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

Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes, including vasodilatation and macrophage‐mediated immunity. Macrophages express inducible NO synthase (iNOS) and produce NO after lipopolysaccharide (LPS) stimulation. Gallotannins are water‐soluble polyphenols with wide‐ranging biological activities. Various chemical structures of gallotannins occurring in medicinal and food plants that are used worldwide showed several remarkable biological and pharmacological activities. In the present study, we examined the inhibitory effects of gallotannin 1,2,3,6‐tetra‐O‐galloyl‐β‐D‐allopyranose (GT24) isolated from Euphorbia jolkini on the LPS‐induced NO production and underlying mechanisms of action. GT24 dose‐dependently decreased LPS‐induced NO production and iNOS expression in J774A.1 macrophages. In addition, GT24 inhibited LPS‐induced activation of nuclear factor (NF)‐κB as indicated by inhibition of degradation of I‐κBα, nuclear translocation of NF‐κB, and NF‐κB dependent gene reporter assay. Our results suggest that GT24 possesses an inhibitory effect on the LPS‐induced inflammatory reaction. Copyright © 2010 John Wiley & Sons, Ltd.     

Nimbolide Inhibits Nuclear Factor‐КB Pathway in Intestinal Epithelial Cells and Macrophages and Alleviates Experimental Colitis in Mice

Nimbolide is a limonoid extracted from neem tree (Azadirachta indica) that has antiinflammatory properties. The effect of nimbolide on the nuclear factor‐kappa B (NF‐κB) pathway in intestinal epithelial cells (IECs), macrophages and in murine colitis models was investigated. The IEC COLO 205, the murine macrophage cell line RAW 264.7, and peritoneal macrophages from interleukin‐10‐deficient (IL‐10^?/?) mice were preconditioned with nimbolide and then stimulated with tumor necrosis factor‐α (TNF‐α) or lipopolysaccharide. Dextran sulfate sodium‐induced acute colitis model and chronic colitis model in IL‐10^?/? mice were used for in vivo experiments. Nimbolide significantly suppressed the expression of inflammatory cytokines (IL‐6, IL‐8, IL‐12, and TNF‐α) and inhibited the phosphorylation of IκBα and the DNA‐binding affinity of NF‐κB in IECs and macrophages. Nimbolide ameliorated weight loss, colon shortening, disease activity index score, and histologic scores in dextran sulfate sodium colitis. It also improved histopathologic scores in the chronic colitis of IL‐10^?/? mice. Staining for phosphorylated IκBα was significantly decreased in the colon tissue after treatment with nimbolide in both models. Nimbolide inhibits NF‐κB signaling in IECs and macrophages and ameliorates experimental colitis in mice. These results suggest nimbolide could be a potentially new treatment for inflammatory bowel disease. Copyright © 2016 John Wiley & Sons, Ltd.     

Synergistic anti‐inflammatory effects of quercetin and catechin via inhibiting activation of TLR4–MyD88‐mediated NF‐κB and MAPK signaling pathways

The synergistic anti‐inflammatory effect of quercetin and catechin was investigated using lipopolysaccharide (LPS)‐stimulated macrophage RAW 264.7 cells. Results showed that the combined treatment of quercetin with catechin synergistically attenuated LPS‐stimulated increase of some proinflammatory molecules, including nitric oxide, tumor necrosis factor α, interleukin‐1β, nitric oxide synthase, and cyclooxygenase‐2. Moreover, it exhibited significantly (p < 0.05) stronger inhibitory effect on nuclear translocation of nuclear factor‐κB (NF‐κB) by suppressing the phosphorylation of NF‐κB p65 and p50 submits and on the phosphorylation of ETS domain‐containing protein and c‐Jun N‐terminal kinase than any of quercetin or catechin alone. Besides, the cotreatment of quercetin with catechin significantly (p < 0.05) restored the impaired expression of toll‐like receptor 4, myeloid differentiation primary response gene 88, and some downstream effectors (IRAK1, TRAF6, and TAK1). These results suggest that quercetin and catechin possessed synergistic anti‐inflammatory effects, which may be attributed to their roles in suppressing the activation of TLR4–MyD88‐mediated NF‐κB and mitogen‐activated protein kinases signaling pathways.     

