Cytokines In Lipopolysaccharide Research Articles

Anthocyanins downregulate lipopolysaccharide-induced inflammatory responses in BV2 microglial cells by suppressing the NF-κB and Akt/MAPKs signaling pathways.

Anthocyanins are naturally occurring polyphenols that impart bright color to fruits, vegetables and plants and have a variety of protective properties, which have generally been attributed to their antioxidant capacity. However, little is known about the molecular mechanisms underlying anti-inflammatory effects of anthocyanins related to neurodegenerative diseases. Therefore, we determined whether anthocyanins isolated from black soybean seed coats would inhibit pro-inflammatory mediators and cytokines in lipopolysaccharide (LPS)-stimulated murine BV2 microglial cells. Our results showed that anthocyanins significantly inhibited LPS-induced pro-inflammatory mediators, such as nitric oxide (NO) and prostaglandin E2, and pro-inflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-1β, without significant cytotoxicity. Anthocyanins also downregulated excessive expression of inducible NO synthase, cyclooxygenase-2, TNF-α, and IL-1β in LPS-stimulated BV2 cells. Moreover, anthocyanins inhibited nuclear translocation of nuclear factor-kappa B (NF-κB) by reducing inhibitor of NF-κB alpha degradation as well as phosphorylating extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, and Akt. These findings suggest that anthocyanins may offer substantial therapeutic potential for treating inflammatory and neurodegenerative diseases accompanied by microglial activation.

Aqueous extracts of Ocimum grasstimum inhibits lipopolysaccharide-induced interleukin-6 and interleukin-8 expression in airway epithelial cell BEAS-2B

To investigate the antiinflammatory activities of aqueous extract of Occimum gratissmium (OGE) with emphasis on expression of proinflammatory cytokines in Lipopolysaccharide (LPS)-stimulated epithelial cell BEAS-2B. Effects of OGE on cell viability were determined by MTT assay. mRNA expression were analyzed by and reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR. Activation of kinase cascades was investigated by immunoblot. Intracellular reactive oxygen species (ROS) was analyzed by flow cytometry. OGE (<200 μg/mL) treatment or pretreatment and following LPS exposure slightly affected viability of BEAS-2B cells. Increase of interleukin (IL)-6 and IL-8 and the elevated level of intracellular ROS in LPS-stimulated BEAS-2B cells were diminished by OGE pretreatment in a dose-dependent manner. OGE suppressed inflammatory response-associated mitogen-activated protein kinases (MAPKs) and Akt activation. Additionally, OGE pretreatment increased level of cellular inhibitor of κBα (IκBα) and inhibited nuclear translocation of nuclear factor kappa B (NF-κB). These findings indicate that significant suppression of IL-6 and IL-8 expressions in LPS-stimulated BEAS-2B cells by OGE may be attributed to inhibiting activation of MAPKs and Akt and consequently suppressing nuclear translocation of NF-κB.

Intravenous immunoglobulin preparation attenuates LPS-induced production of pro-inflammatory cytokines in human monocytic cells by modulating TLR4-mediated signaling pathways

Intravenous immunoglobulin (IVIG) has been used for the treatment of inflammatory and autoimmune diseases. The ability to modulate cytokine production has been formerly described as one of the mechanisms of its action. This study aimed to investigate the effect of IVIG on the production of pro-inflammatory cytokines in lipopolysaccharide (LPS)-stimulated monocytic cells. Peripheral blood mononuclear cells (PBMCs) or THP-1 cells treated with phorbol myristate acetate (PMA) were stimulated with LPS. The protein levels of pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-6, and high-mobility group box 1 (HMGB1)] in the culture supernatants were determined using appropriate enzyme-linked immunosorbent assay kits. The mRNA of TNF-α was determined by reverse transcription-polymerase chain reaction. The phosphorylation of nuclear factor kappa B (NF-κB) and the mitogen-activated protein kinases was examined by Western blot analyses. IVIG suppressed the production of pro-inflammatory cytokines such as TNF-α and IL-6 in LPS-stimulated PBMCs. Furthermore, IVIG inhibited TNF-α, IL-6, and HMGB1 production from LPS-stimulated THP-1 cells treated with PMA. In addition, Fc fragment prepared from the IVIG inhibited production of these cytokines from the cells to the same degree as IVIG, whereas Fab and F(ab')(2) fragments inhibited this only partially. We showed that IVIG and Fc fragments suppressed LPS-induced signal transduction pathways involving phosphorylation of NF-κB, p38, and c-Jun N-terminal kinase (JNK). Taken together, our results suggest that IVIG attenuates LPS-induced cytokine production predominantly mediated by its Fc region. The activity might be regulated by inhibiting NF-κB, p38, and JNK pathways in human monocytic cells.

