Nuclear Translocation Of NF-kappaB Research Articles

Mechanisms of Adiponectin in Regulation of Proinflammatory Cytokine Production and Migration in Macrophages.

IntroductionEvidence indicate that adiponectin may exert pro-inflammatory effects on inflammatory cells. We have found that adiponectin knockout decreased inflammatory reaction and tubular damage in the ischemia-reperfusion kidney in APN-knockout mice. Globular adiponectin and full-length adiponectin (g-APN and f-APN) were used in this study to investigate the effects of adiponectin on proinflammatory cytokines production and migration in Raw 264.7 cells.MethodsProinflammatory cytokines production was detected by real-time RT-PCR. NF-kappaB activation was interrupted through Ad-DN-IκBα or SN-50 to confirm how g-APN induces proinflammatory cytokines production. The siRNA against AdipoR1 and AdipoR2 was investigated to uncover the signaling pathway that may involve in NF-kappaB activation and migration in Raw 264.7 cells.Resultsg-APN, not f-APN, was found triggering the production of inflammatory cytokine MIP-2, IL-6, TNFα, and MCP-1 in Raw 264.7 cells. NF-kappaB Inhibition by Ad-DN-IκBα expression or cell-permeable NF-κB inhibitor SN-50 could decrease NF-kappaB nuclear translocation and subsequently decrease inflammatory cytokine expression triggered by globular ANP. However, AdipoR1 and AdipoR2 were not found involved in NF-kappaB activation in this study. Full-length APN, not g-APN, was involved in the promotion of macrophage migration. The migration induced by full-length APN could be inhibited by knockdown of AdipoR1 expression with siRNA. The migration effect could also be inhibited by PI3Kγ inhibitor, AS-605240.DiscussionThese results suggested that full-length adiponectin increases macrophage migration through Adiponectin-AdipoR1-PI3Kgamma signaling pathway. However, NF-κB activation induced by g-APN in this study was independent of AdipoR1 or AdipoR2. The exact signaling pathway of NF-κB activation by adiponectin should be further studied to find a new anti-inflammatory target.

Resolvin D5, a Lipid Mediator, Inhibits Production of Interleukin-6 and CCL5 Via the ERK-NF-κB Signaling Pathway in Lipopolysaccharide-Stimulated THP-1 Cells.

One of the omega-3 essential fatty acids, docosahexaenoic acid (DHA), is a significant constituent of the cell membrane and the precursor of several potent lipid mediators. These mediators are considered to be important in preventing or treating several diseases. Resolvin D5, an oxidized lipid mediator derived from DHA, has been known to exert anti-inflammatory effects. However, the detailed mechanism underlying these effects has not yet been elucidated in human monocytic THP-1 cells. In the present study, we investigated the effects of resolvin D5 on inflammation-related signaling pathways, including the extracellular signal-regulated kinase (ERK)-nuclear factor (NF)-κB signaling pathway. Resolvin D5 downregulated the production of interleukin (IL)-6 and chemokine (C-C motif) ligand 5 (CCL5). Additionally, these inhibitory effects were found to be modulated by mitogen-activated protein kinase (MAPK) and NF-κB in lipopolysaccharide (LPS)-treated THP-1 cells. Resolvin D5 inhibited the LPS-stimulated phosphorylation of ERK and translocation of p65 and p50 into the nucleus, resulting in the inhibition of IL-6 and CCL5 production. These results revealed that resolvin D5 exerts anti-inflammatory effects in LPS-treated THP-1 cells by regulating the phosphorylation of ERK and nuclear translocation of NF-kappaB.

The Therapeutic Effects of Treadmill Exercise on Osteoarthritis in Rats by Inhibiting the HDAC3/NF-KappaB Pathway in vivo and in vitro.

