DNA-binding Activity Of Nuclear factor-κB Research Articles

β-eudesmol inhibits thymic stromal lymphopoietin through blockade of caspase-1/NF-κB signal cascade in allergic rhinitis murine model

Allergic rhinitis (AR) is a global health problem because of its steadily increasing incidence and prevalence that currently affects about 30% of people worldwide. β-eudesmol has various beneficial effects, including anti-cancer and anti-allergic activities. However, the effects of β-eudesmol on AR have not yet been clarified; thus, we investigated the effects of β-eudesmol in an ovalbumin-induced AR animal model using enzyme-linked immunosorbent assay, histamine assay, Western blotting, and hematoxylin and eosin staining methods. β-eudesmol reduced the nasal rubs score and levels of histamine and immunoglobulin E in serum of AR mouse. In addition, the levels of thymic stromal lymphopoietin, interleukin-1β, tumor necrosis factor-α, and macrophage inflammatory protein-2 were down-regulated and infiltration of eosinophils and the level of intercellular adhesion molecule-1 were inhibited by β-eudesmol administration. β-eudesmol administration also reduced active caspase-1 and nuclear factor-κB DNA binding activity in nasal mucosa tissues of AR mice. Taken together, these results indicate that β-eudesmol would be effective for the treatment of allergic and inflammatory diseases, such as AR.

Cross-talk between Bcl-2 and IKK in 4-hydroxynonenal-induced apoptosis

Apoptosis of macrophage foam cells loaded with modified/oxidized lipids is implicated in destabilization of advanced atherosclerotic plaques in humans. Concentration of HNE, main aldehydic product of plasma LDL peroxidation, elevates in atherosclerotic lesions as well as in cultured cells under oxidative stress. Although this reactive aldehyde has been shown to promote apoptosis with the involvement of p38 MAPK and JNK in various mammalian cell lines, roles of B-cell lymphoma 2 (Bcl-2) family proteins remain to be deciphered. We demonstrated that HNE-induced apoptosis was accompanied by concurrent downregulations of antiapoptotic Bcl-x(L) and Mcl-1 as well as upregulation of proapoptotic Bak. Furthermore, phoshorylation of Bcl-2 at Thr56, Ser70, and probably more phosphorylation sites located on N-terminal loop domain associated with HNE-induced apoptosis in both U937 and HeLa cells while ectopic expression of a phospho-defective Bcl-2 mutant significantly attenuated apoptosis. In parallel to this, HNE treatment caused release of proapoptotic Bax from Bcl-2. Pharmacological inhbition of Inhibitory kappa B kinase (IKK) inhibited HNE-induced Bcl-2 phosphorylation. Similarly, silencing IKKα and -β both ended up with abrogation of Bcl-2 phosphorylation along with attenuation of apoptosis. Moreover, both IKKα and -β coimmunoprecipitated with Bcl-2 and in vitro kinase assay proved the ability of IKK to phosphorylate Bcl-2. In view of these findings and considering HNE inhibits DNA-binding activity of nuclear factor-κB (NF-κB) through prevention of IκB phosphorylation/ubiquitination/proteolysis, IKK appears to directly interfere with Bcl-2 activity through phosphorylation in HNE-mediated apoptosis independent of NF-κB signaling.

Ethanol Extract of Potentilla supina Linne Suppresses LPS-induced Inflammatory Responses through NF-κB and AP-1 Inactivation in Macrophages and in Endotoxic mice.

In this study, we investigated the antiinflammatory effects of ethanol extracts of Potentilla. supina Linne (EPS) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and septic mice. EPS suppressed LPS-induced nitric oxide, prostaglandin E2 , TNF-α, interleukin-6 and interleukin-1β at production and mRNA levels in LPS-induced RAW 264.7 macrophages. Consistent with these observations, EPS attenuated the expressions of inducible nitric oxide synthase and cyclooxygenase-2 by downregulation of their promoter activities. Molecularly, EPS reduced the LPS-induced transcriptional activity and DNA-binding activity of nuclear factor-κB (NF-κB), and this was associated with a decrease of translocation and phosphorylation of p65 NF-κB by inhibiting the inhibitory κB-α degradation and IKK-α/β phosphorylation. Furthermore, EPS inhibited the LPS-induced activation of activator protein-1 by reducing the expression of c-Fos and c-Jun in nuclear. EPS also suppressed the phosphorylation of mitogen-activated protein kinase, such as p38 mitogen-activated protein kinase and c-Jun N-terminal kinase. In an LPS-induced endotoxemia mouse model, pretreatment with EPS reduced the mRNA levels of inducible nitric oxide synthase, cyclooxygenase-2 and proinflammatory cytokines and increased the survival rate of mice. Collectively, these results suggest that the antiinflammatory effects of EPS were associated with the suppression of NF-κB and activator protein-1 activation and support its possible therapeutic role for the treatment of endotoxemia. Copyright © 2017 John Wiley & Sons, Ltd.

