Amino acids stimulate Akt phosphorylation,and reduce IL‐8 production and NF‐κB activity in HepG2 liver cells

Hepatic insulin resistance and inflammatory cytokine production contribute to the manifestation of the metabolic syndrome. As amino acids have been implicated in modulating insulin signaling and inflammation, we have investigated the effects of glutamine, leucine and proline on markers of inflammation and insulin sensitivity in HepG2 liver cells. Cells were incubated with IL‐1β (5 ng/mL) to stimulate IL‐8 production. After 24 h, glutamine inhibited IL‐8 production significantly (p<0.05) at 2, 5 and 10 mM (to 82, 73 and 72% of control), whereas leucine reduced IL‐8 production significantly only at 10 mM (66%) and proline at 5 and 10 mM (71 and 52%). Glutamine, leucine and proline all reduced NF‐κB activity after 3 h of IL‐1β stimulation at 2, 5 and 10 mM (p<0.001). Insulin‐induced (1 nM) Akt phosphorylation was reduced in cells treated with tumour necrosis factor‐α (10 ng/mL) for 24 h, but was partly restored by simultaneous incubation with glutamine, leucine and proline (25 mM). Phosphorylation of glycogen synthase kinase‐3β was unaffected by insulin stimulation and amino acid treatment. Our results indicate that glutamine, leucine and proline attenuate IL‐8 production, probably through inhibition of NF‐κB, and that they increase Akt phosphorylation in HepG2 cells.     

Ginger Extract Suppresses Inflammatory Response and Maintains Barrier Function in Human Colonic Epithelial Caco‐2 Cells Exposed to Inflammatory Mediators

The beneficial effects of ginger in the management of gastrointestinal disturbances have been reported. In this study, the anti‐inflammatory potential of ginger extract was assessed in a cellular model of gut inflammation. In addition, the effects of ginger extract and its major active compounds on intestinal barrier function were evaluated. The response of Caco‐2 cells following exposure to a mixture of inflammatory mediators [interleukin [IL]‐1β, 25 ng/mL; lipopolysaccharides [LPS], 10 ng/mL; tumor necrosis factor [TNF]‐α, 50 ng/mL; and interferon [INF]‐γ, 50 ng/mL] were assessed by measuring the levels of secreted IL‐6 and IL‐8. In addition, the mRNA levels of cyclooxygenase‐2 and inducible nitric oxide synthase were measured. Moreover, the degree of nuclear factor (NF)‐κB inhibition was examined, and the intestinal barrier function was determined by measuring the transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)‐dextran transfer. It was observed that ginger extract and its constituents improved inflammatory responses by decreasing the levels of nitrite, PGE2, IL‐6, and IL‐8 via NF‐κB inhibition. The ginger extract also increased the TEER and decreased the transfer of FITC‐dextran from the apical side of the epithelium to the basolateral side. Taken together, these results show that ginger extract may be developed as a functional food for the maintenance of gastrointestinal health.     

Dietary Trans Fats Enhance Doxorubicin‐Induced Cardiotoxicity in Mice

This study investigated the combined effects of trans fat diet (TFD) and doxorubicin upon cardiac oxidative, inflammatory, and coagulatory stress. TFD increased trans fatty acid deposit in heart (P < 0.05), and decreased protein C and antithrombin‐III activities in circulation (P < 0.05). TFD plus doxorubicin treatment elevated activities of plasminogen activator inhibitor‐1, lactate dehydrogenase, and creatine phosphokinase (P < 0.05). This combination also raised xanthine oxidase activity, and enhanced cardiac levels of reactive oxygen species, interleukin (IL)‐6, IL‐10, tumor necrosis factor‐alpha, and monocyte chemoattractant protein‐1 than TFD or doxorubicin treatment alone (P < 0.05). TFD alone increased cardiac nuclear factor kappa B (NF‐κB) activity (P < 0.05), but failed to affect expression of NF‐κB and mitogen‐activated protein kinase (MAPK) (P > 0.05). Doxorubicin treatment alone augmented cardiac activity, mRNA expression, and protein production of NF‐κB and MAPK (P < 0.05). TFD plus doxorubicin treatment further upregulated cardiac expression of NF‐κB p65, p‐p38, and p‐ERK1/2 (P < 0.05). These findings suggest that TFD exacerbates doxorubicin‐induced cardiotoxicity.     