Myrislignan Attenuates Lipopolysaccharide‐induced Inflammation Reaction in Murine Macrophage Cells Through Inhibition of NF‐κB Signalling Pathway Activation

Myrislignan is a new kind of lignan isolated from Myristica fragrans Houtt. Its antiinflammatory effects have not yet been reported. In the present study, the antiinflammatory effects and the underlying mechanisms of myrislignan in lipopolysaccharide (LPS)‐induced inflammation in murine RAW 264.7 macrophage cells were investigated. Myrislignan significantly inhibited LPS‐induced production of nitric oxide (NO) in a dose‐dependent manner. It inhibited mRNA expression and release of interleukin‐6 (IL‐6) and tumour necrosis factor‐α (TNF‐α). This compound significantly inhibited mRNA and protein expressions of inducible NO synthase (iNOS) and cyclooxygenase‐2 (COX‐2) dose‐dependently in LPS‐stimulated macrophage cells. Further study showed that myrislignan decreased the cytoplasmic loss of inhibitor κB‐α (IκB‐α) protein and the translocation of NF‐κB from cytoplasm to the nucleus. Our results suggest that myrislignan may exert its antiinflammatory effects in LPS‐stimulated macrophages cells by inhibiting the NF‐κB signalling pathway activation. Copyright © 2012 John Wiley & Sons, Ltd.     

A Polyphenol‐rich Pomegranate Fruit Extract Suppresses NF‐κB and IL‐6 Expression by Blocking the Activation of IKKβ and NIK in Primary Human Chondrocytes

Pomegranate fruit extract (PE) rich in polyphenols has been shown to exert chondroprotective effects, but the mechanism is not established. Here, we used an in vitro model of inflammation in osteoarthritis (OA) to investigate the potential of PE to suppress interleukin 1 beta (IL‐1β)‐stimulated expression of inflammatory cytokine IL‐6, generation of reactive oxygen species (ROS) levels, and investigated the mechanism of NF‐κB inhibition by analyzing the activation of the kinases upstream of IκBα in primary human chondrocytes. Total and phosphorylated forms of kinases and expression of IL‐6 were determined at protein and mRNA levels by western immunoblotting and Taqman assay, respectively. Dihydrorhodamine 123 staining estimated ROS generation. Pomegranate fruit extract inhibited the mRNA and protein expression of IL‐6, generation of ROS, and inhibited the IL‐1β‐mediated phosphorylation of inhibitor of nuclear factor kappa‐B kinase subunit beta (IKKβ), expression of IKKβ mRNA, degradation of IκBα, and activation and nuclear translocation of NF‐κB/p65 in human chondrocytes. Importantly, phosphorylation of NF‐κB‐inducing kinase was blocked by PE in IL‐1β‐treated human OA chondrocytes. Taken together, these data suggest that PE exerts the chondroprotective effect(s) by suppressing the production of IL‐6 and ROS levels. Inhibition of NF‐κB activation by PE was blocked via modulation of activation of upstream kinases in human OA chondrocytes. Copyright © 2017 John Wiley & Sons, Ltd.     

Calycosin attenuates MPTP‐induced Parkinson's disease by suppressing the activation of TLR/NF‐κB and MAPK pathways

