Anti-inflammatory potential of Phaseolus calcaratus Roxburgh, a oriental medicine, on LPS-stimulated RAW 264.7 macrophages

Objectives The seed of Phaseolus calcaratus Roxburgh (PHCR) has traditionally been used as a herbal medicine, considered to have anti‐inflammatory potential. Here we examined the ability of PHCR seed extract to inhibit inflammatory responses of macrophages to bacterial toxin and the mechanism involved. Methods In the present study, we prepared four fractions from an ethanol extract of PHCR seed and investigated their effects on the production of nitric oxide and cytokines, and the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophage cells. Key findings The fractions inhibited LPS‐induced nitric oxide production and cyclooxygenase‐2 (COX‐2) expression in the cells. The ethyl acetate fraction at 100 µg/ml almost completely suppressed NO production, iNOS and COX‐2 expression, and TNF‐α and IL‐6 secretion in cells stimulated with LPS. The fraction also inhibited phosphorylation of extracellular signal‐regulated kinase (ERK) and p38 in LPS‐stimulated cells with the attendant suppression of IκBα nuclear translocation and nuclear factor (NF)‐κB activation. Furthermore, PHCR seed extracts contained a large number of phenolic compounds having antioxidant potentials against 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radicals and hydroxyl radicals. We identified catechin‐7‐O‐β‐d ‐glucopyranoside as one of the active compounds responsible for the biological activity of PHCR seed extract. Conclusions These results suggest for the first time that ethanol extracts from PHCR seed have anti‐inflammatory potential on LPS‐stimulated macrophages through the down‐regulation of ERK/p38‐ and NF‐κB‐mediated signalling pathways.     

Suppression of inflammatory response and endothelial nitric oxide synthase downregulation in hyperlipidaemic C57BL/6J mice by eugenosedin-A

Objectives Eugenosedin‐A has been found to ameliorate high‐fat diet (HFD)‐induced hyperglycaemia and hyperlipidaemia in C57BL/6J mice. This study aimed to investigate the mechanisms of action of eugenosedin‐A on endothelial function and inflammation in hyperlipidaemic mice. Methods C57BL/6J mice were randomly divided into two control groups and two treatment groups. The control mice received either a regular diet or HFD, and the treatment groups were fed HFD with either 5 mg/kg eugenosedin‐A or atorvastatin for eight weeks. Key findings Mice fed a HFD had higher concentrations of nitrate (NO) but not prostaglandin E₂ (PGE₂), increased tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ) mRNA and inducible nitric oxide synthase (iNOS) proteins, but decreased endothelial nitric oxide synthase (eNOS) proteins. HFD‐induced upregulation of iNOS is associated with p38, extracellular signal‐regulated kinase (ERK), c‐Jun‐N‐terminal kinase (JNK), PI3K and Akt/IKKα/p65. Eugenosedin‐A and atorvastatin reduced HFD‐induced TNF‐α and IFN‐γ mRNA, NO generation, upregulation of iNOS protein, and down‐regulation of eNOS protein. Both agents inhibited p38, ERK, JNK and Akt/IKKα/p65 protein levels in the aorta. However, eugenosedin‐A did not significantly reduce p38 in the liver. Conclusions Our results showed an association between obesity‐induced inflammation and altered levels of TNF‐α, IFN‐γ, p38, ERK, JNK and Akt/IKKα/p65. Eugenosedin‐A, like atorvastatin, could inhibit p38, ERK, JNK, Akt/IKKα/p65 proteins, as well as TNF‐α and IFN‐γ mRNA during the regulation of the obesity‐induced inflammatory process.     

Inhibitory effect of fucoidan on nitric oxide production in lipopolysaccharide‐activated primary microglia

