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.     

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.     

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.     

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.     

Mogroside IIIE Attenuates LPS‐Induced Acute Lung Injury in Mice Partly Through Regulation of the TLR4/MAPK/NF‐κB Axis via AMPK Activation

Acute lung injury (ALI) often leads to high mortality, and there is as yet no effective drug treatment. The present study aimed to investigate protective effects of mogroside IIIE (MGIIIE, a cucurbitane‐type triterpenoid from Siraitia grosvenorii Fruits) in experimental ALI and its underlying mechanism. MGIIIE (1, 10 0r 20 mg/kg) was orally administered for 1 h before a single intratracheal administration of lipopolysaccharide (LPS, 5 mg/kg). MGIIIE treatment dose‐dependently suppressed pulmonary oedema, pro‐inflammatory mediators (IL‐1β, IL‐6, TNF‐α and HMGB1) release and higher MPO activity in lung tissues induced by LPS challenge. Molecular researches showed that mogroside IIIE (20 mg/kg) not only increased the phosphorylation of adenosine 5′‐monophosphate‐activated protein kinase (AMPK) but suppressed the over‐expression of toll‐like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). In addition, MGIIIE also inhibited the activation of MAPKs and nuclear factor κB (NF‐κB) signalling in lung tissues from LPS‐challenged mice. Similar antiinflammatory effects of MGIIIE were obtained in LPS‐treated macrophages. Compound C (a pharmacological AMPK inhibitor) obviously reversed the antiinflammatory effect of MGIIIE in LPS‐induced ALI mice. Taken together, AMPK activation plays a crucial role in the antiinflammatory effects of MGIIIE in LPS‐induced ALI by down‐regulating TLR4/MAPK/NF‐κB signalling pathways. Copyright © 2017 John Wiley & Sons, Ltd.     

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.     

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.     

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.     

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.     

Hesperidin ameliorates insulin resistance by regulating the IRS1‐GLUT2 pathway via TLR4 in HepG2 cells

The aim of this study was to evaluate the effect and mechanism of hesperidin (HES) on insulin resistance (IR) in the human hepatocellular carcinoma cell line (HepG2 cells). HepG2 cells were induced with lipopolysaccharide (LPS) as a model of IR and treated with HES at three dosages. Next, the levels of interleukin‐6 (IL‐6) and tumor necrosis factor‐α (TNF‐α), the glucose content, and glucose uptake were evaluated by enzyme‐linked immunosorbent assay, glucose oxidase‐peroxidase method (GOD‐POD), or (2‐(N‐(7‐nitrobenz‐2‐oxa‐1, 3‐diazol‐4‐yl)amino)‐2‐deoxyglucose) (2‐NBDG). Moreover, the protein expression of toll‐like receptors 4 (TLR4), insulin receptor substrate 1 (IRS1), nuclear factor kappa B (NF‐κB), and glucose transporter 2 (GLUT2) in HepG2 cells treated with HES were assessed via western blotting analysis. In addition, GLUT2 protein expression exposed to HES was detected following treatment with TLR4 inhibitor (HTA125). Our results demonstrated that HES decreased the levels of TNF‐α and IL‐6, attenuated the glucose content in culture medium and increased glucose uptake in insulin‐resistant HepG2 cells in vitro. Moreover, HES upregulated the expression of IRS1 and GLUT2 protein and downregulated the protein expression of TLR4 and NF‐κB in insulin‐resistant HepG2 cells. The expression of GLUT2 protein had no significant changes when treated with HES after blockade of TLR4. HES attenuated IR in LPS‐inducedinsulin‐resistant HepG2 cells. Therefore, regulating the IRS1‐GLUT2 pathway via TLR4 represents a potential mechanism of HES on IR in HepG2 cells.     

The ability of hydroxysafflor yellow a to attenuate lipopolysaccharide‐induced pulmonary inflammatory injury in mice

Hydroxysafflor yellow A (HSYA) is a component of the flower of Carthamus tinctorius L. The present study investigated whether HSYA could attenuate acute lung injury (ALI) induced by lipopolysaccharide (LPS) administration. Male Kunming mice were pretreated with HSYA 0.5 h prior to intraperitoneal application of LPS. Arterial blood gas, lung water content index, lung tissue myeloperoxidase (MPO) activity, mRNA expression of inflammatory cytokines, NF‐κBp65, p38 mitogen‐activated protein kinase (MAPK) and pathological changes in lung morphology were assessed. After LPS administration, all animals displayed increased arterial carbon dioxide partial pressure (PaCO₂), and decreased arterial oxygen partial pressure (PaO₂), arterial oxygen saturation (SO₂), HCO₃^? concentration and pH, which were ameliorated by pretreating the animals with HSYA. HSYA administration significantly attenuated inflammatory cell infiltration and alleviated pulmonary edema induced by LPS. Moreover, HSYA decreased NF‐κB p65 nuclear translocation, inhibited proinflammatory cytokine TNF‐α, IL‐1β and IL‐6 mRNA expression and promoted antiinflammatory cytokine IL‐10 gene expression following LPS injection. Pulmonary p38 MAPK phosphorylation was upregulated 4 h after LPS treatment, which could be suppressed by pretreatment with HSYA. These findings demonstrated the protective effect of HSYA against LPS‐induced acute lung injury, which is suggested to be associated with the inhibition of p38 MAPK, NF‐κB p65 activation and alteration of inflammatory cytokine expression. Copyright © 2010 John Wiley & Sons, Ltd.     

Retracted: Lipopolysaccharides‐mediated injury to chondrogenic ATDC5 cells can be relieved by Sinomenine via downregulating microRNA‐192

Sinomenine (SIN) is an isoquinoline derived from Caulis Sinomenii that has been used to treat rheumatoid arthritis and osteoarthritis for several decades in China. This study aims to reveal the effects of SIN on mouse chondrogenic ATDC5 cells growth and inflammation. SIN was used to treat ATDC5 cells injured by lipopolysaccharides (LPS). The following parameters were determined for evaluating the treatment effects of SIN: cell viability, apoptosis, reactive oxygen species generation, and pro‐inflammatory cytokines release. Besides, the expression of LPS‐sensitive miRNA (miR‐192) and the activation of NF‐κB and MAPK signaling were studied to explain SIN's function. SIN with concentration of 30 μM significantly attenuated LPS‐induced cell damage via increasing cell viability, inhibiting apoptosis and reactive oxygen species generation, and declining IL‐6 and TNF‐α release. miR‐192 was downregulated by SIN treatment. Restoration of miR‐192 expression by miRNA transfection could significantly impede SIN's protective action. Besides, the inhibitory effects of SIN on the activation of NF‐κB and MAPK signaling were attenuated by miR‐192 overexpression. Furthermore, GDF11 was found to be a target gene of miR‐192. LPS‐mediated injury to chondrogenic ATDC5 cells can be relieved by SIN via downregulating miR‐192 and subsequently repressing the activation of NF‐κB and MAPK signaling.     

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.