Esculentoside B inhibits inflammatory response through JNK and downstream NF-κB signaling pathway in LPS-triggered murine macrophage RAW 264.7 cells

Natural compound esculentoside B (EsB), (2S,4aR,6aR,6aS,6bR,8aR,9R,10R,11S,12aR,14bS)-11-hydroxy-9-(hydroxymethyl)-2 methoxycarbonyl-2,6a,6b,9,12a-pentamethyl-10-[(2S,3R,4S,5R)-3,4,5-trihydroxyoxan-2-yl]oxy-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid with molecular weight of 664.833, isolated from roots of Phytolacca acinosa Roxb has been widely used as a constituent of traditional Chinese medicine (TCM). However, the anti-inflammatory capacity of EsB has not been reported yet. Therefore, the objective of this study was to investigate anti-inflammatory activities of EsB in LPS-treated macrophage RAW 264.7 cells. EsB could inhibit nitric oxide (NO) production. EsB also suppressed gene and protein expression levels of inducible isoform of NO synthase (NOS) and cyclooxygenase-2 in a dose-dependent manner. In addition, EsB decreased gene expression and protein secretion levels of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6. EsB remarkably suppressed nuclear translocation of nuclear factor kappa-B (NF-κB) from cytosolic space. Phosphorylation of IκB was also inhibited by EsB. Moreover, EsB specifically down-regulated phospho-c-Jun N-terminal kinase (p-JNK), but not p-p38 or phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2). Taken together, these results suggest that EsB has inhibitory effect on inflammatory response by inactivating NF-κB and p-JNK. It could be used as a new modulatory drug for effective treatment of inflammation-related diseases.     

IFN-τ inhibits S. aureus-induced inflammation by suppressing the activation of NF-κB and MAPKs in RAW 264.7 cells and mice with pneumonia

Staphylococcus aureus (S. aureus), a significant cause of pneumonia, leads to severe inflammation. Few effective treatments or drugs have been reported for S. aureus infection. Interferon tau (IFN-τ) is a type I interferon with low cellular toxicity even at high doses. Previous studies have reported that IFN-τ could significantly mitigate tissue inflammation; however, IFN-τ treatment in S. aureus-induced pneumonia has not been well reported. Thus, the aim of this study was to identify the anti-inflammatory mechanism of IFN-τ in S. aureus-induced pneumonia in mice. A S. aureus-induced pneumonia model and RAW 264.7 cells were used in this research. The histopathological as well as lung wet to dry ratio (W/D) and myeloperoxidase (MPO) activity results showed that IFN-τ could protect the lung from S. aureus damage. In addition, ELISA and qPCR revealed that IFN-τ treatment led to a decreased expression of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in both the cells and mouse model, but IL-10 was increased. TLR2, which is involved in the response during S. aureus infection, was also down-regulated by IFN-τ treatment and directly affected NF-κB and MAPK pathway activation. Then, we examined the phosphorylation of IκBα, NF-κB p65 and MAPKs by western blotting, and the results displayed that the phosphorylation of IκBα, NF-κB p65 and MAPKs was inhibited upon IFN-τ treatment in both the cells and mouse model. These findings indicate that IFN-τ has anti-inflammatory properties in vitro and in vivo through the inhibition of NF-κB and MAPK activation, suggesting that IFN-τ may have potential as a therapeutic agent against S. aureus-induced inflammatory diseases.     

Theophylline inhibits NF-κB activation and IκBα degradation in human pulmonary epithelial cells

Previous studies demonstrated that theophylline modulates NF-kappaB activation in mast cells and pulmonary epithelial cells. We examined whether or not this modulation of NF-kappaB activation by theophylline is due to inhibition of the degradation of the IKBalpha protein, which suppresses NF-kappaB activation. TNF-alpha-induced NF-kappaB activation in a human pulmonary epithelial cell line (A549) was evaluated by Western blotting and a chloramphenicol acetyltransferase (CAT) assay. Expression of the IkappaBalpha protein was evaluated by Western blotting. Western blotting of nuclear extracts of A549 cells demonstrated that theophylline suppresses NF-kappaB-p65 nuclear translocation. The CAT assay indicated that NF-kappaB-dependent reporter gene expression is inhibited in A549 cells pretreated with theophylline. Western blotting of cytoplasmic extracts of A549 cells revealed that this inhibition was linked to theophylline-induced protection of expression of the IkappaBalpha protein. Moreover, theophylline inhibited interleukin-6 production induced by TNF-alpha in A549 cells. These findings are consistent with the idea that theophylline suppresses the production of proinflammatory cytokines via inhibition of NF-kappaB activation through protection of the IkappaBalpha protein.     