Chicoric acid suppresses BAFF expression in B lymphocytes by inhibiting NF-κB activity

B cell activating factor belonging to the TNF family (BAFF) plays a critical role in the pathogenesis of autoimmune diseases. The inhibition of BAFF expression is an emerging therapeutic approach for these disorders. Chicoric acid (CA), a bioactive phytochemical found in several widely used traditional medicinal plants, has significant anti-inflammatory activity and anti-arthritic effects. However, the role of CA in modulation of BAFF expression remains unknown. In this study, we demonstrated that CA reduced BAFF expression in human B lymphocyte cell lines and decreased the DNA-binding activity of nuclear factor-κB (NF-κB) in the BAFF promoter region. Furthermore, CA inhibited both the nuclear translocation of p65 (the subunit of NF-κB) and the phosphorylation of IκBα (inhibitor of NF-κB). These results suggest that CA suppresses BAFF expression by inhibiting NF-κB activity, and CA may serve as a novel therapeutic agent to down-regulate excessive BAFF expression in autoimmune diseases.

An ethanol extract of Aster yomena (Kitam.) Honda inhibits lipopolysaccharide-induced inflammatory responses in murine RAW 264.7 macrophages.

Aster yomena (Kitam.) Honda has been widely used as a traditional herbal medicine for centuries to treat cough, asthma, insect bites, etc. Recent reports indicate that A. yomena possesses a wide spectrum of pharmacological activities; however, few experiments have described its anti-inflammatory properties. The present study examined the anti-inflammatory effects of an ethanol extract of A. yomena leaves (EEAY) on lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophages. Treatment with EEAY significantly reduced the secretion of pro-inflammatory molecules, such as nitric oxide and interleukin-1β, in LPS-stimulated RAW 264.7 cells, without incurring any significant cytotoxicity. These protective effects were accompanied by a marked reduction in the expression of regulatory genes at the transcription level. Treatment with EEAY also inhibited the DNA-binding activity of nuclear factor-κB (NF-κB) by suppression of nuclear translocation of NF-κB and by degradation of the inhibitor of NF-κB; these effects were associated with suppression of the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase signaling pathways. The EEAY treatment also potently suppressed LPS-induced toll like receptor (TLR) 4 expression and attenuated the binding of LPS to the macrophage cell surface. In addition, EEAY treatment markedly inhibited LPS-induced accumulation of intracellular reactive oxygen species in RAW 264.7 macrophages. Therefore, the inhibitory effects of EEAY on LPS-stimulated inflammatory responses in RAW 264.7 macrophages were apparently associated with suppression of the TLR-mediated NF-κB signaling pathway. More work is needed to fully understand the critical role and clinical usefulness of EEAY treatment, but the findings of the present study provide some insights into the potential of EEAY as a therapeutic agent for treatment of inflammatory disorders.

Fulvic acid promotes extracellular anti-cancer mediators from RAW 264.7 cells, causing to cancer cell death in vitro