1. Microglial activation plays an important role in the pathogenesis of neurodegenerative diseases by producing various pro‐inflammatory cytokines. Microglia‐derived nitric oxide (NO) is critical for the lipopolysaccharide (LPS)‐induced selective loss of dopaminergic neurons. 2. Fucoidan is a sulphated polysaccharide extracted from brown seaweeds. It has a variety of biological actions, including anticoagulant, antiviral and anti‐inflammatory effects. The aim of the present study was to investigate the effects of fucoidan on LPS‐induced cellular activation in microglia and to evaluate the inhibitory mechanisms involved. 3. To investigate the effects of fucoidan on LPS‐induced cellular activation in microglia, primary microglial cells were preincubated with fucoidan (31.25, 62.5 and 125 μg/mL) for 10 min, followed by stimulation with LPS (0.01 μg/mL). Then, cell shape and NO production were determined 24 h after LPS stimulation, whereas inducible nitric oxide synthase (iNOS) mRNA and protein expression were determined at 6 and 18 h after LPS stimulation, respectively. To evaluate the inhibitory mechanisms involved, mitogen‐activated protein kinase (MAPK) activation was also evaluated. 4. Lipopolysaccharide transformed cells into an amoeboid shape, whereas 62.5 μg/mL fucoidan inhibited this activation. Moreover, 125 μg/mL fucoidan significantly inhibited microglial NO production to 75% of that in LPS‐treated group and also significantly diminished the expression of iNOS mRNA and protein by nearly 50%. Fucoidan (125 μg/mL) also suppressed phosphorylation of p38 and extracellular signal‐regulated kinase (ERK) by approximately 50%, but not that of c‐Jun N‐terminal kinase. 5. The results provide the first evidence that fucoidan has a potent inhibitory effect against LPS‐induced NO production by microglia. The results also suggest that this inhibitory action of fucoidan involves suppression of p38 and ERK phosphorylation.     

Anti‐inflammatory activity and possible mechanism of extract from Mikania laevigata in carrageenan‐induced peritonitis

Objectives The aim was to test the potential use of an extract of Mikania laevigata (popularly known in Brazil as guaco), made from leaves harvested in different months of the year, on neutrophil migration after an inflammatory stimulus and investigate the underlying molecular mechanisms. Methods We examined the effect of guaco on vascular permeability and leucocyte function in carrageenan‐induced peritonitis in mice. Key findings Our results demonstrated that guaco extract administered subcutaneously (3 mg/kg) decreased the vascular permeability and also leucocyte rolling and adhesion to the inflamed tissues by a mechanism dependent on nitric oxide. Specifically, inhibitors of nitric oxide synthase remarkably abrogated the guaco extract‐mediated suppression of neutrophil migration to the inflammatory site. In addition, guaco extract‐mediated suppression of neutrophil migration appeared to be dependent on the production of the cytokines interleukin‐1β and tumour necrosis factor‐α. One of the major constituents of the guaco extract, coumarin, was able to inhibit the neutrophil migration towards the inflammatory focus. Conclusions In conclusion the anti‐inflammatory effect induced by guaco extract may be by inhibition of pro‐inflammatory cytokine production at the inflammatory site.     

Dexmedetomidine protects mice against myocardium ischaemic/reperfusion injury by activating an AMPK/PI3K/Akt/eNOS pathway

Acute myocardial ischaemia/reperfusion (MIR) injury leads to severe arrhythmias and has a high rate of lethality. In the present study, we aim to determine the effect of dexmedetomidine (Dex) on heart injury parameters following MIR surgery. We examined the effects of Dex on heart function parameters and infarct size following MIR surgery. Proinflammatory cytokines, oxidative products and anti‐oxidative enzymes in the myocardium were measured to evaluate the anti‐inflammatory and anti‐oxidative effects of Dex. The role of the adenosine 5′‐monophosphate (AMP)‐activated protein kinase (AMPK)/phosphatidylino‐sitol 3‐kinase (PI3k)/Akt/endothelial nitric oxide synthase (eNOS) pathway was investigated using their inhibitors. The alteration of haemodynamic parameters, histopathological results, and infarct size caused by MIR was attenuated by Dex. The interleukine‐1 beta (IL‐1β), IL‐6, tumour necrosis factor‐a (TNF‐α) and myeloperoxidase (MPO) were all significantly decreased. Anti‐oxidative enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were restored by Dex. Oxidative products8‐OHdG, MDA and protein carbonyl were all decreased by Dex (P<.05). Dex activated AMPK expression, eNOS and Akt phosphorylation. The influence of Dex on cardiac function was reversed by the inhibitors of the eNOS, AMPK and PI3K/Akt pathways. These results indicate that Dex protected the cardiac functional, histological changes, inflammation and oxidative stress induced by MIR. Our results present a novel signalling mechanism that Dex protects MIR injury by activating an AMPK/PI3K/Akt/eNOS pathway.     