The postprandial inflammatory response after ingestion of heated oils in obese persons is reduced by the presence of phenol compounds

Scope Heating during the process of cooking alters the chemical properties of foods and may affect subsequent postprandial inflammation. We tested the effects of four meals rich in different oils subjected to heating on the postprandial inflammatory metabolism of peripheral blood mononuclear cells (PBMCs). Methods and results Twenty obese participants received four breakfasts following a randomized crossover design, consisting of milk and muffins made with different oils (virgin olive oil (VOO), sunflower oil (SFO), and a mixture of seeds oil (SFO/canola oil) with added either dimethylpolysiloxane (SOD), or natural antioxidants from olive mill wastewater alperujo (phenols; SOP)), previously subjected to 20 heating cycles. Postprandial inflammatory status in PBMCs was assessed by the activation of nuclear NF‐κB, the concentration in cytoplasm of the NF‐κB inhibitor (IκB‐α), the mRNA levels of NF‐κB subunits and activators (p65, IKKβ, and IKKα) and other inflammatory molecules (TNF‐α, IL‐1β, IL‐6, MIF, and JNK), and lipopolysaccharide (LPS) levels. VOO and SOP breakfasts reduced NF‐κB activation, increased IκB‐α, and decreased LPS plasma concentration. SFO increased IKKα, IKKβ, p65, IL‐1b, IL‐6, MIF, and JNK mRNA levels, and plasma LPS Conclusion Oils rich in phenols, whether natural (VOO) or artificially added (SOP), reduce postprandial inflammation, compared with seed oil (sunflower).     

Salvianolic Acid B Inhibits High‐Fat Diet‐Induced Inflammation by Activating the Nrf2 Pathway

Salvianolic acid B (Sal B) is a major water‐soluble bioactive component of Salvia miltiorrhiza, which is a traditional Chinese medicine. We investigated the ways in which Sal B affects high‐fat diet (HFD)‐induced immunological function disorder remission using a C57BL/6 mouse model. We gave groups of C57BL/6 mice a normal diet (Control), a normal diet supplemented with Sal B (Control + Sal B), a high‐fat diet (HF), and a high‐fat diet supplemented with Sal B (HF + Sal B) for 10 wk. Sal B supplementation decreased the body weight and plasma lipids, increased the fecal excretion of lipids, prevented the accumulation of chronic oxidative stress, and reversed the disproportionality of CD3⁺CD4⁺ and CD3⁺CD8⁺ T lymphocytes compared to HFD. We found an increase in IL‐6 and TNF‐α, while IL‐10 decreased in plasma after the HFD and Sal B reversed the deregulation of the Thl/Th2 ratio. In addition, HFD‐induced inflammation was stopped by Sal B through the downregulation of nuclear factor‐κB (NF‐κB), cyclooxygenase‐2 (COX‐2), and inducible NO synthesis (iNOS), and the upregulation of nuclear factor‐erythroid 2‐related factor 2 (Nrf2)‐regulated genes. These findings demonstrated that Sal B could effectively attenuate inflammation by activating the Nrf2‐mediated antioxidant defense system.     

Anti‐Diabetic Effects of Gynura Bicolor Aqueous Extract in Mice

The effects of Gynura bicolor aqueous extract (GAE) upon glycemic control, coagulation disorder, lipid accumulation, and glycative, oxidative, and inflammatory stresses in diabetic mice were investigated. Mice were treated with streptozotocin to induce type 1 diabetes. Diabetic mice were divided into four groups, consumed GAE at 0%, 0.25%, 0.5%, or 1%. Normal group consumed standard mouse basal diet. After 8‐week treatments, mice were sacrificed after overnight fasting. Results showed that GAE supplement at 0.5% and 1% decreased plasma glucose level and increased plasma insulin level. Diabetes lowered plasma level of protein C and anti‐thrombin III; and raised plasminogen activator inhibitor‐1 activity and fibrinogen level in plasma. GAE supplement at 0.5% and 1% reversed these alterations. Histological data, assayed by Oil Red O stain, indicated that GAE supplement decreased lipid accumulation in liver. GAE supplement at 0.5% and 1% reduced aldose reductase activity in heart and kidney; and lowered the levels of carboxymethyllysine and pentosidine in plasma and two organs. Diabetes decreased glutathione content, and increased reactive oxygen species, interleukin (IL)‐1β, IL‐6, and tumor necrosis factor‐α production in heart and kidney. GAE supplement at three test doses reversed these changes. Diabetes upregulated the mRNA expression of p38 and nuclear factor kappa (NF‐κ)B in heart and kidney. GAE supplement suppressed the mRNA expression of both p38 and NF‐κB. These novel findings suggest that Gynura bicolor is a potent functional food for diabetic prevention or alleviation.     