Nodakenin Suppresses Lipopolysaccharide-Induced Inflammatory Responses in Macrophage Cells by Inhibiting Tumor Necrosis Factor Receptor-Associated Factor 6 and Nuclear Factor-κB Pathways and Protects Mice from Lethal Endotoxin Shock

Nodakenin, a coumarin isolated from the roots of Angelicae gigas, has been reported to possess neuroprotective, antiaggregatory, antibacterial, and memory-enhancing effects. In the present study, we investigated the anti-inflammatory effects of nodakenin by examining its in vitro inhibitory effects on inducible nitric-oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and proinflammatory cytokines in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and mouse peritoneal macrophages and its in vivo effects on LPS-induced septic shock in mice. Our results indicate that nodakenin concentration-dependently inhibits iNOS and COX-2 at the protein, mRNA, and promoter binding levels, and these inhibitions cause attendant decreases in the production of nitric oxide (NO) and prostaglandin E₂ (PGE₂). Furthermore, we found that nodakenin inhibits the production and mRNA expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β induced by LPS. Molecular data revealed that nodakenin suppressed the transcriptional activity and translocation of nuclear factor-κB (NF-κB) by inhibiting inhibitory κB-α degradation and IκB kinase-α/β phosphorylation. In addition, nodakenin was found to significantly inhibit the LPS-induced binding of transforming growth factor-β-activated kinase 1 to tumor necrosis factor receptor-associated factor 6 (TRAF6) by reducing TRAF6 ubiquitination. Pretreatment with nodakenin reduced the serum levels of NO, PGE₂, and proinflammatory cytokines and increased the survival rate of mice with LPS-induced endotoxemia. Taken together, our data suggest that nodakenin down-regulates the expression of the proinflammatory iNOS, COX-2, TNF-α, IL-6, and IL-1β genes in macrophages by interfering with the activation of TRAF6, thus preventing NF-κB activation.

Sulfated Derivative of 20(S)-Ginsenoside Rh2 Inhibits Inflammatory Cytokines Through MAPKs and NF-kappa B Pathways in LPS-Induced RAW264.7 Macrophages

In the previous study, we found that sulfated derivative B2 of ginsenoside Rh2 (Rh2-B2) has greater anti-inflammatory effects than 20(S)-ginsenoside Rh2. However, the anti-inflammatory mechanism of Rh2-B2 remains unclear. We therefore assessed the effects of Rh2-B2 on inflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. We found that Rh2-B2 (1-5mg/L) significantly inhibited tumor necrosis factor alpha, interleukin (IL)-6, IL-1β, and increased IL-10 production from protein and mRNA levels. Furthermore, Rh2-B2 significantly inhibited the phosphorylation of p38 and c-Jun N-terminal kinase as well as decreased p65 nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) translocation into the nucleus by nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha degradation. The present results indicate that Rh2-B2 inhibits the production of inflammatory cytokines induced by LPS through blocking mitogen-activated protein kinases and NF-κB signaling pathways.

유색미 겨 아라비녹실레인과 운동트레이닝이 Lipopolysaccharide 처치된 흰쥐의 면역인자 및 염증억제에 미치는 영향

Arabinoxylan (Ara) rice bran has been shown to be a potent biological response modifier as manifested by stimulation of different arms of the immune system. We examined the effects of Ara rice bran and exercise on the immune function and cytokines in lipopolysaccharide (LPS)-stimulated rats. As the results, tumor necrosis factor-α as representative inflammatory cytokines showed a significantly lower in Ara supplement group, thus the Ara rice bran had a higher inhibitory activity than the both exercise and control group. However, 4 weeks of exercise training significantly increased inflammatory reactions rather than treatment with Ara in LPS-treated rats. The Ara rice bran acted to decrease the inflammatory reaction. These results suggest that the supplement of Ara rice bran is likely contribute to inflammation response and the Ara rice bran can be used as a possible safe alternative to the immunotherapeutic modalities.