Parkinson is the second common neurodegenerative disease. The characteristics of Parkinson's disease (PD) are the dopamin neurons loss caused by neuroinflammation responses. C alycosin, an isoflavone phytoestrogen isolated from Astragalus membranaceus, has multiple pharmacological activities, such as anti‐inflammation, anti‐tumor, and neuroprotective effects. However, it is unknown whether calycosin can mitigate PD symptoms. This study aims to explore whether calycosin can alleviate PD symptoms and the underlying mechanisms. PD was induced in mice by 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) injection, and calycosin was given intracerebroventricularly to these mice. A cell model of nerve inflammation was established by BV2 microglia cells injected with lipopolysaccharide (LPS). The motor states were evaluated by stepping, whisker, and cylinder experiments. The states of dopaminergic neurons and microglia were detected by immunostainning of tyrosine hydroxylase and cluster of differentiation molecule 11b (CD11b). The expression levels of inflammatory factors were detected by qPCR. Toll‐like receptor (TLR)/nuclear factor kappa B (NF‐κB) and mitogen‐activated protein kinase (MAPK) pathways were investigated by western blot. We found that calycosin treatment mitigated the behavioral dysfunctions and inflammatory responses in MPTP‐induced PD mice. The TLR/NF‐κB and MAPK pathways in MPTP‐induced PD mice were inhibited by calycosin treatment, which was coincident with experiments in LPS‐induced BV2 cells. Above all, calycosin mitigates PD symptoms through TLR/NF‐κB and MAPK pathways in mice and cell lines.     

Withanolides against TLR4‐Activated Innate Inflammatory Signalling Pathways: A Comparative Computational and Experimental Study

Innate inflammations are dominant causes of poor health and high mortality. The pathogen‐associated molecular pattern and lipopolysaccharide (LPS) are sensed by immune cells through activation of toll‐like receptor 4 leading to mitogen‐activated protein kinases (MAPKs) and NF‐κB activations. Controlled MAPK and Nf‐κB inhibitors have been proposed as potential antiinflammatory drugs. Withania somnifera is an important medicinal herb with known antiinflammatory activity. In this study, the selected Withania somnifera extracts and withanolides were analysed on LPS‐induced macrophages comparatively. Molecular docking analysis revealed withaferin A, withanone and withanolide A as effective withanolides against inflammatory target molecules. In experiments, withaferin A and withanone treatment had prominent suppressions on LPS‐induced expression of pro‐inflammatory cytokines in bone marrow‐derived macrophages. Withaferin A regulated all the major four pathways (MAPKs and NF‐κB) involved in innate inflammations. Similarly among the Withania extracts analysed, the in vitro propagated leaf and field grown root extracts containing high withaferin A content suppressed the inflammatory molecules through NF‐κB and MAPK pathways. Withaferin A was found to be best in suppressing the activated inflammatory pathways among all the analysed withanolides. Therefore, withaferin A and extracts with high withaferin A content can be used as promising drug candidates against innate inflammations. Copyright © 2016 John Wiley & Sons, Ltd.     

Isoforskolin and forskolin attenuate lipopolysaccharide‐induced inflammation through TLR4/MyD88/NF‐κB cascades in human mononuclear leukocytes

The principal active component of isoforskolin (ISOF) is from the plant Coleus forskohlii, native to China, which has attracted much attention for its biological effects. We hypothesize that ISOF and forskolin (FSK) pretreatment attenuates inflammation induced by lipopolysaccharide (LPS) related to toll‐like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor kappa B (NF‐κB) signaling. Mononuclear leukocytes (MLs) from healthy donors' blood samples were separated by using density gradient centrifugation. Protein levels of TLR4, MyD88, and NF‐κB were detected using western blot and inflammatory cytokines interleukin (IL) 1β, IL‐2, IL‐6, IL‐21, IL‐23, tumor necrosis factor (TNF) α, and TNF‐β were tested by enzyme‐linked immunosorbent assay and Quantibody array in MLs. Our results showed that LPS augmented the protein levels of TLR4, MyD88, and NF‐κB in MLs and the production of IL‐1β, IL‐2, IL‐6, IL‐21, IL‐23, TNF‐α, and TNF‐β in supernatants of MLs. Despite treatment with ISOF and FSK prior to LPS, the protein levels of TLR4, MyD88, NF‐κB, IL‐1β, IL‐2, IL‐6, IL‐21, IL‐23, TNF‐α, and TNF‐β in MLs were apparently decreased. roflumilast (RF) and dexamethasone (DM) had a similar effect on MLs with ISOF and FSK. Our results, for the first time, have shown that ISOF and FSK attenuate inflammation in MLs induced by LPS through down‐regulating protein levels of IL‐1β and TNF‐α, in which TLR4/MyD88/NF‐κB signal pathway could be involved.     