Osteoarthritis (OA) is a disease characterized by non-bacterial inflammation. Histone deacetylase 3 (HDAC3) is a crucial positive regulator in the inflammation that leads to the development of non-OA inflammatory disease. However, the precise involvement of HDAC3 in OA is still unknown, and the underlying mechanism of exercise therapy in OA requires more research. We investigated the involvement of HDAC3 in exercise therapy-treated OA. Expression levels of HDAC3, a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), matrix metalloproteinase-13 (MMP-13), HDAC3 and nuclear factor-kappaB (NF-kappaB) were measured by western blotting, reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. Cartilage damage and OA evaluation were measured by hematoxylin and eosin staining and Toluidine blue O staining according to the Mankin score and OARSI score, respectively. We found that moderate-intensity treadmill exercise could relieve OA. Meanwhile, the expression of HDAC3, MMP-13, ADAMTS-5 and NF-kappaB decreased, and collagen II increased in the OA + moderate-intensity treadmill exercise group (OAM) compared with the OA group (OAG) or OA + high- or low-intensity treadmill exercise groups (OAH or OAL). Furthermore, we found the selective HDAC3 inhibitor RGFP966 could also alleviate inflammation in OA rat model through inhibition of nuclear translocation of NF-kappaB. To further explore the relationship between HDAC3 and NF-kappaB, we investigated the change of NF-kappaB relocation in IL-1β-treated chondrocytes under the stimulation of RGFP966. We found that RGFP966 could inhibit the expression of inflammatory markers of OA via regulation of HDAC3/NF-kappaB pathway. These investigations revealed that RGFP966 is therefore a promising new drug for treating OA.

Long non-coding RNA NKILA inhibits migration and invasion of non-small cell lung cancer via NF-\u03baB/Snail pathway

BackgroundNumerous studies have shown that long non-coding RNAs (lncRNAs) play key roles during multiple cancer processes, such as cell proliferation, apoptosis, migration and invasion. The previous studies found that NKILA interacted with and suppressed the nuclear translocation of NF-KappaB, which influenced metastasis and prognosis in breast cancer. However the clinical significance and biological role of NKILA in non-small cell lung cancer (NSCLC) remains unknown.MethodsWe examined expression levels of NKILA in 106 pairs of NSCLC tissues and cell lines. The expression level of NKILA after TGF-β1 stimulation also was examined by qRT-PCR and validated by Chromatin immunoprecipitation (ChIP). Gain-of-function and loss-of-function assays were performed to examine the effect of NKILA on proliferation, migration and invasion of NSCLC cells. RNA immunoprecipitation (RIP), western blot and rescue experiments were carried out to reveal the interrelation between NKILA, NF-κB and EMT signal pathway.ResultsThe expression of NKILA was down-regulated in NSCLC cancer tissues compared with matched adjacent noncancerous tissues, and lower NKILA expression in tumor tissues were significantly correlated with lymph node metastasis and advanced TNM stage. We found that the expression of NKILA was mainly regulated by classical TGF-β signal pathway in NSCLC cells rather than NF-κB pathway reported in breast cancer. Gain and loss of function assays found that NKILA inhibited migration, invasion and viability of NSCLC cells. Mechanistic study showed that NKILA attenuated Snail expression via inhibiting the phosphorylation of IκBα and NF-κB activation, subsequently suppressed the expression of markers of epithelial-mesenchymal transition process.ConclusionsThe present study found that the expression of NKILA was downregulated in tumor tissues of NSCLC, which improved the metastasis of NSCLC patients. In vitro studies further clarified that the expression of NKILA was regulated through classical TGF-β signal pathway, which subsequently inhibited migration and invasion of NSCLC cells through interfering NF-κB/Snail signal pathway in NSCLC cells.

A liposomal formulation of the synthetic curcumin analog EF24 (Lipo-EF24) inhibits pancreatic cancer progression: towards future combination therapies.

BackgroundPancreatic cancer is one of the most lethal of human malignancies known to date and shows relative insensitivity towards most of the clinically available therapy regimens. 3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24), a novel synthetic curcumin analog, has shown promising in vitro therapeutic efficacy in various human cancer cells, but insufficient water solubility and systemic bioavailability limit its clinical application. Here, we describe nano-encapsulation of EF24 into pegylated liposomes (Lipo-EF24) and evaluation of these particles in preclinical in vitro and in vivo model systems of pancreatic cancer.ResultsTransmission electron microscopy and size distribution studies by dynamic light scattering confirmed intact spherical morphology of the formed liposomes with an average diameter of less than 150 nm. In vitro, treatment with Lipo-EF24 induced growth inhibition and apoptosis in MIAPaCa and Pa03C pancreatic cancer cells as assessed by using cell viability and proliferation assays, replating and soft agar clonogenicity assays as well as western blot analyses. Lipo-EF24 potently suppressed NF-kappaB nuclear translocation by inhibiting phosphorylation and subsequent degradation of its inhibitor I-kappa-B-alpha. In vivo, synergistic tumor growth inhibition was observed in MIAPaCa xenografts when Lipo-EF24 was given in combination with the standard-of-care cytotoxic agent gemcitabine. In line with in vitro observations, western blot analysis revealed decreased phosphorylation of I-kappa-B-alpha in excised Lipo-EF24-treated xenograft tumor tissues.ConclusionDue to its promising therapeutic efficacy and favorable toxicity profile Lipo-EF24 might be a promising starting point for development of future combinatorial therapeutic regimens against pancreatic cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0209-6) contains supplementary material, which is available to authorized users.