Protein Kinase C and its Inhibitors in the Regulation of Inflammation: Inducible Nitric Oxide Synthase as an Example

Protein kinase C (PKC) is a family of ten isoenzymes that play a crucial role in cellular signal transduction. Studies with PKC knockout animals have revealed that many of the isoenzymes are involved in cell growth, proliferation and differentiation. Several PKC isoenzymes have also been shown to be important mediators in inflammation and immunity, particularly in lymphocyte responses. However, less is known about the role of PKC in the regulation of the expression of inflammatory genes. In inflammatory processes, nitric oxide is primarily produced by inducible nitric oxide synthase (iNOS) in inflammatory cells, such as macrophages. In innate immunity, nitric oxide functions as an effector molecule towards the infectious organisms. Increased levels of nitric oxide are also produced by inflammatory and tissue cells in inflammatory diseases, such as asthma and arthritis. In this MiniReview, the role of PKC isoenzymes in the pathogenesis and as a potential drug target in inflammation will be discussed presenting iNOS as an example of an inflammatory gene regulated by the pleiotropic PKC signalling pathway.     

Myrrh mediates haem oxygenase-1 expression to suppress the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages

Objectives To elucidate a novel anti‐inflammatory mechanism of myrrh against lipopolysaccharide (LPS)‐induced inflammation. Methods RAW264.7 macrophages were cultured in DMEM and then cells were treated with LPS or LPS plus a myrrh methanol extract (MME) for 24 h. The culture medium was collected for determination of nitric oxide (NO), prostaglandin (PG)E₂, interleukin (IL)‐1β, and tumour necrosis factor (TNF)‐α, and cells were harvested by lysis buffer for Western blot analysis. Key findings Our data showed that treatment with the MME (1～100 µg/ml) did not cause cytotoxicity or activate haem oxygenase‐1 (HO‐1) protein synthesis in RAW264.7 macrophages. Furthermore, the MME inhibited LPS‐stimulated NO, PGE₂, IL‐1β and TNF‐α release and inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)‐2 protein expression. Zn(II) protoporphyrin IX, a specific inhibitor of HO‐1, blocked the inhibition of iNOS and COX‐2 expression by the MME. Conclusions These results suggest that among mechanisms of the anti‐inflammatory response, the MME inhibited the production of NO, PGE₂, IL‐1β and TNF‐α by downregulating iNOS and COX‐2 gene expression in macrophages and worked through the action of HO‐1.     

Catalpol protects against 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin‐induced cytotoxicity in osteoblastic MC3T3‐E1 cells

2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a well‐known environmental contaminant that produces a wide variety of adverse effects in humans. Catalpol, a major bioactive compound enriched in the dried root of Rehmannia glutinosa, is a major iridoid glycoside that alleviates bone loss. However, the detailed mechanisms underlying the effects of catalpol remain unclear. The present study evaluated the effects of catalpol on TCDD‐induced cytotoxicity in osteoblastic MC3T3‐E1 cells. Catalpol inhibited TCDD‐induced reduction in cell viability and increases in apoptosis and autophagic activity in osteoblastic MC3T3‐E1 cells. Additionally, pretreatment with catalpol significantly decreased the nitric oxide and nitrite levels compared with a control in TCDD‐treated cells and significantly inhibited TCDD‐induced increases in the levels of cytochrome P450 1A1 and extracellular signal‐regulated kinase. Pretreatment with catalpol also effectively restored the expression of superoxide dismutase and extracellular signal‐regulated kinase 1 and significantly enhanced the expression of glutathione peroxidase 4 and osteoblast differentiation markers, including alkaline phosphatase and osterix. Taken together, these findings demonstrate that catalpol has preventive effects against TCDD‐induced damage in MC3T3‐E1 osteoblastic cells.     

Luteolin inhibits lipopolysaccharide actions on human gingival fibroblasts

Periodontal disease comprises a group of infections that lead to inflammation of the gingiva, periodontal tissue destruction, and in severe cases is accompanied by alveolar bone loss with tooth exfoliation. Actinobacillus actinomycetemcomitans is a Gram-negative microorganism, which possesses and produces lipopolysaccharide (LPS) molecules that play a key role in disease development. Human gingival fibroblasts are the major constituents of gingival connective tissue and may interact directly with bacteria and bacterial products including LPS. Flavonoids possess antioxidant and anti-inflammatory properties that reduce inflammatory molecule expression in macrophages and monocytes. In this study, we evaluated the ability of diverse flavonoids to regulate nitric oxide production of LPS-stimulated human gingival fibroblasts, and studied the effect of luteolin on diminish phosphorylation in mitogen-activated protein kinase (MAPK) family members as well as in protein kinase B (Akt), nuclear factor kappa B (NF-kappaB) activation, inducible nitric oxide synthase (NOS) expression, and nitric oxide (NO) synthesis. We also found that pretreatment with three flavonoids, including quercetin, genistein, and luteolin, blocked nitric oxide synthesis in a dose-dependent fashion. Luteolin exerted the strongest blocking action on expression of this inflammatory mediator. Luteolin pretreatment attenuated LPS-induced extracellular signal-regulated kinase, p38, and Akt phosphorylation. LPS treatment of human gingival fibroblasts resulted in NF-kappaB translocation. Cell pretreatment with luteolin abolished LPS effects on NF-kappaB translocation. In addition, luteolin treatment blocked LPS-induced cellular proliferation inhibition without affecting genetic material integrity. We concluded that luteolin interferes with LPS signaling pathways, reducing activation of several mitogen-activated protein kinase family members, and inhibits inflammatory mediator expression.     