Apoptosis Induction by 13‐Acetoxyrolandrolide through the Mitochondrial Intrinsic Pathway

The aim of this study was to evaluate the mechanisms of cytotoxicity of the sesquiterpene lactone 13‐acetoxyrolandrolide, a nuclear factor kappa B (NF‐κB) inhibitor that was previously isolated from Rolandra fruticosa. The effects associated with the inhibition of the NF‐κB pathway included dose‐dependent inhibition of the NF‐κB subunit p65 (RelA) and inhibition of upstream mediators IKKβ and oncogenic Kirsten rat sarcoma (K‐Ras). The inhibitory concentration of 13‐acetoxyrolandrolide on K‐Ras was 7.7 µm . The downstream effects of the inhibition of NF‐κB activation were also investigated in vitro. After 24 h of treatment with 13‐acetoxyrolandrolide, the mitochondrial transmembrane potential was depolarized in human colon cancer (HT‐29) cells. The mitochondrial oxidative phosphorylation was also negatively affected, and reduced levels of nicotinamine adenine dinucleotide phosphate (NAD(P)H) were detected after 2 h of 13‐acetoxyrolandrolide exposure. Furthermore, the expression of the pro‐apoptotic protein caspase‐3 increased in a concentration‐dependent manner. Cell flow cytometry showed that 13‐acetoxyrolandrolide induced cell cycle arrest at G₁, indicating that the treated cells had undergone caspase‐3‐mediated apoptosis, indicating negative effects on cancer cell proliferation. These results suggest that 13‐acetoxyrolandrolide inhibits NF‐κB and K‐Ras and promotes cell death mediated through the mitochondrial apoptotic pathway. Copyright © 2013 John Wiley & Sons, Ltd.     

Nerolidol Protects Against LPS‐induced Acute Kidney Injury via Inhibiting TLR4/NF‐κB Signaling

Acute kidney injury (AKI) is a critical care syndrome, resulting in acute reduction of renal function and up to 22% mortality of hospitalized patients. Nerolidol is a major component in several essential oils that possesses various pharmacological properties. The present study aimed to investigate the potential effect of nerolidol on lipopolysaccharide (LPS)‐induced AKI. Nerolidol dose‐dependently reduced the pathological injuries of kidney induced by LPS in rats. Nerolidol significantly decreased the levels of blood urea nitrogen and creatinine in LPS‐treated rats in a dose‐dependent manner. In addition, nerolidol inhibited LPS‐induced decrease of cell viability in NRK‐52E rat proximal tubular cells, which effect was concentration dependent. Nerolidol notably inhibited the increase of TNFα and IL‐1β in LPS‐treated rats and the mRNA expression of TNFα and IL‐1β in LPS‐treated NRK‐52E cells. Nerolidol suppressed the increase of toll‐like receptor 4 (TLR4) expression, phosphorylation and nuclear translocation of p65 NF‐κB in kidneys of LPS‐treated rats and LPS‐treated NRK‐52E cells. Overexpression of TLR4 and p65 NF‐κB significantly suppressed nerolidol‐induced inhibition of TNFα and IL‐1β expression and increase of cell viability in LPS‐treated cells. In summary, we found that nerolidol played a critical anti‐inflammatory effects through inhibition of TLR4/NF‐κB signaling and protected against LPS‐induced AKI. Copyright © 2017 John Wiley & Sons, Ltd.     

Toll‐like receptor 4‐mediated immunoregulation by the aqueous extract of Mori Fructus