Fulvic acid (FA) is known to promote electrochemical balance as a donor or a receptor possessing many biomedical functions. Nevertheless, the effect of FA on the anti-cancer activity has not been elucidated. In the current study, we first isolated FA from humus and investigated whether FA regulates immune-stimulating functions, such as production of nitric oxide (NO), in RAW 264.7 cells. Our data showed that FA slightly enhances cell viability in a dose-dependent manner and secretion of NO from RAW 264.7 cells. It upregulated the protein and mRNA expression of inducible NO synthesis (iNOS). In addition, FA enhanced the DNA-binding activity of nuclear factor-κB (NF-κB) in RAW 264.7 cells; the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) effectively attenuated the expression of FA-stimulated iNOS, suggesting that FA stimulates NF-κB to promote iNOS and NO production. Finally, FA-stimulated culture media (FA-CM) from RAW 264.7 cells were collected and MCA-102 fibrosarcoma cells were cultured in this media. The FA-CM augmented MCA-102 fibrosarcoma cell apoptosis; however, an NO inhibitor NG-monomethyl-l-arginine (NMMA) slightly inhibited the FA-CM-mediated MCA-102 fibrosarcoma cell apoptosis, which was accompanied by low levels of NO. In the present study, we found that FA induces the generation of NO and iNOS in RAW 264.7 cells by inducing NF-κB activation; however, NO did not significantly stimulate MCA-102 fibrosarcoma cell apoptosis in the current study. In addition, FA-CM enhanced cell death in various human cancer cells such as Hep3B, LNCaP, and HL60. Taken together, FA most likely stimulates immune-modulating molecules such as NO and induces cancer cell apoptosis.

Antagonistic effects of acetylshikonin on LPS-induced NO and PGE2 production in BV2 microglial cells via inhibition of ROS/PI3K/Akt-mediated NF-κB signaling and activation of Nrf2-dependent HO-1.

Although acetylshikonin (ACS) is known to have antioxidant and antitumor activities, whether ACS regulates the expression of proinflammatory mediators in lipopolysaccharide (LPS)-stimulated microglial cells remains unclear. In this study, it was found that ACS isolated from Lithospermum erythrorhizon inhibits LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) release by suppressing the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in BV2 microglial cells. Furthermore, ACS reduced the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) and subsequently suppressed iNOS and COX-2 expression. Consistent with these data, ACS attenuated the phosphorylation of PI3K and Akt and suppressed the DNA-binding activity of NF-κB by inducing the generation of reactive oxygen species (ROS) in LPS-stimulated cells. In addition, ACS enhanced heme oxygenase-1 (HO-1) expression via nuclear factor-erythroid 2-related factor 2 (Nrf2) activation. Zinc protoporphyrin, a specific HO-1 inhibitor, partially attenuated the antagonistic effects of ACS on LPS-induced NO and PGE2 production. By contrast, the presence of cobalt protoporphyrin, a specific HO-1 inducer, potently suppressed LPS-induced NO and PGE2 production. These data indicate that ACS downregulates proinflammatory mediators such as NO and PGE2 by suppressing PI3K/Akt-dependent NF-κB activity induced by ROS as well as inducing Nrf2-dependent HO-1 activity. Taken together, ACS might be a good candidate to regulate LPS-mediated inflammatory diseases.

Shikonin Isolated from Lithospermum erythrorhizon Downregulates Proinflammatory Mediators in Lipopolysaccharide-Stimulated BV2 Microglial Cells by Suppressing Crosstalk between Reactive Oxygen Species and NF-κB.

According to the expansion of lifespan, neuronal disorder based on inflammation has been social problem. Therefore, we isolated shikonin from Lithospermum erythrorhizon and evaluated anti-inflammatory effects of shikonin in lipopolysaccharide (LSP)-stimulated BV2 microglial cells. Shikonin dose-dependently inhibits the expression of the proinflammatory mediators, nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α) as well as their main regulatory genes and products such as inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α in LPS-stimulated BV2 microglial cells. Additionally, shikonin suppressed the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) to regulate the key regulatory genes of the proinflammatory mediators, such as iNOS, COX-2, and TNF-α, accompanied with downregulation of reactive oxygen species (ROS) generation. The results indicate that shikonin may downregulate the expression of proinflammatory genes involved in the synthesis of NO, PGE2, and TNF-α in LPS-treated BV2 microglial cells by suppressing ROS and NF-κB. Taken together, our results revealed that shikonin exerts downregulation of proinflammatory mediators by interference the ROS and NF-κB signaling pathway.

Fexofenadine regulates nuclear factor-κB signaling and endoplasmic reticulum stress in intestinal epithelial cells and ameliorates acute and chronic colitis in mice.