Antihypertensive methyldopa,labetalol, hydralazine,and clonidine reversed tumour necrosis factor‐α inhibited endothelial nitric oxide synthase expression in endothelial‐trophoblast cellular networks

Medications used to control hypertension in pregnancy also improve trophoblast and endothelial cellular interaction in vitro. Tumour necrosis factor‐α (TNF‐α) inhibits trophoblast and endothelial cellular interactions and simultaneously decreases endothelial nitric oxide synthase (eNOS) expression. This study investigated whether antihypertensive medications improved these cellular interactions by modulating eNOS and inducible nitric oxide synthase (iNOS) expression. Human uterine myometrial microvascular endothelial cells (UtMVECs) were pre‐incubated with (or without) low dose TNF‐α (0.5 ng/mL) or TNF‐α plus soluble fms‐like tyrosine kinase‐1 (sFlt‐1) (100 ng/mL). The endothelial cells were cultured on Matrigel. After endothelial cellular networks appeared, trophoblast derived HTR‐8/SVneo cells were co‐cultured in the presence of clinically relevant doses of methyldopa, labetalol, hydralazine or clonidine for 24 hours. Cells were retrieved from the Matrigel to extract mRNA and eNOS and iNOS expression were examined by quantitative PCR. Methyldopa, labetalol, hydralazine and clonidine reversed the inhibitory effect of TNF‐α on eNOS mRNA expression. After pre‐incubating endothelial cells with TNF‐α and sFlt‐1, all the medications except methyldopa lost their effect on eNOS mRNA expression. In the absence of TNF‐α, antihypertensive medications did not change eNOS expression. The mRNA expression of iNOS was not affected by TNF‐α or any medications. This study shows that selected antihypertensive medications used in the treatment of hypertension in pregnancy increase eNOS expression in vitro when induced by the inflammatory TNF‐α. The anti‐angiogenic molecule sFlt‐1 may antagonise the potential benefit of these medications by interfering with the NOS pathway.     

Pharmacokinetics and Pharmacodynamics of Curcumin in regulating anti‐inflammatory and epigenetic gene expression

Chronic inflammation is a key driver of cancer development. Nitrite levels, which are regulated by inducible nitric oxide synthase (iNOS), play a critical role in inflammation. While the anti‐oxidant and anti‐inflammatory effects of curcumin, a natural product present in the roots of Curcuma longa have been studied widely, the acute pharmacokinetics (PK) and pharmacodynamics (PD) of curcumin in suppressing pro‐inflammatory markers and epigenetic modulators remain unclear. This study evaluated the PK and PD of curcumin‐induced suppression of lipopolysaccharide (LPS)‐mediated inflammation in rat lymphocytes. LPS was administered intravenously either alone or with curcumin to female Sprague–Dawley rats. Plasma samples were analysed for curcumin concentration and mRNA expression was quantified in lymphocytes. The relative gene expression of several inflammatory and epigenetic modulators was analysed. To investigate the relationship between curcumin concentration and iNOS, TNF‐α, and IL‐6 gene expression, PK/PD modeling using Jusko's indirect response model (IDR) integrating transit compartments (TC) describing the delayed response was conducted. The concentration–time profile of curcumin exhibited a bi‐exponential decline, which was well described by a two‐compartmental pharmacokinetic model. Importantly the results demonstrate that LPS induced gene expression of pro‐inflammatory markers in lymphocytes, with peak expression at approximately 3 h and curcumin suppressed the gene expression in animals administered with LPS. These effects were well captured using the IDR model and an IDR model with the transit compartments. In summary, the PK/PD modeling approach could potentially provide a robust quantitative framework for evaluating the acute anti‐inflammatory and epigenetic effects of curcumin in future clinical trials.     