In vitro effect of glucocorticoids on nasal polyps

AbstractGlucocorticoids are considered the main treatment option for nasal polyps, but their effect is only recently being understood. AimTo evaluate whether fluticasone propionate (FP) inhibits the inflammatory process induced by TNF-alpha in vitro, and to assess if NF-kappaB is associated to this inhibition. Study DesignExperimental in vitro study. Materials and MethodsNasal polyp fibroblasts were cultured during 24 hours. Three different concentrations of FP (1, 10 and 100 nM, added to TNF-alpha) were compared to negative (without additive) and positive (TNF-alpha) controls. Gene expression (RTQ-PCR) and protein concentration (ELISA) of VCAM-1, ICAM-1, eotaxin and RANTES were measured, as well as the nuclear translocation of NF-kappaB. ResultsTNF-alpha significantly increased protein concentration and RNA expression of all the studied molecules, as well as the nuclear translocation of NF-kappaB, when compared to the negative control. FP decreased these parameters in a dose-dependent manner, statistically different from positive control up to 100nM. ConclusionsFP extensively inhibited inflammatory recruiters, at both protein and RNA levels, confirming the ability of glucocorticoids to modulate the inflammatory process in nasal polyps. This inhibition was associated to decreased NF-kappaB nuclear translocation, demonstrating that this is an important mechanism of glucocorticoids action for nasal polyps.

Diphlorethohydroxycarmalol inhibits interleukin-6 production by regulating NF-κB, STAT5 and SOCS1 in lipopolysaccharide-stimulated RAW264.7 cells.

Diphlorethohydroxycarmalol (DPHC) is a phlorotannin compound isolated from Ishige okamuarae, a brown alga. This study was conducted to investigate the anti-inflammatory effect and action mechanism of DPHC in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We found that DPHC strongly reduces the production of interleukin 6 (IL-6), but not that of tumor necrosis factor-alpha (TNF-α) induced by LPS. DPHC (12.5 and 100 μM) suppressed the phosphorylation and the nuclear translocation of NF-kappaB (NF-κB), a central signaling molecule in the inflammation process induced by LPS. The suppressor of cytokine signaling 1 (SOCS1) is a negative feedback regulator of Janus kinase (Jak)-signal transducer and activator of transcription (STAT) signaling. In this study, DPHC inhibited STAT5 expression and upregulated that of SOCS1 at a concentration of 100 μM. Furthermore, N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (a specific NF-κB inhibitor) and JI (a specific Jak2 inhibitor) reduced the production of IL-6, but not that of tumor necrosis factor-alpha (TNF-α) in LPS-stimulated RAW 264.7 macrophages. These findings demonstrate that DPHC inhibits IL-6 production via the downregulation of NF-κB and Jak2-STAT5 pathway and upregulation of SOCS1.

GABA receptor A α3 subunit can be involved in the pathophysiology of preeclampsia

GABA receptor A α3 subunit can be involved in the pathophysiology of preeclampsia

Decidualized Human Endometrial Stromal Cells Are Sensors of Hormone Withdrawal in the Menstrual Inflammatory Cascade1