The aim of this study was to evaluate the effect of fexofenadine on intestinal inflammation. HCT116 and COLO205 cells were pretreated with fexofenadine and then stimulated with tumor necrosis factor (TNF)-α. Interleukin (IL)-8 expression was determined by real-time reverse-transcription polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay. DNA-binding activity of nuclear factor-κB was assessed by electrophoretic mobility shift assay. The molecular markers of endoplasmic reticulum (ER) stress were evaluated by Western blot analysis and PCR. In the acute colitis model, mice were given 4% dextran sulfate sodium (DSS) for 5 days with or without fexofenadine. IL-10(-/-) mice were used to evaluate the effect of fexofenadine on chronic colitis. Fexofenadine significantly inhibited the upregulated expression of IL-8 in HCT116 and COLO205 cells stimulated with TNF-α. Fexofenadine suppressed nuclear factor-κB DNA-binding activity. C/EBP homologous protein mRNA expression was enhanced in the presence of TNF-α, and it was dampened by pretreatment of fexofenadine. In addition, the induction of ER stress markers caspase-12 and p-eukaryotic initiation factor 2 (eIF2)-α was significantly suppressed by the pretreatment of fexofenadine. Administration of fexofenadine significantly reduced the severity of DSS-induced murine colitis, as assessed by the disease activity index, colon length, and histology. In addition, the DSS-induced phospho-IκB kinase activation was significantly decreased in fexofenadine-pretreated mice. Finally, fexofenadine significantly reduced the severity of colitis and the immunoreactivity of caspase-12 and p-eIF2-α in IL-10(-/-) mice as compared with controls. These results suggest that fexofenadine is a potential therapeutic agent for the treatment of inflammatory bowel disease.

New compound, 5-O-isoferuloyl-2-deoxy-D-ribono-γ-lacton from Clematis mandshurica: Anti-inflammatory effects in lipopolysaccharide-stimulated BV2 microglial cells.

Microglia are main immune cells to exacerbate neural disorders in persistent overactivating. Therefore, it is a good strategy to regulate microglia for the treatment of neural disorders. In the present study, we isolated and characterized a novel compound, 5-O-isoferuloyl-2-deoxy-D-ribono-γ-lacton (5-DRL) from Clematis mandshurica, and evaluated its anti-inflammatory effect in lipopolysaccharide (LPS)-treated BV2 microglial cells. 5-DRL inhibited the expression of LPS-stimulated proinflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2), as well as their regulatory genes inducible NO syntheses (iNOS) and cyclooxygenase-2 (COX-2). 5-DRL also downregulated the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) through suppression of the nuclear translocation of the NF-κB subunits, p65 and p50. Consistent with the inhibition of iNOS and COX-2 via NF-κB activity with 5-DRL, an inhibitor of NF-κB, pyrrolidine dithiocarbamate (PDTC), also led to the suppression of LPS-induced iNOS and COX-2 expression. Additionally, 5-DRL corresponding with antioxidants, N-acetylcysteine (NAC) and glutathione (GSH), remarkably inhibited reactive oxygen species (ROS) generation. Both NAC and GSH, thus attenuated the expression of iNOS and COX-2 by suppressing NF-κB activation, indicating that 5-DRL suppresses LPS-induced iNOS and COX-2 expression through downregulation of the ROS-dependent NF-κB signaling pathway. The present study also indicated that 5-DRL suppresses NO and PGE2 production by inducing heme oxygenase-1 (HO-1) via nuclear factor erythroid 2-related factor 2 (Nrf2). Taken together, the present data indicate that 5-DRL attenuates the production of proinflammatory mediators such as NO and PGE2 as well as their regulatory genes in LPS-stimulated BV2 microglial cells by inhibiting ROS-dependent NF-κB activation and stimulating the Nrf2/HO-1 signal pathway. These data may be implicated in the application of 5-DRL in LPS-stimulated inflammatory disease.