Polygonum viviparum L. inhibits the lipopolysaccharide‐induced inflammatory response in RAW264.7 macrophages through haem oxygenase‐1 induction and activation of the Nrf2 pathway

BACKGROUND: Polygonum viviparum L. (PV) is a member of the family Polygonaceae and is widely distributed in high‐elevation areas. It is used as a folk remedy to treat inflammation‐related diseases. This study was focused on the anti‐inflammatory response of PV against lipopolysaccharide (LPS)‐induced inflammation in RAW264.7 macrophages. RESULTS: Treatment with PV did not cause cytotoxicity at 0–50 µg mL^?1 in RAW264.7 macrophages, and the IC₅₀ value was 270 µg mL^?1. PV inhibited LPS‐stimulated nitric oxide (NO), prostaglandin (PG)E₂, interleukin (IL)‐1β and tumour necrosis factor (TNF)‐α release and inducible NO synthase (iNOS) and cyclooxygenase (COX)‐2 protein expression. In addition, PV suppressed the LPS‐induced p65 expression of nuclear factor (NF)‐κB, which is associated with the inhibition of IκB‐α degradation. These results suggest that, among mechanisms of the anti‐inflammatory response, PV inhibits the production of NO and these cytokines by down‐regulating iNOS and COX‐2 gene expression. Furthermore, PV can induce haem oxygenase (HO)‐1 protein expression through nuclear factor E2‐related factor 2 (Nrf2) activation. A specific inhibitor of HO‐1, zinc(II) protoporphyrin IX, inhibited the suppression of iNOS and COX‐2 expression by PV. CONCLUSION: These results suggest that PV possesses anti‐inflammatory actions in macrophages and works through a novel mechanism involving Nrf2 actions and HO‐1. Thus PV could be considered for application as a potential therapeutic approach for inflammation‐associated disorders. © 2012 Society of Chemical Industry     

Sesamin attenuates intercellular cell adhesion molecule‐1 expression in vitro in TNF‐α‐treated human aortic endothelial cells and in vivo in apolipoprotein‐E‐deficient mice

Sesame lignans have antioxidative and anti‐inflammatory properties. We focused on the effects of the lignans sesamin and sesamol on the expression of endothelial‐leukocyte adhesion molecules in tumor necrosis factor‐α (TNF‐α)‐treated human aortic endothelial cells (HAECs). When HAECs were pretreated with sesamin (10 or 100 μM), the TNF‐α‐induced expression of intercellular cell adhesion molecule‐1 (ICAM‐1) was significantly reduced (35 or 70% decrease, respectively) by Western blotting. Sesamol was less effective at inhibiting ICAM‐1 expression (30% decrease at 100 μM). Sesamin and sesamol reduced the marked TNF‐α‐induced increase in human antigen R (HuR) translocation and the interaction between HuR and the 3'UTR of ICAM‐1 mRNA. Both significantly reduced the binding of monocytes to TNF‐α‐stimulated HAECs. Sesamin significantly attenuated TNF‐α‐induced ICAM‐1 expression and cell adhesion by downregulation of extracellular signal‐regulated kinase 1/2 and p38. Furthermore, in vivo, sesamin attenuated intimal thickening and ICAM‐1 expression seen in aortas of apolipoprotein‐E‐deficient mice. Taken together, these data suggest that sesamin inhibits TNF‐α‐induced extracellular signal‐regulated kinase/p38 phosphorylation, nuclear translocation of NF‐κB p65, cytoplasmic translocalization of HuR and thereby suppresses ICAM‐1 expression, resulting in reduced adhesion of leukocytes. These results also suggest that sesamin may prevent the development of atherosclerosis and inflammatory responses.     

Immunomodulatory properties of the milk whey products obtained by enzymatic and microbial hydrolysis

The objective of this study was to investigate the in vitro immunomodulatory effect of milk whey through enzymatic hydrolysis, microbial fermentation and two‐stage hydrolytic reactions (enzymatic and microbial reactions) by analysing cytokine profiles. Results indicated that the milk whey sample fermented by Lactobacillus kefiranofaciens M1 and two‐stage hydrolytic reactions (hydrolysed by Alcalase and then fermented by L. kefiranofaciens M1) could significantly induce the production of tumour necrosis factor (TNF)‐α and IL‐12 compared with the milk whey control and enzymatic treatments. Further characterisation of the immunomodulatory factor by membrane filtration and mutanolysin hydrolysation, the stimulatory activity for IL‐12 and TNF‐α production was found to be reduced and to be correlated positively with the cell wall components in L. kefiranofaciens M1. In addition, Th2‐polarised splenocytes revealed that L. kefiranofaciens M1 had both IL‐12 inducing and IL‐4 repressing activities. These results suggested that L. kefiranofaciens M1 could direct the Th1/Th2 balance toward Th1.     