Withaferin A inhibits iNOS expression and nitric oxide production by Akt inactivation and down-regulating LPS-induced activity of NF-kappaB in RAW 264.7 cells

Induction of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production is thought to have beneficial immunomodulatory effects in acute and chronic inflammatory disorders. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, withaferin A inhibited LPS-induced expression of both iNOS protein and mRNA in a dose-dependent manner. To investigate the mechanism by which withaferin A inhibits iNOS gene expression, we examined activation of mitogen-activated protein kinases (MAPKs) and Akt in Raw 264.7 cells. We did not observe any significant changes in the phosphorylation of p38 MAPK in cells treated with LPS alone or LPS plus withaferin A. However, LPS-induced Akt phosphorylation was markedly inhibited by withaferin A, while the phosphorylation of p42/p44 extracellular signal-regulated kinases (ERKs) was slightly inhibited by withaferin A treatment. Withaferin A prevented IkappaB phosphorylation, blocking the subsequent nuclear translocation of nuclear factor-kappaB (NF-kappaB) and inhibiting its DNA binding activity. LPS-induced p65 phosphorylation, which is mediated by extracellular signal-regulated kinase (ERK) and Akt pathways, was attenuated by withaferin A treatment. Moreover, LPS-induced NO production and NF-kappaB activation were inhibited by SH-6, a specific inhibitor of Akt. Taken together, these results suggest that withaferin A inhibits inflammation through inhibition of NO production and iNOS expression, at least in part, by blocking Akt and subsequently down-regulating NF-kappaB activity.     

Protective effect of wogonin on endotoxin‐induced acute lung injury via reduction of p38 MAPK and JNK phosphorylation

Acute lung injury (ALI) is a serious inflammatory disorder which remains the primary cause of incidence and mortality in patients with acute pulmonary inflammation. However, there is still no effective medical strategy available clinically for the improvement of ALI. Wogonin, isolated from roots of Scutellaria baicalensis Georgi, is a common medicinal herb which presents biological and pharmacological effects, including antioxidation, anti‐inflammation, and anticancer. Preadministration of wogonin inhibited not only lung edema but also protein leakage into the alveolar space in murine model of lipopolysaccharide (LPS)‐induced ALI. Moreover, wogonin not only reduced the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)?2 but also inhibited the phosphorylation of mitogen‐activated protein kinase (MAPK) induced by LPS. We further found wogonin inhibited the phosphorylation of p38 MAPK and JNK at a concentration lower than ERK. In addition, inhibition of lung edema, protein leakage, expression of iNOS and COX‐2, and phosphorylation of p38 MAPK and JNK were all observed in a parallel concentration‐dependent manner. These results suggest that wogonin possesses potential protective effect against LPS‐induced ALI via downregulation of iNOS and COX‐2 expression by blocking phosphorylation of p38 MAPK and JNK. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 397–403, 2017.     

Ghrelin suppression of <Emphasis Type="Italic">Helicobacter pylori</Emphasis>-induced <Emphasis Type="Italic">S</Emphasis>-nitrosylation-dependent Akt inactivation exerts modulatory influence on gastric mucin synthesis

Loss of mucus coat integrity and the impairment in its mucin component as well as the disturbance in nitric oxide (NO) generation are well-recognized features of gastric disease associated with H. pylori infection. As ghrelin plays a major role in the regulation of nitric oxide synthase system, we investigated the influence of this hormone on H. pylori LPS-induced interference with gastric mucin synthesis. The results revealed that the LPS-induced impairment in mucin synthesis and accompanied induction in inducible nitric oxide synthase (iNOS) expression, were associated with the suppression in Akt kinase activity and the impairment in constitutive nitric oxide synthase (cNOS) phosphorylation. The LPS effect on Akt inactivation was manifested in the kinase protein S-nitrosylation and a decrease in its phosphorylation at Ser⁴⁷³. Further, we show that the countering effect of ghrelin, on the LPS-induced impairment in mucin synthesis was reflected in the suppression of iNOS and the increase in Akt activation, associated with the loss in S-nitrosylation and the increase in phosphorylation, as well as cNOS activation through phosphorylation. Our findings demonstrate that up-regulation in iNOS with H. pylori infection and subsequent Akt kinase inactivation through S-nitrosylation exerts the detrimental effect on the processes dependent on Akt activation, including that of cNOS activation and mucin synthesis. We also show that ghrelin protection against H. pylori-induced impairment in mucin synthesis is intimately linked to the events of Akt activation and reflected in a decrease in the kinase S-nitrosylation and the increase in its phosphorylation.