Wogonin prevents lipopolysaccharide‐induced acute lung injury and inflammation in mice via peroxisome proliferator‐activated receptor gamma‐mediated attenuation of the nuclear factor‐kappaB pathway

Acute lung injury (ALI) from a variety of clinical disorders, characterized by diffuse inflammation, is a cause of acute respiratory failure that develops in patients of all ages. Previous studies reported that wogonin, a flavonoid-like chemical compound which was found in Scutellaria baicalensis, has anti-inflammatory effects in several inflammation models, but not in ALI. Here, the in vivo protective effect of wogonin in the amelioration of lipopolysaccharide (LPS) -induced lung injury and inflammation was assessed. In addition, the in vitro effects and mechanisms of wogonin were studied in the mouse macrophage cell lines Ana-1 and RAW264.7. In vivo results indicated that wogonin attenuated LPS-induced histological alterations. Peripheral blood leucocytes decreased in the LPS-induced group, which was ameliorated by wogonin. In addition, wogonin inhibited the production of several inflammatory cytokines, including tumour necrosis factor-α, interleukin-1β (IL-1β) and IL-6, in the bronchoalveolar lavage fluid and lung tissues after LPS challenge, while the peroxisome proliferator-activated receptor γ (PPARγ) inhibitor GW9662 reversed these effects. In vitro results indicated that wogonin significantly decreased the secretion of IL-6, IL-1β and tumour necrosis factor-α in Ana-1 and RAW264.7 cells, which was suppressed by transfection of PPARγ small interfering RNA and GW9662 treatment. Moreover, wogonin activated PPARγ, induced PPARγ-mediated attenuation of the nuclear translocation and the DNA-binding activity of nuclear factor-κB in vivo and in vitro. In conclusion, all of these results showed that wogonin may serve as a promising agent for the attenuation of ALI-associated inflammation and pathology by regulating the PPARγ-involved nuclear factor-κB pathway.

Fluoxetine inhibits hyperresponsive lamina propria mononuclear cells and bone marrow-derived dendritic cells, and ameliorates chronic colitis in IL-10-deficient mice.

We aimed to study the effect of fluoxetine on chronic colitis and its anti-inflammatory mechanism in interleukin-10-deficient (IL-10(-/-)) mice. IL-10(-/-) mice were administered with either the vehicle or one of the two dosages of fluoxetine (1 or 5 mg/kg) by oral gavage daily for 2 weeks. Lamina propria mononuclear cells (LPMCs) were isolated from IL-10(-/-) mice treated with or without fluoxetine, and cytokine expression was measured. Bone marrow-derived dendritic cells (BMDCs) isolated from IL-10(-/-), and wild-type mice were pretreated with fluoxetine and then stimulated with lipopolysaccharide. IL-12p40 and tumor necrosis factor (TNF)-α gene expression was determined by real-time PCR. DNA-binding activity of nuclear factor-κB (NF-κB) was evaluated by electrophoretic mobility shift assay. Fluoxetine significantly reduced intestinal inflammation as assessed by body weight, colon length, and histopathological grading, and it inhibited the level of TNF-α gene expression in IL-10(-/-) mice. LPMCs were isolated from IL-10(-/-) mice, and cellular expression of IL-12p40 and interferon-γ was suppressed by fluoxetine treatment. Fluoxetine significantly inhibited IL-12p40 and TNF-α gene expression and protein secretion in BMDCs from IL-10(-/-) mice. DNA-binding activity of NF-κB was suppressed by fluoxetine pretreatment. These results indicate that fluoxetine ameliorates intestinal inflammation in IL-10(-/-) mice and inhibits hyperresponsive LPMCs and BMDCs, which suggests that fluoxetine could be a potential therapeutic agent for inflammatory bowel disease.

The influence of dietary taurine and reduced housing density on hepatic functions in laying hens