Resveratrol Regulates Activated Hepatic Stellate Cells by Modulating NF‐κB and the PI3K/Akt Signaling Pathway

In the present study, we investigated whether resveratrol could suppress the hepatic fibrogenesis in activated hepatic stellate cells. The immortalized rat hepatic stellate cells, t‐HSC/Cl‐6, were treated with resveratrol 1 h prior to lipopolysaccharide (LPS, 1 μg/mL). Resveratrol decreased t‐HSC/Cl‐6 cell viability at much lower concentrations within 24 h. Resveratrol pretreatment also decreased the LPS‐induced protein expression of α‐SMA and collagen I. In addition, resveratrol significantly reduced the protein expression of Toll‐like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88), and the expression of phosphorylated phosphatidylinositol 3‐kinase (PI3K) and phosphorylated serine/threonine kinase B (Akt). Moreover, resveratrol markedly blocked the translocation of nuclear factor (NF)‐κB in LPS‐activated HSCs. Furthermore, resveratrol inhibited HSCs activation through stimulating LXRβ, but did not influence LXRα. Overall, we conclude that the antifibrotic effect of resveratrol is the result of blocking NF‐κB activation and PI3K/Akt phosphorylation, which inhibits HSC activation to obstruct liver fibrosis. Thus, resveratrol may be a natural agent for preventing hepatic fibrosis.     

Suppressive effects of extracts from the aerial part of Coriandrum sativum L. on LPS‐induced inflammatory responses in murine RAW 264.7 macrophages

BACKGROUND: Coriandrum sativum is used not only as a spice to aid flavour and taste values in food, but also as a folk medicine in many countries. Since little is known about the anti‐inflammatory ability of the aerial parts (stem and leaf) of C. sativum, the present study investigated the effect of aerial parts of C. sativum on lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophages. We further explored the molecular mechanism underlying these pharmacological properties of C. sativum. RESULTS: Ethanolic extracts from both stem and leaf of C. sativum (CSEE) significantly decreased LPS‐induced nitric oxide and prostaglandin E₂ production as well as inducible nitric oxide synthase, cyclooxygenase‐2, and pro‐interleukin‐1β expression. Moreover, LPS‐induced IκB‐α phosphorylation and nuclear p65 protein expression as well as nuclear factor‐κB (NF‐κB) nuclear protein–DNA binding affinity and reporter gene activity were dramatically inhibited by aerial parts of CSEE. Exogenous addition of CSEE stem and leaf significantly reduced LPS‐induced expression of phosphorylated mitogen‐activated protein kinases (MAPKs). CONCLUSION: Our data demonstrated that aerial parts of CSEE have a strong anti‐inflammatory property which inhibits pro‐inflammatory mediator expression by suppressing NF‐κB activation and MAPK signal transduction pathway in LPS‐induced macrophages. Copyright © 2010 Society of Chemical Industry     

Bovine casein peptides co‐stimulate naive macrophages with lipopolysaccharide for proinflammatory cytokine production and nitric oxide release

Three bovine casein peptides, ie LLY, PGPIPN and TTMPLW, were used to investigate their effects on cytokine (TNF‐α and IL‐6) production and nitric oxide (NO) release by murine bone marrow macrophages (BMMs). The results showed that these peptides alone were incapable of stimulating cytokine production or NO release in naive or IFN‐γ‐primed BMMs. However, when BMMs were co‐incubated with the peptides at a concentration of 1.0 µM and lipopolysaccharide (LPS; 100 ng ml⁻¹), an augmentative effect on TNF‐α, IL‐6 and NO production was observed. Of the three peptides, TTMPLW had the greatest augmentative effect on NO production by LPS‐stimulated BMMs and induced the highest amount of TNF‐α production at a concentration of 1.0 µM . All the peptides at a concentration of 1.0 µM stimulated IL‐6 production by BMMs. TNF‐α was neutralised by anti‐TNF‐α monoclonal antibody and the release of NO was reduced by about 33.3% (p < 0.01). These results demonstrate that bovine casein peptides can co‐stimulate naive macrophages with LPS for proinflammatory cytokine production and NO release and may play a role in host defence against pathogens. © 2000 Society of Chemical Industry     

Mung Bean Protein Hydrolysate Modulates the Immune Response Through NF‐κB Pathway in Lipopolysaccharide‐Stimulated RAW 264.7 Macrophages