Menstruation is a complex process dependent on premenstrual release of inflammatory mediators and proteolytic enzymes from endometrial cells. Endometrial leukocytes are traditionally considered to be the major source of the inflammatory factors. However, evidence is emerging to suggest a role for decidualized endometrial stromal cells in the premenstrual inflammatory cascade. We sought to determine if withdrawal of hormone support (estrogen and progesterone) from decidualized endometrial stromal cells, in a model mimicking the precise timing leading to menstruation, activated inflammatory signaling pathways and downstream release of inflammatory mediators. Human endometrial stromal cells decidualized gradually over 12 days of estradiol and progestin treatment as evidenced by an increase in prolactin secretion. Withdrawal of hormone support from decidualized stromal cells resulted in a decrease in cytoplasmic IkappaB and a progressive increase in nuclear accumulation of NF-kappaB, as demonstrated by Western immunoblot and immunocytochemical analyses. Concomitant with nuclear translocation of NF-kappaB, hormone withdrawal led to production of a host of inflammatory mediators by the decidualized stromal cells, including IFN-alpha, IL-6, CCL11, GM-CSF, CCL2, IL1-RA, CXCL10, CXCL8, IL-12, IL-15, VEGF, and CCL5. Elevation of inflammatory mediators was not observed, however, upon hormone withdrawal in cells treated with the NF-kappaB inhibitor BAY 11-7085. Decidualized stromal cells are likely highly sensitive sensors of changing hormone levels. This provides a mechanism by which decidualized stromal cells may recruit inflammatory leukocytes into the premenstrual endometrium and contribute to the intense inflammation underlying this unique physiological process.

Lipopolysaccharide-induced cross-tolerance against renal ischemia–reperfusion injury is mediated by hypoxia-inducible factor-2α-regulated nitric oxide production

Although the protective effect of lipopolysaccharide (LPS) pretreatment on renal ischemia/reperfusion injury is known, a link to hypoxia-inducible factors (HIFs) has not been established. Here we show that LPS treatment led to HIF-2α accumulation in mouse kidneys and endothelial cells, a result of nuclear factor-κB activation. Inactivation of HIF-2α, rather than HIF-1α, completely negated LPS-mediated protection against renal ischemia/reperfusion injury. LPS-stimulated renoprotection was related to inducible/endothelial nitric oxide synthase (iNOS/eNOS) expression, increased production of nitric oxide, and enhanced postischemic microcirculatory recovery. All these effects were lost in HIF-2α knockout mice. Preischemic administration of a nitric oxide donor, rather than erythropoietin, restored the lost preconditioning effect of LPS in HIF-2α knockout mice. In vitro and in vivo studies demonstrated that HIF-2α in endothelial cells, rather than myeloid cells or hepatocytes, was responsible for the LPS-mediated effects. Thus, our results demonstrated that LPS preconditioning protected against renal ischemia/reperfusion injury by HIF-2α activation in endothelial cells that subsequently improved renal microvascular perfusion and reduced ischemic tubular damage.

Suppression of IL-12 production by TTP through blocking NF-kappaB nuclear translocation (P3008)

Abstract Tristetraprolin (TTP) plays a significant role in regulating the expression of AREs containing mRNAs. TTP-/- Mice develop a systemic autoimmune inflammatory syndrome characterized by cachexia, conjunctivitis and dermatitis. IL-12 plays a crucial role in immune defense against infectious and malignant diseases. In the present study, we found increased production of IL-12 during endotoxic shock and enhanced Th1 cells in TTP knockout mice. The levels of IL-12 p70 and p40 protein as well as p40 and p35 mRNA were also increased in activated macrophages deficient of TTP. In line with these findings, overexpression of TTP suppressed IL-12 p35 and p40 expression at the mRNA and promoter level, while surprisingly had no effects on their mRNA stability. Our data showed that the inhibitory effects of TTP on p35 gene transcription were completely rescued by overexpression of NF-kappaB p65 and c-Rel but not the p50 in activated macrophages. Our data further indicated that TTP acquired its inhibition on IL-12 expression through blocking nuclear translocation of NF-kappaB p65 and c-Rel while enhancing p50 upon stimulation. In summary, our study reveals a novel pathway through which TTP suppresses IL-12 production in macrophages, resulting in suppression of Th1 cell differentiation. This study may provide us with therapeutic targets for treatment of inflammatory and autoimmune disorders

Angiotensin-(1–7) regulates Angiotensin II-induced VCAM-1 expression on vascular endothelial cells