Rheosmin,a naturally occurring phenolic compound inhibits LPS-induced iNOS and COX-2 expression in RAW264.7 cells by blocking NF-κB activation pathway

Inflammation is part of the host defense mechanism against harmful matters and injury; however, aberrant inflammation is associated to the development of chronic disease such as cancer. Raspberry ketone is a natural phenolic compound. It is used in perfumery, in cosmetics, and as a food additive to impart a fruity odor. In this study, we evaluated whether rheosmin, a phenolic compound isolated from pine needles regulates the expression of iNOS and COX-2 protein in LPS-stimulated RAW264.7 cells. Rheosmin dose-dependently inhibited NO and PGE₂ production and also blocked LPS-induced iNOS and COX-2 expression. Rheosmin potently inhibited the translocation of NF-κB p65 into the nucleus by IκB degradation following IκB-α phosphorylation. This result shows that rheosmin inhibits NF-κB activation. In conclusion, our results suggest that rheosmin inhibits LPS-induced iNOS and COX-2 expression in RAW264.7 cells by blocking NF-κB activation pathway.     

Desoxyrhapontigenin,a potent anti-inflammatory phytochemical,inhibits LPS-induced inflammatory responses via suppressing NF-κB and MAPK pathways in RAW 264.7 cells

This study investigates the anti-inflammatory effects of a stilbene compound, desoxyrhapontigenin, which was isolated from Rheum undulatum. To determine the anti-inflammatory effects of this compound, lipopolysaccharide (LPS)-induced RAW 264.7 macrophages were treated with different concentrations of six stilbene derivatives. The results indicated that compared with other stilbene compounds, desoxyrhapontigenin (at 10, 30 and 50 μM concentrations) significantly inhibited nitric oxide (NO) production, nuclear factor kappa B (NF-κB) activation, the protein expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression. Therefore, the anti-inflammatory mechanism of desoxyrhapontigenin was investigated in detail. The results of this investigation demonstrated that desoxyrhapontigenin suppressed not only LPS-stimulated pro-inflammatory cytokine secretions, including the secretions of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), but also PGE₂ release. As assayed by electrophoretic mobility shift assays (EMSAs), desoxyrhapontigenin also produced the dose-dependent inhibition of the LPS-induced activation of NF-κB and AP-1. Moreover, desoxyrhapontigenin inhibited the protein expression of myeloid differentiation primary response gene 88 (MyD88), IκB kinase (IKK) phosphorylation and the degradation of IκBα. Activations of p-JNK1 and p-Akt were also significantly inhibited, and phosphorylation of p38 and ERK was down-regulated. A further study revealed that desoxyrhapontigenin (5 and 25 mg/kg, i.p.) reduced paw swelling in carrageenan-induced acute inflammation model in vivo. On the whole, these results indicate that desoxyrhapontigenin showed anti-inflammatory properties by the inhibition of iNOS and COX-2 expression via the down-regulation of the MAPK signaling pathways and the inhibition of NF-κB and Akt activation.     

Luteolin and chicoric acid synergistically inhibited inflammatory responses via inactivation of PI3K-Akt pathway and impairment of NF-κB translocation in LPS stimulated RAW 264.7 cells

Synergistic anti-inflammatory effects of luteolin and chicoric acid, two abundant constituents of the common dandelion (Taraxacum officinale Weber), were investigated in lipopolysaccharide (LPS) stimulated RAW 264.7 cells. Co-treatment with luteolin and chicoric acid synergistically reduced cellular concentrations of nitric oxide (NO) and prostaglandin E2 (PGE2) and also inhibited expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In addition, co-treatment reduced the levels of proinflammatory cytokines, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β. Both luteolin and chicoric acid suppressed oxidative stress, but they did not exhibit any synergistic activity. Luteolin and chicoric acid co-treatment inhibited phosphorylation of NF-κB and Akt, but had no effect on extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38. This anti-inflammatory signaling cascade coincides with that affected by luteolin treatment alone. These results suggest that luteolin plays a central role in ameliorating LPS-induced inflammatory cascades via inactivation of the NF-κB and Akt pathways, and that chicoric acid strengthens the anti-inflammatory activity of luteolin through NF-κB attenuation.     