The aqueous extract of Mori Fructus (MF) exerts a change of phenotype and a cytoprotective effect in macrophages. The present study was carried out to investigate the immunomodulating activity of MF on the expression of nitric oxide (NO), tumor necrosis factor alpha (TNF‐α), co‐stimulatory molecules and also interferon‐gamma (IFN‐γ) in macrophages and splenocytes. Toll‐like receptor 4 (TLR4) is a promising molecular target for immune‐modulating drugs. It was hypothesized that one possible upstream signaling pathway leading to immunoregulation of MF may be mediated by TLRs. Multiple signaling molecules (NF‐κB, ERK1/2, p38 and JNK) of the TLR4 signaling pathway were also detected. It was found that MF increased NO production and TNF‐α secretion in RAW 264.7 and peritoneal macrophages, co‐stimulatory molecules expression in peritoneal macrophages and IFN‐γ expression in splenocytes. Further studies indicated that MF could significantly induce the phosphorylation of signal molecules of MAPKs and the degradation of IκBα which finally led to the activation and nuclear translocation of nuclear factor‐κB (NF‐κB) for the target gene expression. All those notions disclosed that the aqueous extract MF is a new TLR4 activator, which induces a Th1 immune response as a consequence of induction of cytokines secretion, especially TNF‐α and IFN‐γ. Copyright © 2009 John Wiley & Sons, Ltd.     

Down‐regulatory effect of usnic acid on nuclear factor‐κB‐dependent tumor necrosis factor‐α and inducible nitric oxide synthase expression in lipopolysaccharide‐stimulated macrophages RAW 264.7

The purpose of this study was to investigate the molecular mechanisms that are responsible for the antiinflammatory effect of usnic acid (UA). UA is one of the most common and abundant lichen metabolites. The present study examined the effects of UA on the tumor necrosis factor‐α (TNF‐α) and nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW264.7 macrophages and the underlying molecular mechanisms. UA decreased the TNF‐α level in LPS‐stimulated RAW264.7 macrophages in dose‐dependent manner, the IC₅₀ value was 12.8 µM. RT‐PCR analysis indicated that it inhibited TNF‐α mRNA expression. Furthermore, it inhibited NO production in LPS‐activated RAW264.7 macrophages, the IC₅₀ value was 4.7 µM. Western blot analysis showed that UA attenuated LPS‐induced synthesis of iNOS protein and nuclear translocation of NF‐κB p65 in the macrophages, in parallel. UA also inhibited LPS‐mediated I‐κBα degradation. Taken together, this suggests that UA has an antiinflammatory effect by inhibiting TNF‐α and iNOS expression, possibly through suppression of nuclear translocation of NF‐κB p65 and I‐κBα degradation. Copyright © 2008 John Wiley & Sons, Ltd.     

Anti‐inflammatory Activities of Gouania leptostachya Methanol Extract and its Constituent Resveratrol

Gouania leptostachya DC. var. tonkinensis Pitard. Rhamnaceae is a traditional medicinal plant used in Thailand for treating various inflammatory symptoms. However, no systematic studies have been performed concerning the anti‐inflammatory effects or molecular mechanisms of this plant. The immunopharmacological activities of a methanol extract from the leaves and twigs of G. leptostachya (Gl‐ME) were elucidated based on the gastritis symptoms of mice treated with HCl/EtOH and the inflammatory responses, such as nitric oxide (NO) release and prostaglandin E₂ (PGE₂) production, from RAW264.7 cells and peritoneal macrophages. Moreover, inhibitory target molecules were also assessed. Gl‐ME dose‐dependently diminished the secretion of NO and PGE₂ from LPS‐stimulated RAW264.7 cells and peritoneal macrophages. The gastritis lesions of HCl/EtOH‐treated mice were also attenuated after Gl‐ME treatment. The extract (50 and 300 µg/mL) clearly reduced mRNA expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)‐2, nuclear translocation of p65/nuclear factor (NF)‐κB, phosphorylation of p65‐activating upstream enzymes, such as protein kinase B (AKT), inhibitor of κBα kinase (IKK), and inhibitor of κB (IκBα), and the enzymatic activity of Src. By HPLC analysis, one of the major components in the extract was revealed as resveratrol with NO and Src inhibitory activities. Moreover, this compound suppressed NO production and HCl/EtOH‐induced gastric symptoms. Therefore, these results suggest that Gl‐ME might be useful as an herbal anti‐inflammatory medicine through the inhibition of Src and NF‐κB activation pathways. The efficacy data of G. leptostachya also implies that this plant could be further tested to see whether it can be developed as potential anti‐inflammatory preparation. Copyright © 2014 John Wiley & Sons, Ltd.     