To investigate the influence of dietary taurine and reduced housing density on hepatic functions in laying hens, green-shell laying hens were randomly assigned to 3 groups: a free-range group, a caged group with low-density, and a caged group with high-density. Each group was further divided into the control (C) and taurine-treatment (T) groups. All the test birds were fed the same basic diet, except that the T groups were supplemented with 0.1% taurine. After 15 d, sera and liver were aseptically collected. The results show that dietary taurine supplementation and reduced housing density significantly attenuated physiopathological changes in the liver. When compared with the free-range group, serum alanine aminotransterase and aspartate aminotransterase in the caged hens were significantly higher and were deceased by taurine (P < 0.05). Serum inducible nitric oxide synthase activity in caged hens was higher than that in free-range hens, and taurine reduced serum inducible nitric oxide synthase activities in the low-density group (P < 0.05). Nuclear factor-κB DNA-binding activity increased significantly in the high-density housing group when compared with the other 2 housing patterns and was decreased by taurine (P < 0.05). Taurine reduced the expression of tumor necrosis factor-α mRNA in all 3 rearing patterns, IL-4 mRNA expression in the high-density group, and IL-10 in the low-density group (P < 0.05). Malondialdehyde levels decreased in serum and liver from T groups and serum total antioxidation capability levels increased significantly (P < 0.05) in the low-density group. Dietary taurine supplementation decreased acetyl-CoA and sterol regulatory element-binding protein-1c mRNA expression in the high-density groups (P < 0.05). Taurine significantly increased lipoprotein lipase mRNA expression in the high-density group and peroxisome proliferator receptor mRNA expression both in the low- and high-density groups (P < 0.05). Taurine supplementation reduced total cholesterol levels in the low- and high-density groups, decreased triglyceride and low-density lipoprotein cholesterol levels in high-density groups, and increased high-density lipoprotein cholesterol levels in all 3 rearing patterns (P < 0.05). Our data demonstrate that dietary taurine and reduced housing density offer significant protection from hepatic damage in laying hens.

Potential chemoprevention of LPS-stimulated nitric oxide and prostaglandin E2 production by α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranosyl-3-indolecarbonate in BV2 microglial cells through suppression of the ROS/PI3K/Akt/NF-κB pathway

α-l-Rhamnopyranosyl-(1→6)-β-d-glucopyranosyl-3-indolecarbonate (RG3I) is a chemical constituent isolated from the commonly used Asian traditional medicinal plant, Clematis mandshurica; however, no studies have been reported on its anti-inflammatory properties. In the present study, we found that RG3I attenuates the lipopolysaccharide (LPS)-induced DNA-binding activity of nuclear factor-κB (NF-κB) via the dephosphorylation of PI3K/Akt in BV2 microglial cells, leading to a suppression of nitric oxide (NO) and prostaglandin E2 (PGE2) production, along with that of their regulatory genes, inducible NO synthase (iNOS) and cyclooxygenase-2 (Cox-2). Further, the PI3K/Akt inhibitor, LY294002 diminished the expression of LPS-stimulated iNOS and COX-2 genes by suppressing NF-κB activity. Moreover, RG3I significantly inhibited LPS-induced reactive oxygen species (ROS) generation similar to the ROS inhibitors, N-acetylcysteine (NAC) and glutathione (GSH). Notably, NAC and GSH abolished the LPS-induced expression of iNOS and Cox-2 in BV2 microglial cells by inhibiting NF-κB activity. Taken together, our data indicate that RG3I suppresses the production of proinflammatory mediators such as NO and PGE2 as well as their regulatory genes in LPS-stimulated BV2 microglial cells by inhibiting the PI3K/Akt- and ROS-dependent NF-κB signaling pathway, suggesting that RG3I may be a good candidate to regulate LPS-induced inflammatory response.

Anti-inflammatory effects of β-hydroxyisovalerylshikonin in BV2 microglia are mediated through suppression of the PI3K/Akt/NF-kB pathway and activation of the Nrf2/HO-1 pathway

In the present study, we investigated whether β-hydroxyisovalerylshikonin (β-HIVS) affects the production of proinflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2) in BV2 microglial cells. Our data showed that β-HIVS inhibited secretion of NO and PGE2 and downregulated expression of their main regulatory genes, inducible NO synthesis (iNOS) and cyclooxygenase-2 (COX-2). β-HIVS also reduced the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) by suppressing nuclear translocation of the NF-κB subunits and inhibiting the degradation and phosphorylation of IκBα. Furthermore, an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), attenuated LPS-stimulated iNOS and COX-2 expression, suggesting that NF-κB inhibition is a main effector in the expression of iNOS and COX-2. We also found that LPS-induced NF-κB activation is regulated through inhibition of PI3K/Akt phosphorylation in response to β-HIVS. Additionally, β-HIVS caused the induction of heme oxygenase-1 (HO-1) via upregulation of nuclear factor-erythroid 2-related factor 2 (Nrf2), both of which are involved in the secretion of proinflammatory mediators such as NO and PGE2. Taken together, our data indicate that β-HIVS diminishes the proinflammatory mediators NO and PGE2 and the expression of their regulatory genes, iNOS and COX-2, in LPS-stimulated BV2 microglial cells by inhibiting PI3K/Akt-dependent NF-κB activation and inducing Nrf2-mediated HO-1 expression.