Neocryptotanshinone inhibits lipopolysaccharide-induced inflammation in RAW264.7 macrophages by suppression of NF-κB and iNOS signaling pathways

Neocryptotanshinone (NCTS) is a natural product isolated from traditional Chinese herb Salvia miltiorrhiza Bunge. In this study, we investigated its anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW264.7) cells. MTT results showed that NCTS partly reversed LPS-induced cytotoxicity. Real-time PCR results showed that NCTS suppressed LPS-induced mRNA expression of inflammatory cytokines, including tumor necrosis factor α (TNFα), interleukin-6 (IL-6) and interleukin-1β (IL-1β). Moreover, NCTS could decrease LPS-induced nitric oxide (NO) production. Western blotting results showed that NCTS could down-regulate LPS-induced expression of inducible nitric oxide synthase (iNOS), p-IκBα, p-IKKβ and p-NF-κB p65 without affecting cyclooxygenase-2 (COX-2). In addition, NCTS inhibited LPS-induced p-NF-κB p65 nuclear translocation. In conclusion, these data demonstrated that NCTS showed anti-inflammatory effect by suppression of NF-κB and iNOS signaling pathways.KEY WORDS: Neocryptotanshinone, Inflammation, NF-κB, Inducible nitric oxide synthase     

Tetrandrine down-regulates ERK/NF-κB signaling and inhibits activation of mesangial cells

Objectives

Tetrandrine (TET), a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra S. Moore of the Menispermaceae, possesses anti-inflammatory activity. We examined the effect of tetrandrine on interleukin-1β (IL-1β)-provoked inflammatory response in mesangial cells.

Materials and methods

Primary rat mesangial cells (PRMCs) were treated with IL-1β to induce inflammation to resemble glomerulonephritis. Cell viability, morphology and NO production were evaluated. Western blotting was applied for expression of matrix metalloproteinase-9 (MMP-9), inducible NO synthase (iNOS), extracellular signal-regulated kinase (ERK) and NF-κB-related molecules. Electrophoretic mobility shift assay was performed to examine the DNA-binding activity of NF-κB.

Results

TET, at concentrations up to 10 μg/ml, had no significant effect on viability of PRMCs. At non-toxic concentrations, TET inhibited expression of phosphorylated ERK as well as phosphorylated IKK, enhanced degradation of IκBα and reduced the DNA-binding activity of NF-κB in IL-1β-primed PRMCs, suggesting an inhibitory effect on ERK/NF-κB signaling. TET attenuated the IL-1β-provoked expression of iNOS and release of NO. Moreover, both the protein expression and gelatinase activity of MMP-9, but not MMP-2, were markedly suppressed by TET.

Significance

TET down-regulated ERK/NF-κB signaling and inhibited the expression of inflammatory mediators NO and MMP-9. Since these mediators appear to activate mesangial cells, TET may play an important role in prevention of glomerulonephritis.     

Anti-inflammatory effects of sophocarpine in LPS-induced RAW 264.7 cells via NF-κB and MAPKs signaling pathways

Sophocarpine, a tetracyclic quinolizidine alkaloid, is one of the most abundant active ingredients in Sophora alopecuroides L. Our previous studies have showed that sophocarpine exerts anti-inflammatory activity in animal models. In the present study, anti-inflammatory mechanisms of sophocarpine were investigated in lipopolysaccharide (LPS)-induced responses in RAW 264.7 cells. Furthermore, the cytotoxicity of sophocarpine was tested. The results indicated that sophocarpine could increase the LDH level and inhibit cell viability up to 800μg/ml, and which was far higher than that of the plasma concentration of sophocarpine in clinical effective dosage. The results also demonstrated that sophocarpine (50 and 100μg/ml) suppressed LPS-stimulated NO production and pro-inflammatory cytokines secretion, including tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). These were associated with the decrease of the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, sophocarpine inhibited LPS-mediated nuclear factor-κB (NF-κB) activation via the prevention of inhibitor κB (IκB) phosphorylation. Sophocarpine had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2), whereas it attenuated the phosphorylation of p38 mitogen-activated protein (MAP) kinase and c-Jun NH(2)-terminal kinase (JNK). Our data suggested that sophocarpine exerted anti-inflammatory activity in vitro, and it might attribute to the inhibition of iNOS and COX-2 expressions via down-regulation of the JNK and p38 MAP kinase signal pathways and inhibition of NF-κB activation.     

