObjectiveThe aim of this study was to examine whether resveratrol might represent a promising therapeutic tool with which to combat adipose tissue chronic inflammation in a model of genetic obesity and to link its anti-inflammatory activity with its effect on body fat reduction. MethodsTwenty 6-wk-old male Zucker (fa/fa) rats were randomly distributed into two experimental groups. Resveratrol (RSV) was given orally (15 mg/kg body weight/d in RSV group) by means of an orogastric catheter for 6 wk. Enzyme activities were measured spectrophotometrically or fluorimetrically. Gene and protein expressions were analyzed by reverse transcriptase polymerase chain reaction and Western blot respectively. Cytokine concentrations and the activity of nuclear factor κ-light-chain-enhancer of activated β cells (NF-κB) were measured by using commercial kits. ResultsRSV reduced the weight of internal adipose tissues. In epididymal depot glucose-6P-dehydrogenase, acetyl-CoA carboxylase activities, as well as lipoprotein lipase expression and activity were reduced by RSV. The expression of hormone-sensitive lipase was increased, and that of the cluster of differentiation 36 was reduced. Serum concentrations of tumor necrosis factor-α, monocyte chemoattractant protein 1, and C-reactive protein were lower in the RSV-treated group than in the control group. Protein expression of interleukin-6 and the activity of NF-κB, were decreased by RSV. ConclusionThe present results provide evidence that fatty acid uptake and lipolysis are metabolic pathways involved in the response of adipose tissue to RSV. This polyphenol modulates plasma cytokine levels partially by reducing macrophage infiltration in adipose tissue and inhibiting NF-κB activity.