The objective of this study was to evaluate the immunomodulatory activity of mung bean protein hydrolysate (MBPH) in lipopolysaccharide (LPS)‐induced RAW 264.7 cells and discuss the possible immune regulatory mechanism. MBPH was prepared by alcalase, trypsin, neutrase, and flavourzyme. The 3‐h alcalase‐hydrolyzed hydrolysate with a molecular weight less than 1,450 Da was selected for the immunological tests. Results showed that MBPH possessed strong suppressing activity to proinflammatory mediators in a dose‐dependent manner. Compared to the LPS alone group, MBPH (200 µg/mL) significantly reduced nitric oxide (NO), inducible nitric oxide synthase, interleukin (IL)‐6, and IL‐1β secretion levels by 52.6%, 53.2%, 48.4%, and 49.7%, respectively, in LPS‐induced macrophages. It also enhanced IL‐10 secretion from 789 to 3,678 pg/mL. MBPH blocked nuclear factor‐kappa B (NF‐κB) translocation in LPS‐induced macrophages through the prevention of IκBα phosphorylation, and this process further prevented p65 translocation into the nucleus. A possible mechanism of MBPH is that it regulated the expression of inflammatory factors via the NF‐κB pathway, thus inhibiting inflammatory reactions. The results suggested that MBPH is of application potential in the development of immunomodulatory functional food to ameliorate immunosuppression.     

The preventive role of breadfruit against inflammation‐associated epithelial carcinogenesis in mice

Artocarpus communis has been identified as a rich source of flavonoids and has been gaining attention for its potential chemopreventive abilities. In this study, methanol extracts from the fruit of A. communis (MEFA) and leaf of A. communis (MELA) were prepared, and their effects on inflammation‐associated skin tumorigenesis were assessed using mouse models, including 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) induced cutaneous inflammation as well as 7,12‐dimethylbenz[α]anthracene (DMBA) initiated and TPA‐promoted skin tumorigenesis. According to the results, both MEFA and MELA decreased the intensity of leukocyte infiltration in mouse dorsal skin and cutaneous edema induced by TPA, which appeared to be mediated by inhibition of proinflammatory genes (inducible nitric oxide synthase, cyclooxygenase‐2 (COX‐2), tumor necrosis factor‐α (TNF‐α), IL‐1β, and IL‐6) and proinflammatory mediators (TNF‐α, IL‐1β, and Prostaglandin E₂). In addition, topical application with MEFA or MELA effectively attenuated tumor incidence, multiplicity, volume, malignancy as well as angiogenesis of TPA‐stimulated skin tumor promotion in DMBA‐initiated mice. Notably, immunohistochemical stain showed that MEFA and MELA attenuated COX‐2 expression of both skin and tumor tissues in different animal tests, which may be closely related to the suppression of nuclear factor kappa B/activator protein signaling networks. These findings first demonstrate that flavonoid‐rich A. communis may exert potent anti‐inflammatory activity through modulation of COX‐2 in TPA‐activated skin and tumor tissues.     

Carbonyl compounds methylglyoxal and glyoxal affect interleukin‐8 secretion in intestinal cells by superoxide anion generation and activation of MAPK p38

The carbonyl compounds methylglyoxal (MG) and glyoxal (GL) are reactive intermediates of glucose degradation pathways and capable of inducing cellular damage. Although immune‐stimulating activity has been investigated in endothelial cells, little is known about the signaling pathways of cytokine induction of these compounds in the intestine. Hence, we investigated the impact of mitogen‐activated protein kinases (MAPK) and nuclear factor kappa B (NF‐κB) on IL‐8 production by human intestinal cells (Caco‐2 and HT‐29) after stimulation by MG and GL. Both compounds induced a dose‐dependent enhancement of IL‐8 secretion in human intestinal cells. MAPK p38 and extracellular signal‐regulated kinase (ERK) were phosphorylated in these cells after having been stimulated by MG and GL. Furthermore, inhibitors of MAPK p38 (SB 203580 and 239063), ERK1/2 (PD 98059) and NF‐κB activation (SM‐7368 and SC‐514) reduced IL‐8 secretion. The most important mechanism by which MG and GL induced IL‐8 secretion was the generation of superoxide anions which was confirmed by the inhibition of the cytosolic NADPH oxidase with diphenyl iodonium (DPI) or by application of superoxide dismutase (SOD). Our data suggest that multiple pathways were simultaneously activated; however, superoxide dependent MAPK p38 activation seems to be the most dominant pathway for IL‐8 secretion in intestinal cells.