Isotrifoliol inhibits pro-inflammatory mediators by suppression of TLR/NF-κB and TLR/MAPK signaling in LPS-induced RAW264.7 cells

Soybeans, produced by Glycine max (L.) Merr., contain high levels of isoflavones, such as genistein and daidzein. However, soy leaves contain more diverse and abundant flavonol glycosides and coumestans, as compared to the soybean. This study investigated the anti-inflammatory effects of the major coumestans present in soy leaf (coumestrol, isotrifoliol, and phaseol) in lipopolysaccharide (LPS)-induced RAW264.7 cells. Coumestans significantly reduced LPS-induced nitric oxide (NO), prostaglandin E2 (PGE₂), and reactive oxygen species (ROS) production; isotrifoliol had the most potent anti-inflammatory activity. Isotrifoliol reduced LPS-mediated induction of mRNA expression of inducible nitric-oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-1β, IL-6, tumor necrosis factor alpha (TNFα), and chemokines, such as chemokine (C-C motif) ligand (CCL) 2, CCL3, and CCL4. Isotrifoliol prevented NF-κB p65 subunit activation by reducing the phosphorylation and degradation of the inhibitor of NF-κB. And isotrifoliol significantly suppressed phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). Furthermore, isotrifoliol suppressed LPS-induced Toll-like Receptor (TLR) signaling pathway, including mRNA expression of TNF receptor associated factor 6, transforming growth factor beta-activated kinase 1 (TAK1), TAK1 binding protein 2 (TAB2), and TAB3. These results demonstrate that isotrifoliol exerts an anti-inflammatory effect by suppressing the expression of inflammatory mediators via inhibition of TLR/NF-κB and TLR/MAPK signaling in LPS-induced RAW264.7 macrophages. Therefore, isotrifoliol can be used as an anti-inflammatory agent, and coumestan-rich soy leaf extracts may provide a useful dietary supplement.     

Vitisin A suppresses LPS-induced NO production by inhibiting ERK,p38, and NF-κB activation in RAW 264.7 cells

Vitisin A, a resveratrol tetramer isolated from Vitis vinifera roots, exhibits antioxidative, anticancer, antiapoptotic, and anti-inflammatory effects. It also inhibits nitric oxide (NO) production. Here, we examined the mechanism by which vitisin A inhibits NO production in lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells. Vitisin A dose dependently inhibited LPS-induced NO production and inducible NO synthase (iNOS) expression. In contrast, the production of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) was not altered by vitisin A. To investigate the signaling pathway for NO inhibition by vitisin A, we examined nuclear factor-κB (NF-κB) activation in the mitogen-activated protein kinase (MAPK) pathway, an inflammation-induced signal pathway in RAW 264.7 cells. Vitisin A inhibited LPS-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 phosphorylation and suppressed LPS-induced NF-κB activation in RAW 264.7 cells. This suggests that vitisin A decreased NO production via downregulation of ERK1/2 and p38 and the NF-κB signal pathway in RAW 264.7 cells.     

Berberine suppresses LPS-induced inflammation through modulating Sirt1/NF-κB signaling pathway in RAW264.7 cells

Chronic inflammation is a major contributing factor in the pathogenesis of many diseases. Natural product berberine (BBR) exhibits potent anti-inflammatory effect in vitro and in vivo, while the underlying mechanisms remain elusive. Sirt1, a NAD⁺-dependent protein deacetylase, was recently found to play an important role in modulating the development and progression of inflammation. Thus, we speculate that Sirt1 might mediate the inhibitory effect of BBR on inflammation. In LPS-stimulated RAW264.7 macrophages, BBR treatment significantly downregulated the expression of proinflammatory cytokines such as MCP-1, IL-6 and TNF-α. Importantly, BBR potently reversed LPS-induced down-regulation of Sirt1. Consistently, the inhibitory effects of BBR on proinflammatory cytokines expression was largely abrogated by Sirt1 inhibition either by EX527, a Sirt1 inhibitor or Sirt1 siRNA. Further mechanistic studies revealed that BBR-induced inhibition of NF-κB is Sirt1-dependent, as either pharmacologically or genetically inactivating Sirt1 enhanced the IκΒα degradation, IKK phosphorylation, NF-κB p65 acetylation and DNA-binding activity. Taken together, our results provide the first evidence that BBR potently suppressed inflammatory responses in macrophages through inhibition of NF-κB signaling via Sirt1-dependent mechanisms.     

Protective effect of (+)–catechin against lipopolysaccharide-induced inflammatory response in RAW 264.7 cells through downregulation of NF-κB and p38 MAPK

Inflammopharmacology - Catechin, a flavonol belonging to the flavonoid group of polyphenols is present in many plant foods. The present study was done to evaluate the effect of catechin on various...     