Inhibitory effects of methanolic extract of Salvia fruticosa Mill. on pro-inflammatory cytokines production in RAW 264.7 in vitro cellular model and in Balb/c mice in vivo animal model

The aim of this study is to elucidate the anti-inflammatory effects of methanolic extract (MeOH) of Salvia fruticosa Mill. on the production of pro-inflammatory cytokines in Lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and in Balb/c mice. Tumor necrosis factor –α (TNF-α), Interlukin-6 (IL-6), and Interlukin 1β (IL-1β) production in RAW 264.7 cells and in Balb/c mice were evaluated. The extract of S. fruticosa exhibited potent inhibitory effects on pro-inflammatory cytokines production in both cellular and animal models stimulated by LPS. Our data suggested that, the methanolic extract of S. fruticosa could be developed as a potential anti-inflammatory candidate for the treatment of inflammatory diseases mediated by overproduction of pro-inflammatory cytokines such as rheumatoid arthritis.

Anti-inflammatory effect of methanol extract of Keum-Ryung-Ja-San in mouse macrophages

Objective : The aim of this study was to determine whether methanol extract of Keum-Ryung-Ja-San (KRJS) inhibit production of NO, , iNOS, COX-2 and pro-inflammatory cytokines in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. Methods : Cytotoxic activity of extracts on RAW 264.7 cells was measured using 5-(3-caroboxymeth-oxyphenyl)-2H-tetra-zolium inner salt (MTS) assay. The nitric oxide (NO) production was measured by Griess reagent system. And proinflammatory cytokines and PGE2 were measured by ELISA kit. The levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2(COX-2), and nuclear NF- p65 expression were detected by western blot. Results : Our results indicated that methanol extract of KRJS significantly inhibited the LPS-induced NO, production and iNOS, COX-2 expression accompanied by an attenuation of TNF-, IL- and IL-6 production in RAW 264.7 cells. Moreover, methanol extract of KRJS treatment also blocked LPS-induced NF- activation. Conclusion : These findings indicate that methanol extract of KRJS inhibits the production of pro-inflammatory mediators and cytokines via suppression of NF- activation. Take together, these results indicate that methanol extract of KRJS has the potential for use as an agent of anti-chronic inflammatory diseases.

Hexane fraction of Zingiberis Rhizoma Crudus extract inhibits the production of nitric oxide and proinflammatory cytokines in LPS-stimulated BV2 microglial cells via the NF-kappaB pathway

Excessive production of inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), and proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) from activated microglia contributes to uncontrolled inflammation in neurodegenerative diseases. It seems possible that treatment with anti-inflammatory agents, including plants used in Oriental medicine, might delay the progression of neurodegeneration through the inhibition of microglial activation. The present study is focused on the inhibitory effect of the rhizome hexane fraction extract of Zingiber officinale Roscoe (ginger hexan extract; GHE) on the production of inflammatory mediators such as NO, PGE2, and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV-2 cells, a mouse microglial cell line. GHE significantly inhibited the excessive production of NO, PGE2, TNF-α, and IL-1β in LPS-stimulated BV2 cells. In addition, GHE attenuated the mRNA expressions and protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and proinflammatory cytokines. The molecular mechanisms that underlie GHE-mediated attenuation are related to the inhibition of the phosphorylation of three mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK), and the activation of nuclear factor-kappaB (NF-κB). Our results indicate that GHE exhibits anti-inflammatory properties by suppressing the transcription of inflammatory mediator genes through the MAPK and NF-κB signaling pathways. The anti-inflammatory properties of GHE may make it useful as a therapeutic candidate for the treatment of human neurodegenerative diseases.

