A Methanol Extract of Adansonia digitata L. Leaves Inhibits Pro-Inflammatory iNOS Possibly via the Inhibition of NF-kappaB Activation

This study examined the total polyphenol content of eight wild edible plants from Ethiopia and their effect on NO production in Raw264.7 cells. Owing to its relatively high polyphenol concentration and inhibition of NO production, the methanol extract of Adansonia digitata L. leaf (MEAD) was subjected to detailed evaluation of its antioxidant and anti-inflammatory effects. Antioxidant effects were assessed by measuring free-radical-scavenging activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and oxygen-radical-absorbance capacity (ORAC) assays, while anti-inflammatory effects were assessed by measuring inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In the ORAC assay, MEAD was 10.2 times more potent than vitamin C at eliminating peroxyl radicals. In DPPH assay, MEAD also showed a strong ROS scavenging effect. MEAD significantly inhibited iNOS activity (IC50=28.6 microg/ml) of LPS-stimulated Raw264.7 cells. We also investigated the relationship between iNOS expression and nuclear factor kappa B (NF-kappaB) activation. MEAD inhibited IkappaBa degradation and NF-kappaB translocation from the cytosol to the nucleus in LPS-induced RAW264.7 cells without significant cytotoxic effects, as confirmed by MTT assay. These results suggest that MEAD inhibits anti-inflammatory iNOS expression, which might be related to the elimination of peroxyl radicals and thus the inhibition of IkappaBa-mediated NF-kappaB signal transduction.

Glatiramer acetate inhibits the activation of NFkappaB in the CNS of experimental autoimmune encephalomyelitis / 대한수의학회지

Glatiramer acetate (GA; Copaxone) has been shown to be effective in preventing and suppressing experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). It has been recently shown that GA-reactive T cells migrate through the blood-brain barrier, accumulate in the central nervous system (CNS), secrete antiinflammatory cytokines and suppress production of proinflammatory cytokines of EAE and MS. Development of EAE requires coordinated expression of a number of genes involved in the activation and effector functions of inflammatory cells. Activation of inflammatory cells is regulated at the transcriptional level by several families of transcription factors. One of these is the nuclear factor kappa B (NFkappaB) family which is present in a variety of cell types and involved in the activation of immune-relative genes during inflammatory process. Since it is highly activated at site of inflammation, NFkappaB activation is also implicated in the pathogenesis of EAE. In this study, we examined whether the inhibition of NFkappaB activation induced by GA can have suppressive therapeutic effects in EAE mice. We observed the expression of NFkappaB and phospho-IkappaB proteins increased in GA-treated EAE mice compared to EAE control groups. The immunoreactivity in inflammatory cells and glial cells of NFkappaB and phospho-IkappaB significantly decreased at the GA-treated EAE mice. These results suggest that treatment of GA in EAE inhibits the activation of NFkappaB and phophorylation of IkappaB in the CNS. Subsequently, the inhibition of NFkappaB activation and IkappaB phosphorylation leads to the anti-inflammatory effects thereby to reduce the progression and severity of EAE.

S-Adenosyl-L-methionine ameliorates TNFalpha-induced insulin resistance in 3T3-L1 adipocytes

An association between inflammatory processes and the pathogenesis of insulin resistance has been increasingly suggested. The IkappaB kinase-beta (IKK-beta)/ nuclear factor-kappaB (NF-kappaB) pathway is a molecular mediator of insulin resistance. S-Adenosyl-L-methionine (SAM) has both antioxidative and anti-inflammatory properties. We investigated the effects of SAM on the glucose transport and insulin signaling impaired by the tumor necrosis factor alpha (TNFalpha) in 3T3-L1 adipocytes. SAM partially reversed the basal and insulin stimulated glucose transport, which was impaired by TNFalpha. The TNFalpha-induced suppression of the tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1) and Akt in 3T3-L1 adipocytes was also reversed by SAM. In addition, SAM significantly attenuated the TNFalpha-induced degradation of IkappaB-alpha and NF-kappaB activation. Interestingly, SAM directly inhibited the kinase activity of IKK-beta in vitro. These results suggest that SAM can alleviate TNFalpha mediated-insulin resistance by inhibiting the IKK-beta/NF-kappaB pathway and thus can have a beneficial role in the treatment of type 2 diabetes mellitus.