Glycoprotein isolated from Cudrania tricuspidata Bureau inhibits iNO and COX-2 expression through modulation of NF-κB in LPS-stimulated RAW 264.7cells

Glycoprotein of Cudrania tricuspidata Bureau (CTB glycoprotein) was isolated from CTB fruits which have been used to heal various disorders of the injury and lung as an herbal agent in Korea since long time ago. The CTB glycoprotein was identified to have a molecular weight of 75kDa and consists of carbohydrate (72.5%) and protein moiety (27.5%). To know inhibitory ability of CTB glycoprotein for inflammation mediated by reactive oxygen radicals, firstly we tested about anti-oxidative activity (DPPH, superoxide anion, and hydroxyl radicals) in cell-free system, and then evaluated changes of inflammation-related signals [intracellular reactive oxygen species (iROS), nitric oxide (NO), nuclear factor-kappa B (NF-κB), COX-2, and iNOS] in the LPS (1μg/ml)-treated RAW 264.7cells. The results in this study showed that CTB glycoprotein (100μg/ml) has a strong scavenging activity against DPPH, superoxide anion, and hydroxyl radicals without any pro-oxidant activity in vitro. In the inflammation-related signals, expression of iROS, NO, NF-κB, COX-2, and iNOS were inhibited by treatment with CTB glycoprotein (50μg/ml) in the presence of LPS (1μg/ml). Taken together, our data obtained from these experiments indicated that CTB glycoprotein suppresses expression of the inflammatory-related proteins (iNOS and COX-2) through regulation of NF-κB. Thus, we speculate that CTB glycoprotein may have therapeutic potential for inflammation-associated disorders.     

Total glucosides of paeony (TGP) inhibits the production of inflammatory cytokines in oral lichen planus by suppressing the NF-κB signaling pathway

Total glucosides of paeony (TGP) is a bioactive compound extracted from paeony roots and has been widely used to ameliorate inflammation in several autoimmune and inflammatory diseases. However, the anti-inflammatory effect of TGP on oral lichen planus (OLP), a chronic inflammatory oral condition characterized by T-cell infiltration and abnormal epithelial keratinization cycle remains unclear. In this study, we found that TLR4 was highly expressed and activation of the NF-κB signaling pathway was obviously observed in the OLP tissues. Moreover, there was significant higher mRNA expression of inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in OLP keratinocytes than normal oral epithelial keratinocytes. With the help of the cell culture model by stimulating the keratinocyte HaCaT cells with lipopolysaccharides (LPS), we mimicked the local inflammatory environment of OLP. And we further confirmed that TGP could inhibit LPS-induced production of IL-6 and TNF-α in HaCaT cells via a dose-dependent manner. TGP treatment decreased the phosphorylation of IκBα and NF-κB p65 proteins, thus leading to less nuclear translocation of NF-κB p65 in HaCaT cells. Therefore, our data suggested that TGP may be a new potential candidate for the therapy of OLP.     

Mitochondrial division inhibitor (mdivi-1) inhibits proliferation and epithelial-mesenchymal transition via the NF-κB pathway in thyroid cancer cells

Excessively fragmented mitochondria have been reported in thyroid cancer (TC). Mitochondrial division inhibitor (mdivi-1), a putative inhibitor of dynamin-related protein 1 (Drp1), prevents mitochondrial fission and thereby restricts cell proliferation across several types of primary cancer. However, the role of mdivi-1 on TC has not been sufficiently studied. This research is intended to explore the therapeutic effect of mdivi-1 in TC cells. Results demonstrated that highly invasive TC cells displayed excessive mitochondrial fission with more fragmented mitochondria. Treatment with mdivi-1 inhibited mitochondrial fission in 8505C cells as indicated by transmission electron microscope (TEM). It also impaired the proliferation and increased apoptosis in 8505C and K1 cells as shown by plate cloning assay, cell viability assay, and apoptosis assay. Mdivi-1 treatment also attenuated migratory and invasive abilities in 8505C and K1 cells as shown by the transwell assay and the wound healing assay. And we noticed the same inhibition of mdivi-1 in cell migration and cell viability after the knockdown of Drp1 in 8505C cells. This demonstrated that mdivi-1 exerted an anti-tumor effect independently of Drp1 in 8505C cells. Moreover, mdivi-1 treatment reversed epithelial-mesenchymal transition (EMT) by inhibiting the NF-κB pathway in 8505C cells. The present findings demonstrate that mdivi-1 has a therapeutic role in thyroid carcinoma.