Praeruptorin a inhibits lipopolysaccharide‐induced inflammatory response in murine macrophages through inhibition of NF‐κB pathway activation

Praeruptorin A (PA) is a pyranocoumarin compound isolated from the dried root of Peucedanum praeruptorum Dunn (Umbelliferae). However, the antiinflammatory effect of PA has not been reported. The present study investigated the antiinflammatory effect of PA in lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophage cells. PA significantly inhibited the LPS‐induced production of nitric oxide (NO), interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α). The mRNA and protein expressions of inducible nitric oxide synthase (iNOS), IL‐1β and TNF‐α were also suppressed by this compound. Further study showed that PA decreased the cytoplasmic loss of inhibitor κB‐α (IκB‐α) protein and inhibited the translocation of NF‐κB from cytoplasm to nucleus. Taken together, the results suggest that PA may exert antiinflammatory effects in vitro in LPS‐stimulated RAW 264.7 macrophages through inhibition of NF‐κB signal pathway activation. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

Ameliorative effects of Magnolia sieboldii buds hexane extract on experimental reflux esophagitis

Gastroesophageal reflux disease (GERD) is a disease that stomach contents continually refluxing into esophagus causes symptoms and/or complications. The study was working to find natural plant extracts with good effects and small side effects to treat reflux esophagitis (RE). The anti‐inflammatory effects of hexane extract of Magnolia sieboldii (MsHE) were conducted on lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophage cells. The ameliorative effects of MsHE on esophageal damage in rats induced by gastric acid reflux was explored in vivo. The results showed that MsHE decreased the production of nitric oxide (NO) and expression levels of iNOS, COX‐2 and TNF‐α on LPS‐stimulated RAW 264.7 cells and MsHE treatment ameliorated the rats' esophageal tissue damage induced by gastric acid and inhibited the increase of inflammatory mediators and pro‐inflammatory cytokines by regulating NF‐κB signaling pathway. In addition, MsHE protected the function of barrier of epithelial cells against inflammatory conditions through increasing the expression of tight junctions. Furthermore, liquid chromatography‐mass spectrometry analysis was used for determine the active ingredients contained in MsHE. The results show that MsHE can alleviate experimental rat RE by regulating NF‐κB signaling pathway. In summary, MsHE may be used as a source material of drug candidate for the treatment of RE.     

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.     

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.     

Ethanol extract of Prunella vulgaris var. lilacina inhibits HMGB1 release by induction of heme oxygenase-1 in LPS-activated RAW 264.7 cells and CLP-induced septic mice

The ethanol extract of the flower of P. vulgaris var. lilacina (EEPV) has been used traditionally as an antiinflammatory agent in many countries. Inducers of heme oxygenase‐1 (HO‐1) reduce high mobility group box 1 (HMGB1), a late phase cytokine, in sepsis. Although EEPV has been used as an antiinflammatory agent, no report is available as to whether it modifies HMGB1 in sepsis due to HO‐1 induction. It was found that EEPV increased HO‐1 protein expression in RAW 264.7 cells, which was significantly inhibited by LY294002, but not PD98059, SB203580 or SP600125. In addition, EEPV activated NF‐E2‐related factor (Nrf2) to move from the cytosol to the nucleus, and EEPV‐induced HO‐1 and activation of ARE‐luciferase activity were significantly reduced by siNrf2 transfection and LY294002 but not SB203508. EEPV also significantly inhibited NF‐κB luciferase activity, and decreased both iNOS/NO and COX‐2/PGE₂ production in lipopolysaccharide (LPS)‐stimulated macrophages which was reversed by siHO‐1 RNA transfection. Importantly, EEPV inhibited HMGB1 release in LPS‐activated macrophages in a PI3K‐sensitive manner and reduced serum HMGB1 level and lung HMGB1 expression in cecal ligation and puncture (CLP)‐induced septic mice. It is concluded that EEPV induces HO‐1 expression through PI3K/Nrf2 signal pathways, which may be beneficial for the treatment of sepsis due to a reduction of HMGB1 release. Copyright © 2011 John Wiley & Sons, Ltd.     

Anti‐inflammatory Effects of Ginsenosides Rg5, Rz1, and Rk1: Inhibition of TNF‐α‐induced NF‐κB,COX‐2, and iNOS transcriptional expression

In the course of this experiment on the anti‐inflammatory effect of ginsenosides, protopanaxdiol ginsenosides have shown inhibition activities in inflammatory responses: NF‐κB, COX‐2, and iNOS were induced by TNF‐α. The responses of this experiment were evaluated by NF‐κB‐luciferase assay and RT‐PCR experiment of COX‐2 and iNOS genes. The NF‐κB expressions were inhibited by ginsenosides Rd, Rg₅, Rz₁, and Rk₁ in a dose‐dependent manner. The IC₅₀ values were 3.47, 0.61, 0.63, and 0.75 μM, respectively. Particularly, ginsenosides Rg₅, Rz₁, and Rk₁ as converted ginsenosides from primary protopanaxdiol ginsenosidess significantly inhibited COX‐2 and iNOS gene expression. These inhibition levels were similar to sulfasalazine as reference material. Copyright © 2014 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.     

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.     

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.     