Glucocorticoid Mechanism of Inhibition of the Inflammatory Cells in Lumbar Intervertebral Disc Cells Stimulated by TNF-alpha Production of Nuclear Factor-kappaB / 대한정형외과연구학회지

PURPOSE: To analyze the action mechanism of NF-kappaB, IkappaB-alpha and effect of the Dexamethasone (DEXA) in mediating this inflammation, after stimulating cultured herniated intervertebral disc cells with TNF-alpha. MATERIALS AND METHODS: After cultured human intervertebral disc cells passaged three times, they were divided into four groups: A control group (A), DEXA treatment group (B), TNF-alpha treated group (C), TNF-alpha and DEXA were treated at the same time (D). IL-6 and IL-1beta gene expression were measured with semi-quantitative RT-PCR. Western blot analysis was performed to measure protein expression of IkappaB-alpha in the above groups for 10 minutes, 1 hour, 2 hours. In addition, in order to explain the mechanism of NF-kappaB nuclear binding for each group, the nuclear amount of NF-kappaB binding in the nucleus is measured by EMSA. RESULTS: In RT-PCR, expression of IL-6 and IL-1beta was greatest in group C, followed by group D, group A. IkappaB-alpha expression of the group treated with DEXA was not detected in Western blot results within 10 minutes. However, if stimulated by TNF-alpha, the DEXA was not inhibited of IkappaB-alpha concentration. After 1 hour and 2 hours, IkappaB-alpha levels were expressed by cells autonomously (autoregulatory induction). EMSA results expression levels in nuclear protein was maintained in accordance with protein expression. CONCLUSIONS: Our study shows that DEXA inhibits the production of mediators such as inflammatory IL-6 and IL-1beta, however, may not inhibit the transcription of NF-kappaB stimulated by TNF-alpha.

The inhibition of inflammatory molecule expression on 3T3-L1 adipocytes by berberine is not mediated by leptin signaling

In our previous study, we have shown that berberine has both anti-adipogenic and anti-inflammatory effects on 3T3-L1 adipocytes, and the anti-adipogenic effect is due to the down-regulation of adipogenic enzymes and transcription factors. Here we focused more on anti-inflammatory effect of berberine using real time RT-PCR and found it changes expressions of adipokines. We hypothesized that anti-adipogenicity of berberine mediates anti-inflammtory effect and explored leptin as a candidate mediator of this signaling. We studied this hypothesis by western blot analysis, but our results showed that berberine has no effect on the phosphorylations of STAT-3 and ERK which have important roles on leptin signaling. These results led us to conclude that the anti-inflammatory effect of berberine is not mediated by the inhibition of leptin signal transduction. Moreover, we have found that berberine down-regulates NF-kappaB signaling, one of the inflammation-related signaling pathway, through western blot analysis. Taken together, the anti-inflammatory effect of berberine is not mediated by leptin, and berberine induces anti-inflammatory effect independent of leptin signaling.

Influences of Particles on Pro-inflammatory Signalings in Pre-osteoclasts / 대한정형외과연구학회지

PURPOSE: To evaluate the roles of macrophages and their influences on the signal transduction in the periprosthetic osteolysis, the pro-inflammatory signals were analyzed in particles-stimulated macrophages. MATERIALS AND METHODS: Raw 264.7 cell line derived from mice macrophages was used as pre-osteoclasts. To increase the stimulatory effects, the particles were composed of PMMA and polyethylene. Under the similar conditions as osteoclast differentiation, we examined the effect of particles on the pro-inflammatory signals in macrophage: the production of TNF-alpha, the activity of MAPKs (mitogen-activated phosphorylation kinase), the expression of I-kappaB (Inhibitory (B) and the production of H2O2 and nitric oxide. RESULTS: The particles stimulated the secretion of TNF-alpha and increased the phosphorylation of p38 and ERK in course of time. The concentration of H2O2 was increased; however the nitric oxide formation was not increased by particle treatment. In addition, the production of H2O2 was synergistically increased by suboptimal stimulation with PMA (phorbol 12-myristate 13-acetate). Expressions of I-kappaB were inhibited by particles. CONCLUSION: The particles may stimulate the activation of MAPKs, the production of TNF-alpha, reactive oxygen species (ROS) and the activation of NF-kappaB in the pre-osteoclasts. We speculate that particles may mediate the pro-inflammatory signal cascade by the activation of NF-kappaB through ROS in pre-osteoclasts, rather than reactive nitrogen species (RNS). Therefore we suggest that the macrophages in inflammatory osteolysis may have the characteristics of pre-osteoclast as well as pro-inflammatory cell. Further researches should be recommended.