Angiotensin II (Ang II) and Angiotensin-(1–7) (Ang-(1–7)) are key effector peptides in the renin–angiotensin system. Increased circulatory Ang II level is associated with the development of hypertension and atherosclerosis, whereas Ang-(1–7) is a counter-regulatory mediator of Ang II which appears to be protective against cardiovascular disease. However, whether Ang-(1–7) regulates the action of Ang II on vascular endothelial cells (EC) remains unclear. We investigated the effects of Ang II and Ang-(1–7) in the context of atherogenesis, specifically endothelial cell VCAM-1 expression that is implicated in early plaque formation. The results show that Ang II increased VCAM-1 mRNA expression and protein displayed on EC surface, while Ang-(1–7) alone exerted no effects. However, Ang-(1–7) significantly suppressed Ang II-induced VCAM-1 expression. Ang-(1–7) also inhibited the Ang II-induced VCAM-1 promoter activity driven by transcription factor NF-KappaB. Furthermore, immunofluorescence assay and ELISA showed that Ang II facilitated the nuclear translocation of NF-kappaB in ECs, and this was attenuated by the presence of Ang-(1–7). The inhibitory effects of Ang-(1–7) on Ang II-induced VCAM-1 promoter activity and NF-kappaB nuclear translocation were all reversed by the competitive antagonist of Ang-(1–7) at the Mas receptor. Our results suggest that Ang-(1–7) mediates its affects on ECs through the Mas receptor, and negatively regulates Ang II-induced VCAM-1 expression by attenuating nuclear translocation of NF-kappaB.

Bmi-1 promotes the aggressiveness of glioma via activating the NF-kappaB/MMP-9 signaling pathway

BackgroundThe prognosis of human glioma is poor, and the highly invasive nature of the disease represents a major impediment to current therapeutic modalities. The oncoprotein B-cell-specific Moloney murine leukemia virus integration site 1 protein (Bmi-1) has been linked to the development and progression of glioma; however, the biological role of Bmi-1 in the invasion of glioma remains unclear.MethodsA172 and LN229 glioma cells were engineered to overexpress Bmi-1 via stable transfection or to be silenced for Bmi-1 expression using RNA interfering method. Migration and invasiveness of the engineered cells were assessed using wound healing assay, Transwell migration assay, Transwell matrix penetration assay and 3-D spheroid invasion assay. MMP-9 expression and activity were measured using real-time PCR, ELISA and the gelatin zymography methods. Expression of NF-kappaB target genes was quantified using real-time PCR. NF-kappaB transcriptional activity was assessed using an NF-kappaB luciferase reporter system. Expression of Bmi-1 and MMP-9 in clinical specimens was analyzed using immunohistochemical assay.ResultsEctopic overexpression of Bmi-1 dramatically increased, whereas knockdown of endogenous Bmi-1 reduced, the invasiveness and migration of glioma cells. NF-kappaB transcriptional activity and MMP-9 expression and activity were significantly increased in Bmi-1-overexpressing but reduced in Bmi-1-silenced cells. The reporter luciferase activity driven by MMP-9 promoter in Bmi-1-overexpressing cells was dependent on the presence of a functional NF-kappaB binding site, and blockade of NF-kappaB signaling inhibited the upregulation of MMP-9 in Bmi-1 overexpressing cells. Furthermore, expression of Bmi-1 correlated with NF-kappaB nuclear translocation as well as MMP-9 expression in clinical glioma samples.ConclusionsBmi-1 may play an important role in the development of aggressive phenotype of glioma via activating the NF-kappaB/MMP-9 pathway and therefore might represent a novel therapeutic target for glioma.

Haemozoin Induces Early Cytokine-Mediated Lysozyme Release from Human Monocytes through p38 MAPK- and NF-kappaB- Dependent Mechanisms

Malarial pigment (natural haemozoin, HZ) is a ferriprotoporphyrin IX crystal produced by Plasmodium parasites after haemoglobin catabolism. HZ-fed human monocytes are functionally compromised, releasing increased amounts of pro-inflammatory molecules, including cytokines, chemokines and cytokine-related proteolytic enzyme Matrix Metalloproteinase-9 (MMP-9), whose role in complicated malaria has been recently suggested. In a previous work HZ was shown to induce through TNFalpha production the release of monocytic lysozyme, an enzyme stored in gelatinase granules with MMP-9. Here, the underlying mechanisms were investigated. Results showed that HZ lipid moiety promoted early but not late lysozyme release. HZ-dependent lysozyme induction was abrogated by anti-TNFalpha/IL-1beta/MIP-1alpha blocking antibodies and mimicked by recombinant cytokines. Moreover, HZ early activated either p38 MAPK or NF-kappaB pathways by inducing: p38 MAPK phosphorylation; cytosolic I-kappaBalpha phosphorylation and degradation; NF-kappaB nuclear translocation and DNA-binding. Inhibition of both routes through selected molecules (SB203580, quercetin, artemisinin, parthenolide) prevented HZ-dependent lysozyme release. These data suggest that HZ-triggered overproduction of TNFalpha, IL-1beta and MIP-1alpha mediates induction of lysozyme release from human monocytes through activation of p38 MAPK and NF-kappaB pathways, providing new evidence on mechanisms underlying the HZ-enhanced monocyte degranulation in falciparum malaria and the potential role for lysozyme as a new affordable marker in severe malaria.