Methanol Extract of Biophytum sensitivum Alters the Cytokine Profile and Inhibits iNOS and COX-2 Expression in LPS/Con A Stimulated Macrophages

Biophytum sensitivum has been used in traditional folk medicine to treat numerous diseases. The molecular mechanism of B. sensitivum pharmacological and biochemical actions of macrophages in inflammation has not been clearly elucidated. We examined how the methanol extract of B. sensitivum regulates the production of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, and nitric oxide (NO) in vitro and in vivo. The extract inhibits the production of NO and proinflammatory cytokines in lipopolysaccharide (LPS) or Concanavalin (Con) A-stimulated primary macrophages. In vitro L929 bioassay revealed the inhibition of TNF-α production by B. sensitivum treatment. Moreover, the extract could suppress the inducible nitric oxide synthase and cyclo-oxygenase-2 mRNA expression in LPS or Con A-stimulated macrophages. These findings provide evidence that B. sensitivum possesses potential anti-inflammatory activity and may be beneficial for the treatment of endotoxin shock or sepsis.

The expression of nuclear factor-kappa B in lipopolysaccharide-induced keratitis of rats and the effect of pyrrolidine dithiocarbamate on its expression

To investigate the expression of nuclear factor-kappa B (NF-kappaB) and cytokines in lipopolysaccharide (LPS)-induced keratitis of rats, and the effect of pyrrolidine dithiocarbamate (PDTC) on its expression. A LPS-induced keratitis model was established in Wistar rats. Thirty minutes before LPS exposure, PDTC and normal saline were injected into subconjunctival tissues separately. At 0.5, 1.0, 3.0, 6.0, 12.0, 24.0 and 72.0 h after LPS exposure, the rats were examined with slit-lamp microscope. Then they were sacrificed and the corneas were excised for routine histological analysis. Immunohistochemical staining with an antibody against activated NF-kappaB was performed to detect the expression of NF-kappaB. The change of TNF-alpha mRNA expression was identified by reverse transcriptase polymerase chain reaction (RT-PCR). Histology findings demonstrated that corneas exposed to LPS showed significant changes in corneal structure, edema and pronounced inflammatory cells infiltration were observed. Both symptoms and damages of the cornea were lesser in the rats of PDTC group than that of the control keratitis group. Compared with PDTC group, the activation of NF-kappaB and the expression of TNF-alpha mRNA were significantly upregulated after LPS challenge 0.5 - 24.0 h (P < 0.01), separately; peak expression of NF-kappaB and TNF-alpha mRNA were observed at 3.0 - 12.0 h after LPS exposure. The expression of NF-kappaB and TNF-alpha mRNA induced by NF-kappaB plays an important role in the pathogenesis of keratitis. The inhibitor of NF-kappaB, PDTC, can relieve the cornea from damages produced by keratitis.

Lipopolysaccharide regulated protein expression is only partly impaired in monocytes from patients with type I diabetes

BackgroundMonocytes play an important role in innate immunity and atherosclerosis. A disturbed secretion of cytokines in lipopolysaccharide (LPS) activated monocytes from type 1 diabetes (T1D) patients has been described and may contribute to the impaired inflammatory response in these individuals. In the present study the influence of LPS on five different proteins with a function in immunity and atherosclerosis was analyzed in monocytes from controls and T1D patients.MethodsMonocytes were isolated from controls and T1D patients and the LPS-stimulated increase of IL-6, CXCL8, monocyte chemotactic protein 1 (CCL2, MCP-1) and superoxide dismutase (SOD 2), as well as the LPS-mediated decrease of apolipoprotein E (Apo E) in primary human monocytes from controls and T1D patients was determined.ResultsCCL2 and IL-6 secretion in response to LPS was found significantly reduced in monocytes from T1D patients when compared to controls whereas basal CCL2 release was similar in control and T1D cells. In contrast, CXCL8 and apolipoprotein E secretion and SOD 2 expression upon LPS stimulation is similar from T1D and control monocytes.ConclusionThese data indicate that LPS-mediated protein expression is only partly disturbed in monocytes from T1D patients. Reduced secretion of IL-6 and CCL2 in activated monocytes of these patients may contribute to an impaired inflammatory response and vascular disease.