Forsythoside B protects against experimental sepsis by modulating inflammatory factors

The present study investigated the effects of Forsythoside B on an experimental model of sepsis induced by caecal ligation and puncture (CLP) in rats and elucidated the potential mechanism in cultured RAW 264.7 cells. Results showed that Forsythoside B concentration‐dependently down‐regulated the levels of TNF‐α, IL‐6 and high‐mobility group‐box 1 protein (HMGB1) in lipopolysaccharide (LPS)‐stimulated RAW264.7 cells, inhibited the IκB kinase (IKK) pathway and modulated nuclear factor (NF)‐ κB. Intravenous injection (i.v.) of Forsythoside B alone or plus Imipenem reduced serum levels of TNF‐α, IL‐6, HMGB1, triggering receptor expressed on myeloid cells (TREM‐1) and endotoxin, while the serum level of IL‐10 was up‐regulated and myeloperoxidase (MPO) in lung, liver and small intestine was reduced. Meanwhile, i.v. of Forsythoside B alone or plus Imipenem reduced CLP‐induced lethality in rats. These data indicated that the antisepsis effect of Forsythoside B is mediated by decreasing local and systemic levels of a wide spectrum of inflammatory mediators. Its antisepsis mechanism may be that Forsythoside B binds to LPS and reduces the biological activity of serum LPS, and inhibits NF‐κB activition. Our studies enhance the case for the use of Forsythoside B in sepsis. Forsythoside B itself has promise as a therapy for the treatment of sepsis in humans. Copyright © 2011 John Wiley & Sons, Ltd.     

Rhynchophylline Attenuates LPS‐induced Pro‐inflammatory Responses through Down‐regulation of MAPK/NF‐κB Signaling Pathways in Primary Microglia

Excessive activation of microglial cells has been implicated in various types of neuroinflammation. Suppression of microglial activation would have therapeutic benefits, leading to the alleviation of the progression of neurodegeneration. In this study, the inhibitory effects of rhynchophylline (RIN), a tetracyclic oxindole alkaloid component isolated from Uncaria rhynchophylla (Miq.) Jacks., on the production of pro‐inflammatory mediators were investigated in lipopolysaccharide (LPS)‐stimulated microglia. The results showed that RIN markedly reduced the production of nitric oxide (NO), prostaglandins E₂ (PGE₂), monocyte chemoattractant protein (MCP‐1), tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) in LPS‐activated microglia. The mRNA expression levels of iNOS and COX‐2 were also depressed by RIN in a concentration‐dependent manner. Further studies revealed that RIN blocked IκBα phosphorylation and degradation, inhibited the phosphorylation of mitogen‐activated protein kinases (MAPKs). In summary, these data suggest that RIN suppresses inflammatory responses of microglia and may act as a potential therapeutic agent for various neurodegenerative diseases involving neuroinflammation. Copyright © 2012 John Wiley & Sons, Ltd.     

Triptolide down-regulates COX-2 expression and PGE2 release by suppressing the activity of NF-κB and MAP kinases in lipopolysaccharide-treated PC12 cells

As an active compound extracted from the Chinese herb Tripterygium wilfordii, triptolide (TP) was demonstrated to have potent antiinflammatory and immunosuppressive properties in previous studies. Recently, it has been shown that TP prevented the loss of dopaminergic neurons in the substantia nigra of rats in a model of Parkinson's disease, but little is known about the precise neuroprotective mechanism of TP. This study was designed to elucidate whether the neuroprotective effect of TP is partially based on its direct inhibition of inflammatory molecules by investigating the effects of TP on the expression of cyclooxygenase (COX)‐2 and prostaglandin E2 (PGE2) related to the nuclear factor (NF)‐κB pathway in lipopolysaccharide (LPS)‐stimulated PC12 cells. The activation of related upstream molecules such as NF‐κB, P38, extracellular signal‐regulated kinase (ERK)1/2, and beta‐alanyl‐alpha‐ketoglutarate transaminase (AKT), in PC12 cells were investigated by real time polymerase chain reaction (PCR), western blotting and enzyme‐linked immunosorbent assay (ELISA). Our results showed that TP directly inhibited the expression of both mRNA and protein of COX‐2 (p < 0.01), decreased PGE2 production (p < 0.01) in a dose‐dependent manner, down‐regulated NF‐κB activity (p < 0.01), and significantly inhibited the phosphorylation of p38, ERK1/2 (p42/p44) and AKT in PC12 cells after LPS challenge. This suggests that the neuroprotective effects of TP may be partially mediated by direct inhibition of the expression of COX‐2, activation of NF‐κB, and phosphorylation of p38, ERK1/2 (p42/p44) and AKT proteins of neuronal cells. Copyright © 2011 John Wiley & Sons, Ltd.     

Anti-inflammatory effect of Citrus Unshiu peel in LPS-stimulated RAW 264.7 macrophage cells

Citrus Unshiu peel (CUP) has been traditionally used in East Asia as a drug for the treatment of vomiting and dyspepsia. However, its effects on inflammation remain unknown. In this study, we investigated the effects of CUP on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The research focused on determining whether CUP could inhibit the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and the activation of nuclear factor (NF)-κB, mitogen-activated protein kinases (MAPKs), as well as the secretion of nitric oxide (NO), prostaglandin (PG) E(2), tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in LPS-stimulated RAW 264.7 cells. We found that CUP represses LPS-induced iNOS and COX-2 gene expression as well as NO, PGE(2), TNF-α and IL-6 production. Additionally, CUP inhibited the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun NH(2)-terminal kinase (JNK) MAPK, and suppressed IκBα degradation and nuclear translocation of NF-κB. Collectively, our results indicate that CUP inhibits the production of various inflammatory mediators via blockade of MAPK phosphorylation pursuant to the inhibition of IκBα degradation and the nuclear translocation of NF-κB. These findings are the first to clarify the mechanism underlying the anti-inflammatory effect exerted by CUP in RAW 264.7 macrophage cells stimulated by inflammatory agents.