NF-kappaB p65 modulates the telomerase reverse transcriptase in the HepG 2 hepatoma cell line

Nuclear factor-kappa B (NF-kappaB) regulates the expression of various genes, several genes involved in inflammation and tumorigenesis, including those of the liver. A role for NF-kappaB has been implicated in the pathogenesis of hepatocellular carcinoma. This transcription factor can regulate hTERT gene transcription. Expression of hTERT was found to be at high levels in hepatocellular carcinoma. However, positive effects of NF-kappaB on hTERT protein synthesis in HepG 2 cells are unknown. In this study, we show that LPS (specific binding to TLR4 to activate NF-kappaB) was positive for NF-kappaB p65 mRNA expression and activation, and also up-regulated hTERT mRNA and protein expressions at 36 h in a dose-dependent manner. In contrast, MG-132 (blocking the activity of 26S proteasome and thereby preventing nuclear translocation of NF-kappaB) significantly inhibited activation of NF-kappaB and mRNA expression. And also reduced the expression of hTERT at both mRNA and protein levels at 36 h in a dose-dependent manner. Furthermore, dexamethasone inhibited LPS-induced activation of NF-kappaB and expression of the hTERT in HepG 2 cells. These findings suggest that NF-kappaB may modulate hTERT mRNA level, importantly, in protein level in HepG 2 cells and dexamethasone inhibits LPS-induced hTERT via blocking NF-kappaB.

Protective effect of hydroxysafflor yellow A on endothelial cell injury induced by lipopolysaccharide

To observe the protective effect of hydroxyl safflor yellow A (HSYA) on endothelial cell (EC). It has been observed by RT-PCR that HSYA can inhibit the elevation of TNF-alpha, IL-6, ICAM-1 and VCAM-1 mRNA level induced by LPS. The result of immunofluorescence test suggested that HSYA can alleviate p65 subgroup of NF-kappaB nuclear translocation. The experiment on EA-HY926 cell line proved that HSYA can protect EC against inflammation injury.

In vitro and in vivo role of SIgA in the interaction of commensals bacteria with the intestinal epithelium (59.9)

Abstract The precise mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple consequences that remain poorly understood at the molecular level. Deciphering such events can provide valuable information as to the mode of action of commensal microorganisms in the gastrointestinal environment. Using human intestinal epithelial Caco-2 cell grown as polarized monolayers, we found that association of a Lactobacillus or a Bifidobacterium with nonspecific secretory IgA (SIgA) enhanced probiotic adhesion, increased the level of nuclear translocation of NF-kappaB and production of epithelial polymeric Ig receptor as compared to bacteria alone, whereas transepithelial electrical resistance remained stable. Following administration in mouse ligated ileal loops containing a Peyer’s patch, it was found that SIgA bound to Lactobacillus or Bifidobacterium is involved in translocation to underlying dendritic cells residing in the subepithelial dome (SED) region. This phenomenon was mediated by passage across M cells, and required exogenously delivered or endogenous SIgA, as free bacteria was not observed in the SED region. These findings add to the multiple functions of SIgA and underscore a novel role of the antibody in its complex cross-talk with the intestinal epithelium.