Innate production of interleukin-10 and tumor necrosis factor affects the risk of multiple sclerosis

Multiple sclerosis (MS) typically presents with a relapsing-remitting onset. This can be distinguished from primary progressive MS. Typical MS is characterized by a profound inflammatory reaction in which anti-inflammatory cytokine interleukin-10 (IL-10) and pro-inflammatory cytokine tumor necrosis factor (TNF) may play a pivotal role. We tested the hypothesis that patients with MS have a distinct innate cytokine production that contributes to the susceptibility for and outcome of MS. The innate cytokine production of patients was estimated as the average production of cytokines in lipopolysaccharide -stimulated whole-blood cultures of 2 to 5 first-degree healthy family members. A total of 126 family members of 50 patients with typical MS, 61 family members of 25 patients with primary progressive MS, and 129 control subjects of 54 families were enrolled in this study. We found that members of families with low IL-10 and high TNF production had a fourfold increased risk of developing typical MS compared with members of families with high IL-10 and low TNF production. Patients with MS were eightfold more likely to develop typical MS than primary progressive MS when they belonged to families with low IL-10 and high TNF production. The presence of human leukocyte antigen–DR2 was associated with MS but not with TNF production. This study shows that typical MS is associated with an innate pro-inflammatory cytokine profile in contrast to primary progressive MS. Ann Neurol 2000;48:641–646

Innate production of interleukin-10 and tumor necrosis factor affects the risk of multiple sclerosis.

Multiple sclerosis (MS) typically presents with a relapsing-remitting onset. This can be distinguished from primary progressive MS. Typical MS is characterized by a profound inflammatory reaction in which anti-inflammatory cytokine interleukin-10 (IL-10) and pro-inflammatory cytokine tumor necrosis factor (TNF) may play a pivotal role. We tested the hypothesis that patients with MS have a distinct innate cytokine production that contributes to the susceptibility for and outcome of MS. The innate cytokine production of patients was estimated as the average production of cytokines in lipopolysaccharide -stimulated whole-blood cultures of 2 to 5 first-degree healthy family members. A total of 126 family members of 50 patients with typical MS, 61 family members of 25 patients with primary progressive MS, and 129 control subjects of 54 families were enrolled in this study. We found that members of families with low IL-10 and high TNF production had a fourfold increased risk of developing typical MS compared with members of families with high IL-10 and low TNF production. Patients with MS were eightfold more likely to develop typical MS than primary progressive MS when they belonged to families with low IL-10 and high TNF production. The presence of human leukocyte antigen-DR2 was associated with MS but not with TNF production. This study shows that typical MS is associated with an innate pro-inflammatory cytokine profile in contrast to primary progressive MS.

INHIBITORY EFFECT OF FERULIC ACID ON MACROPHAGE INFLAMMATORY PROTEIN-2 PRODUCTION IN A MURINE MACROPHAGE CELL LINE, RAW264.7

We investigated time-related productions of certain cytokines in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, a murine macrophage cell line, by enzyme-linked immunosorbent assay. Macrophage inflammatory protein-2 (MIP-2) levels became detectable after 2 h and markedly increased over the first 8 h. Thereafter, this level remained at the same level between 10 and 16 h, and them increased again until 24 h, showing a tendency of biphasic pattern. Tumour necrosis factor (TNF)-α was detectable at 2 h and then increased sharply until 6 h at which it attained its peak. A low but recognizable level of interleukin (IL)-1α/β was also detectable. When the inhibitory effect of ferulic acid (FA), an active component of the Rhizoma ofCimicifugasp. used frequently as anti-inflammatory drug in Japanese Oriental medicines, was compared with that of dexamethasone (DX) on MIP-2 and TNF-α productions in response to LPS, both FA and DX could reduce the production of these cytokines in a dose-dependent manner. Concerning TNF-α, however, the inhibitory effect of FA was very weak compared with that of DX. In addition, FA as well as DX reduced MIP-2 production induced by TNF-α. These data suggest that MIP-2 might be induced by a direct effect of LPS and in part indirect one via initial induction of other cytokines such as TNF-α, leading a tendency of biphasic pattern. Comparing DX, FA is considered to be a novel and unique drug inhibiting MIP-2 production more selectively.