3,3'-Diindolylmethane Inhibits Lipopolysaccharide-Induced Microglial Hyperactivation and Attenuates Brain Inflammation

Recent studies have revealed that microglial hyperactivation and neuroinflammation are implicated in development and progression of neurodegenerative diseases. In this study, we examined the beneficial effects of 3,3'-diindolylmethane (DIM) and indole-3-carbinol (I3C), dietary components found in cruciferous vegetables, on brain inflammation. DIM, a major metabolite of I3C, suppressed lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 in BV-2 microglia, but I3C did not. DIM, but not I3C, attenuated DNA-binding activity of nuclear factor-κB (NF-κB) and phosphorylation of inhibitor of κB, suggesting that DIM might inhibit microglial hyperactivation by attenuating inflammatory transcription factor NF-κB. In addition, DIM, but not I3C, protected primary cortical neurons from inflammatory toxicity induced by the conditioned media from LPS-stimulated BV-2 microglia, indicating that DIM might attenuate microglial hyperactivation-mediated neuronal death. In an in vivo model of neuroinflammation, DIM suppressed LPS-induced brain inflammation in mouse hippocampus, as determined by the number of Iba-1-positive cells and the mRNA expression of F4/80. Taken together, these results suggest that DIM may have beneficial potential against brain inflammation and neurodegenerative diseases through the negative regulation of the NF-κB signal pathway in microglia.

Methanol extract of the aerial parts of barley (Hordeum vulgare) suppresses lipopolysaccharide-induced inflammatory responses in vitro and in vivo

Context: Recently, there has been renewed interest in barley (Hordeum vulgare L. Poaceae) as a functional food and for its medicinal properties.Objective: This study examines the anti-inflammatory potential of the active fractions of barley and the mechanisms involved.Materials and methods: The macrophages were exposed to 100 μg/mL of each of the barley extracts in the presence of 1 μg/mL lipopolysaccharide (LPS) and after 24 or 48 h of incubation, cells or culture supernatants were analyzed by various assays. The anti-inflammatory potential of barley fractions was also investigated using the LPS-injected septic mouse model. The active constituents in the fractions were identified using gas chromatography-mass spectrometry (GC-MS).Results: The active fractions, named F4, F7, F9 and F12, inhibited almost completely the LPS-induced production of nitric oxide (NO) and inducible NO synthase. Pre-treatment with these fractions at 100 μg/mL diminished the tumor necrosis factor-α (TNF-α) levels to 19.8, 3.5, 1.2 and 1.7 ng/mL, respectively, compared to LPS treatment alone (41.5 ng/mL). These fractions at 100 μg/mL also suppressed apparently the secretion of interleukin (IL)-6 and IL-1β and the DNA-binding activity of nuclear factor-κB in LPS-stimulated cells. Mice injected intraperitoneally with LPS (30 mg/kg BW) showed 20% survival at 48 h after injection, whereas oral administration of the fractions improved the survival rates to 80%. GC-MS analysis revealed the presence of the derivatives of benzoic and cinnamic acids and fatty acids in the fractions.Discussion and conclusion: The aerial parts of barley are useful as functional food to prevent acute inflammatory responses.

Isorhamnetin inhibits Prevotella intermedia lipopolysaccharide‐induced production of interleukin‐6 in murine macrophages via anti‐inflammatory heme oxygenase‐1 induction and inhibition of nuclear factor‐κB and signal transducer and activator of transcription 1 activation