1,2,3,6‐tetra‐O‐galloyl‐β‐D‐allopyranose gallotannin isolated, from Euphorbia jolkini, attenuates LPS‐induced nitric oxide production in macrophages

Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes, including vasodilatation and macrophage-mediated immunity. Macrophages express inducible NO synthase (iNOS) and produce NO after lipopolysaccharide (LPS) stimulation. Gallotannins are water-soluble polyphenols with wide-ranging biological activities. Various chemical structures of gallotannins occurring in medicinal and food plants that are used worldwide showed several remarkable biological and pharmacological activities. In the present study, we examined the inhibitory effects of gallotannin 1,2,3,6-tetra-O-galloyl-beta-D-allopyranose (GT24) isolated from Euphorbia jolkini on the LPS-induced NO production and underlying mechanisms of action. GT24 dose-dependently decreased LPS-induced NO production and iNOS expression in J774A.1 macrophages. In addition, GT24 inhibited LPS-induced activation of nuclear factor (NF)-kappaB as indicated by inhibition of degradation of I-kappaBalpha, nuclear translocation of NF-kappaB, and NF-kappaB dependent gene reporter assay. Our results suggest that GT24 possesses an inhibitory effect on the LPS-induced inflammatory reaction.

Regulation of Hepatocyte Lipid Metabolism and Inflammatory Response by 25‐Hydroxycholesterol and 25‐Hydroxycholesterol‐3‐sulfate

Dysregulation of lipid metabolism is frequently associated with inflammatory conditions. The mechanism of this association is still not clearly defined. Recently, we identified a nuclear oxysterol, 25-hydroxycholesterol-3-sulfate (25HC3S), as an important regulatory molecule involved in lipid metabolism in hepatocytes. The present study shows that 25HC3S and its precursor, 25-hydroxycholesterol (25HC), diametrically regulate lipid metabolism and inflammatory response via LXR/SREBP-1 and IkappaBalpha/NFkappaB signaling in hepatocytes. Addition of 25HC3S to primary rat hepatocytes decreased nuclear LXR and SREBP-1 protein levels, down-regulated their target genes, acetyl CoA carboxylase 1 (ACC1), fatty acid synthase (FAS), and SREBP-2 target gene HMG reductase, key enzymes involved in fatty acid and cholesterol biosynthesis. 25HC3S reduced TNFalpha-induced inflammatory response by increasing cytoplasmic IkappaBalpha levels, decreasing NFkappaB nuclear translocation, and consequently repressing expression of NFkappaB-dependent genes, IL-1beta, TNFalpha, and TRAF1. NFkappaB-dependent promoter reporter gene assay showed that 25HC3S suppressed luciferase activity in the hepatocytes. In contrast, 25HC elicited opposite effects by increasing nuclear LXR and SREBP-1 protein levels, and by increasing ACC1 and FAS mRNA levels. 25HC also decreased cytoplasmic IkappaBalpha levels and further increased TNFalpha-induced NFkappaB activation. The current findings suggest that 25HC and 25HC3S serve as potent regulators in cross-talk of lipid metabolism and inflammatory response in the hepatocytes.

Activation of toll‐like receptors 2, 3 or 5 induces matrix metalloproteinase‐1 and ‐9 expression with the involvement of MAPKs and NF‐κB in human epidermal keratinocytes

Toll-like receptors (TLRs) on epidermal keratinocytes are the first line of defense against microbe invasion, and matrix metalloproteases (MMPs) regulate inflammation, cell migration and wound healing. In this study, we demonstrate that the mRNA and protein expressions of MMP-1 and MMP-9 in human epidermal keratinocytes are induced by ligands for TLR2, TLR3 and TLR5 [Pam3CSK4, Poly(I:C) and flagellin, respectively] in a dose-dependent manner. We also found that the ligands for TLR2, TLR3 and TLR5 activate the MAP kinases, JNK and p38 MAPK, but not ERK1/2. Furthermore, treatment with the ligands for TLR2, TLR3 and TLR5 also induced the degradation of IkappaB-alpha and activated the nuclear translocation of NF-kappaB. MMP-1 induction by the ligands for TLR2, TLR3 and TLR5 was inhibited by pretreatment with BAY11-7082 (NF-kappaB inhibitor) or SP600125 (JNK inhibitor), whereas MMP-9 expression was inhibited by pretreatment with BAY11-7082, SP600125 or SB203580. These findings demonstrate that the activation of TLR2, TLR3 or TLR5 induces the expression of MMP-1 and MMP-9 in human epidermal keratinocytes. In addition, NF-kappaB or JNK mediated the MMP-1 expression induced by TLR2, TLR3 and TLR5, whereas NF-kappaB, JNK or p38 MAPK mediated the MMP-9 expression induced by TLR2, TLR3 and TLR5.