Down-regulation of IL-1beta-induced COX-2 Expression in A549 Lung Cancer Cells at Transcriptional Level by Leptomycin B Involves Inhibition of the IkappaB-alpha/NF-kappaB Pathway but Independent of CRM1

PURPOSE: Overexpression of COX-2, an enzyme responsible fro the synthesis of prostaglandins, is well linked to human chronic lung diseases. The mechanism by which COX-2 expression is increased or enhanced in cancer cells remains largely unknown. Any compound which can reduce COX-2 expression may be considered as an anti-cancer agent. MATERIALS AND METHODS: Leptomycin B (LMB) is a metabolite of Streptomyces and a specific inhibitor of CRM1 nuclear export receptor. A549 is a human lung cancer cell line. To evaluate the effect of LMB on COX-2 expression induced by IL-1beta, a pro-inflammatory cytokine, in A549 cells, Western blot and RT-PCR assays were applied to measure COX-2 protein and mRNA expressions in response to IL-1beta, respectively. Luciferase experiments were done to measure promoter activity of COX-2, NF-kappaB or AP-1. CRM1 siRNA trasfection experiment was performed to knock-down endogenous CRM1. Biochemical protein fractionation method was also carried out to see intracellular localization of proteins. RESULTS: LMB at 9 nM strongly suppressed IL-1beta-induced expression of COX-2 protein that was attributable to decreased COX-2 transcript and promoter activity, but not mRNA stability. Distinctly, knock-down of CRM1 had no effect on COX-2 expression by IL-1beta. Moreover, LMB did not affect IL-1beta-induced phosphorylation of ERK-1/2, JNK- 1/2, and p38 MAPK or AP-1 promoter activity. In contrast, LMB blocked IL-1beta- mediated cytosolic IkappaB-alpha degradation, p65 NF-kappaB nuclear translocation, and NF-kappaB promoter activity. CONCLUSION: LMB potently down-regulates IL-1beta- induced COX-2 at transcriptional level in A549 cells, in part, through modulation of the IkappaB-alpha/NF-kappaB pathway but independent of CRM1, MAPKs and AP-1.

Gliotoxin Protects Against TNBS-induced Colitis Via Down-regulation of NF B Activation / 대한해부학회지

During inflammation of the colon, cells of the gut mucosa express numerous inflammatory mediators including interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta(IL-1beta). These cytokines have been implicated as contributing factors in the inflammatory process, which may result in colitis during inflammatory bowel disease (IBD). Gliotoxin is a fungal metabolite of an epipolythiodioxopiperazine analogue with immunosup-pressive properties in vivo and in vitro, but the effects of gliotoxin on IBD have not been largely evaluated. Therefore, this study evaluated the potential of gliotoxin to protect against TNBS-induced colitis. One microgram of gliotoxin in 100microliter of vehicle was intra-rectally administered into mice exhibiting trinitrobenzene sulfonic acid (TNBS)-induced colitis. IL-8 secretion was measured using an enzyme-linked immu-nosorbent assay (ELISA), myeloperoxidase (MPO) activity was evaluated spectrophotometically, and IkappaB degradation was analyzed on Western blots. Gliotoxin treatment of mice bearing TNBS-induced colitis improved macro-and micro-pathological findings and dramatically decreased MPO activity, a marker of leukocyte infiltration. Furthermore, gliotoxin decreased IkappaB degradation and IL-8 induction caused by TNF-alpha or IL-1beta in HT-29 cells. These findings suggest that gliotoxin partially protects against TNBS-induced colitis through the sup-pression of IL-8 induction and IkappaB degradation by inflammatory mediators such as TNF-alpha or IL-1beta.

Effect of Steroid Administration Ex Vivo on the IkappaB/NF-kappaB Pathway in Human Peripheral Blood Monocytes / 결핵

BACKGROUND: Synthetic glucocorticoids are widely used in many chronic inflammatory diseases because of their excellent anti-inflammatory activity. Enhancing the transcription of IkappaB and preventing activated NF-kappaB from binding to kappaB sites are thought to be the underlying mechanisms. But these data are largely derived from in vitro studies using cell lines. In this study, after administrating a steroid to volunteers, we evaluated the effect on the NF-kappaB system. METHODS: Prednisolone(0.5mg/kg/d) was orally administered to 5 healthy volunteers for 7 days. Before and after the administration, we sampled their peripheral blood monocytes, and performed western blot analysis both with stimulation, using IL-1beta, LPS, TNF, and without stimulation(baseline). We also performed EMSA after stimulation with LPS. RESULTS: After ingestion of the steroid, baseline expressions of I(kappa)B(alpha) were increased in two of the subjects, while suppressed degradations of I(kappa)B(alpha) to stimulations were observed in all five. In addition, the binding capacity of NF-kappaB after the administration was decreased. CONCLUSION: Steroid plays such roles as enhancing the transcription of I(kappa)B(alpha), suppressing the DNA binding capacity of NF-kappaB, and suppressing the degradation of I(kappa)B(alpha).