Tectorigenin protects against experimental fulminant hepatic failure by regulating the TLR4/mitogen‐activated protein kinase and TLR4/nuclear factor‐κB pathways and autophagy

Tectorigenin has received attention due to its antiproliferation, anti‐inflammatory, and antioxidant activities. In this study, we investigated the effects of tectorigenin on lipopolysaccharide (LPS)/D‐galactosamine(D‐GalN)‐induced fulminant hepatic failure (FHF) in mice and LPS‐stimulated macrophages (RAW 264.7 cells). Pretreatment with tectorigenin significantly reduced the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), histological injury, apoptosis, and the mortality of FHF mice, by suppressing the production of inflammatory cytokines such as TNF‐α and IL‐6. Tectorigenin also suppressed the activation of the inflammatory response in LPS‐stimulated RAW 264.7 cells. Tectorigenin‐induced protection is mediated through its mitigation of TLR4 expression, inhibition of mitogen‐activated protein kinase (MAPK) and nuclear factor‐κB (NF‐κB) pathway activation, and promotion of autophagy in FHF mice and LPS‐stimulated RAW 264.7 cells. Therefore, tectorigenin has therapeutic potential for FHF in mice via the regulation of TLR4/MAPK and TLR4/NF‐κB pathways and autophagy.     

Chelidonine inhibits TNF‐α‐induced inflammation by suppressing the NF‐κB pathways in HCT116 cells

Nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) is a complex that regulates several hundreds of genes, including those involved in immunity and inflammation, survival, proliferation, and the negative feedback of NF‐κB signaling. Chelidonine, a major bioactive, isoquinoline alkaloid ingredient in Chelidonium majus, exhibits antiinflammatory pharmacological properties. However, its antiinflammatory molecular mechanisms remain unclear. In this work, we explored the effect of chelidonine on TNF‐induced NF‐κB activation in HCT116 cells. We found chelidonine inhibited the phosphorylation and degradation of the inhibitor of NF‐κB alpha and nuclear translocation of RELA. Furthermore, by inhibiting the activation of NF‐κB, chelidonine downregulated target genes involved in inflammation, proliferation, and apoptosis. Chelidonine also inhibited mitogen‐activated protein kinase pathway activation by blocking c‐Jun N‐terminal kinase and p38 phosphorylation. These results suggest that chelidonine may be a potential therapeutic agent against inflammatory diseases in which inhibition of NF‐κB activity plays an important role.     

Berberine alleviates oxidized low‐density lipoprotein‐induced macrophage activation by downregulating galectin‐3 via the NF‐κB and AMPK signaling pathways

Macrophage activation plays a central role in neoatherosclerosis and in‐stent restenosis after percutaneous coronary intervention (PCI). Galectin‐3, mainly expressed on macrophages, is an important regulator of inflammation. This study aimed to investigate the effects of berberine (BBR) on oxidized low‐density lipoprotein (ox‐LDL)‐induced macrophage activation and galectin‐3 expression and their underlying mechanisms. THP‐1‐derived macrophages were pretreated with BBR prior to stimulation with ox‐LDL. Galectin‐3 expression was measured by real‐time PCR, Western blotting, and confocal microscopy. Macrophage activation was assessed by lipid accumulation, expression of inflammatory cytokines, and CD11b and CD86. Plasma galectin‐3 levels were measured in patients undergoing PCI at baseline and after BBR treatment for 3 months. BBR suppressed ox‐LDL‐induced upregulation of galectin‐3 and macrophage activation. Overexpression of galectin‐3 intervened the inhibitory effect of BBR on macrophage activation. BBR activated phospho‐AMPK and inhibited phospho‐NF‐κB p65 nuclear translocation. AMPK inhibition and NF‐κB activation abolished the inhibitory effects of BBR on galectin‐3 expression and macrophage activation. Combination of BBR and rosuvastatin exerted greater effects than BBR or rosuvastatin alone. However, BBR treatment did not further reduce plasma galectin‐3 after PCI in patients receiving standard therapy. In conclusion, BBR alleviates ox‐LDL‐induced macrophage activation by downregulating galectin‐3 via the NF‐κB and AMPK signaling pathways.