Interleukin-6 (IL-6) is a key proinflammatory cytokine that has been considered to be important in the pathogenesis of periodontal disease. Therefore, host-modulatory agents directed at inhibiting IL-6 appear to be beneficial in terms of attenuating periodontal disease progression and potentially improving disease susceptibility. In the current study, we investigated the effect of the flavonoid isorhamnetin on the production of IL-6 in murine macrophages stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in inflammatory periodontal disease, and its mechanisms of action. Lipopolysaccharide from P.intermedia ATCC 25611 was isolated using the standard hot phenol-water method. Culture supernatants were collected and assayed for IL-6. We used real-time PCR to quantify IL-6 and heme oxygenase-1 (HO-1) mRNA expression. The expression of HO-1 protein and the levels of signaling proteins were monitored using immunoblot analyses. The DNA-binding activity of nuclear factor-κB (NF-κB) was analyzed using ELISA-based assay kits. Isorhamnetin significantly down-regulated P.intermedia LPS-induced production of IL-6 as well as its mRNA expression in RAW264.7 cells. Isorhamnetin up-regulated the expression of HO-1 at both gene transcription and translation levels in cells stimulated with P.intermedia LPS. In addition, inhibition of HO-1 activity by tin protoporphyrin IX blocked the inhibitory effect of isorhamnetin on IL-6 production. Isorhamnetin failed to prevent LPS from activating either c-Jun N-terminal kinase or p38 pathways. Isorhamnetin did not inhibit NF-κB transcriptional activity at the level of inhibitory κB-α degradation. Isorhamnetin suppressed NF-κB signaling through inhibition of nuclear translocation and DNA binding activity of NF-κB p50 subunit and attenuated signal transducer and activator of transcription 1 signaling. Although further research is required to clarify the detailed mechanism of action, we propose that isorhamnetin may contribute to blockade of the host-destructive processes mediated by IL-6 and could be a highly efficient modulator of the host response in the treatment of inflammatory periodontal disease. Further research in animal models of periodontitis is required to better evaluate, the potential of isorhamnetin as a novel agent for treating periodontal disease.

Paclitaxel Ameliorates Lipopolysaccharide-Induced Kidney Injury by Binding Myeloid Differentiation Protein-2 to Block Toll-Like Receptor 4–Mediated Nuclear Factor-κB Activation and Cytokine Production

Recent studies suggest that paclitaxel, an anticancer agent, may modulate the injury and inflammatory responses in normal tissues. However, the underlying mechanism is not fully understood. Here we have examined the effect of paclitaxel on lipopolysaccharide (LPS)-induced acute kidney injury (AKI) in mice and further studied the mechanism. At relatively low doses, paclitaxel protected against LPS-induced AKI and improved animal survival. The beneficial effects of paclitaxel were accompanied by the downregulation of tumor necrosis factor-α, interleukin-1, and interleukin-6 production. In cultured renal tubular HK-2 cells, paclitaxel decreased the DNA-binding activity of nuclear factor-κB (NF-κB) during LPS treatment, inhibited the degradation of the inhibitor of κB-α, and blocked the expression and activation of NF-κB p65. At the upstream level, paclitaxel reduced LPS-induced association of myeloid differentiation protein-2 (MD-2) with Toll-like receptor 4 (TLR4). In an in vitro assay, paclitaxel was shown to directly bind recombinant MD-2. The inhibitory effect of paclitaxel on NF-κB activation and cytokine expression disappeared in MD-2 knockdown cells, indicating that paclitaxel acts through MD-2. Collectively, these results suggest that paclitaxel may bind MD-2 to block MD-2/TLR4 association during LPS treatment, resulting in the suppression of NF-κB activation and inhibition of proinflammatory cytokine production.

Water extract of processed Hydrangea macrophylla (Thunb.) Ser. leaf attenuates the expression of pro-inflammatory mediators by suppressing Akt-mediated NF-κB activation

Although Hydrangea macrophylla is native to Northeast Asia and widely cultivated in many parts of the world, no studies on its anti-inflammatory effects have been reported. In this study, we evaluated the anti-inflammatory effect of a water extract of processed H. macrophylla leaf (WH) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. WH inhibited the expression of LPS-stimulated pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α), as well as their regulatory genes inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α without any accompanying cytotoxicity. Moreover, WH significantly suppressed the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB), as well as the nuclear translocation of the NF-κB subunits, p65 and p50 by suppressing of IκBα phosphorylation and degradation. WH also increased Akt dephosphorylation, leading to the suppression of the DNA-binding activity of NF-κB in LPS-stimulated RAW264.7 macrophage cells. Our results indicate that WH downregulates the expression of pro-inflammatory mediators such as NO, PGE2, and TNF-α by suppressing the Akt-mediated NF-κB activity in LPS-stimulated RAW264.7 macrophage cells.