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.     

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.     

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.     

Suppression of IL‐8 Release by Sweet Olive Ethanolic Extract and Compounds in WiDr Colon Adenocarcinoma Cells

Oxidative stress can stimulate the secretion of pro‐inflammatory cytokines. Interleukin‐8 (IL‐8) has been implicated in the pathogenesis of inflammatory bowel disease and the metastatic spread of colorectal cancer. The flowers of Osmanthus fragrans (sweet olive) are used to alleviate dysentery with blood in the bowel, as well as stomach ache and diarrhea. However, the evidence of their therapeutic effects on these symptoms remains unclear. In the present study, the protective effects of sweet olive flower ethanolic extract (OFE) against oxidative stress in WiDr cells was assessed by evaluating its 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) free radical scavenging activity. In addition, cellular IL‐8 secretion was evaluated. Notably, high‐performance liquid chromatography showed verbascoside to be the primary constituent in OFE; it exhibited a DPPH scavenging activity with an IC₅₀ of 8.23 μg/mL. Moreover, OFE (1 to 100 μg/mL) showed a potent, dose‐dependent inhibitory effect on H₂O₂‐induced IL‐8 secretion in WiDr cells. Nine compounds were isolated from OFE based on a protective effect‐guided purification process. Of these compounds, 5 phenolic compounds—verbascoside, phillygenin, tyrosol, methyl 4‐hydroxycinnamate, and eutigoside A—reduced IL‐8 secretion at 10 μg/mL treatment concentrations. Further analysis showed that the anti‐inflammatory effects of OFE likely occurred via nuclear factor‐κB pathway inhibition, which attenuates IL‐8 secretion in cells. Collectively, these data suggest that OFE could be developed as an agent that suppresses IL‐8 secretion to treat chronic inflammatory diseases.     

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.     

Characterization,Anti‐Inflammatory and Antiproliferative Activities of Natural and Sulfonated Exo‐Polysaccharides from Streptococcus thermophilus ASCC 1275

Exo‐polysaccharides (EPS) isolated from Streptococcus thermophilus ASCC 1275 were sulfated (31%). High‐performance liquid chromatography identified that EPS was composed of mannose (30.19%), galactose (20.10%), glucose (18.05%), glucosamine (16.04%), galactosamine (9.06%), glucuronic acid (3.55%), and ribose (3.01%). Pro‐/anti‐inflammatory cytokine secretion ratios (IL‐1β/IL‐10, IL‐6/IL‐10, and TNF‐α/IL‐10) of lipopolysaccharide stimulated RAW 264.7 macrophages were significantly decreased by EPS and S.EPS treatments in a dose dependent manner. Furthermore, anti‐inflammatory activities of S.EPS improved 49.3% and 24.0% than those of EPS before or after LPS treatment. The reactive oxygen species were inhibited by EPS and S.EPS by 49.6% and 55.1% at 50 μg/mL, respectively. Inhibition activities of S.EPS on nitric oxide production were 12.9% and 55.4% higher than those of EPS at 10 and 50 μg/mL. Additionally, S.EPS exhibited stronger antiproliferative activity on Caco‐2 and HepG2 cells. Our results indicated that anti‐inflammatory and antiproliferative activities of EPS were significantly (P < 0.01) improved by sulfonation.     

婴儿源双歧杆菌对人胎结肠上皮细胞的增殖作用及机制研究

分析12株婴儿源双歧杆菌的发酵上清液和破碎物对人胎结肠上皮细胞(CCD841 CoN)的增殖效果。筛选出对细胞增殖促进效果最好的菌株,提取其表面蛋白和分泌蛋白,检测这两类蛋白对细胞增殖、周期及增殖相关基因表达的影响,探讨其对CCD841 CoN细胞增殖促进的作用。结果表明:筛选出双歧杆菌H3-R2,其浓度为50 μg/mL表面蛋白和10 μg/mL分泌蛋白与CCD841 CoN细胞共作24 h后,对细胞有显著增殖促进作用(P<0.05),提高细胞S期比例,降低G0/G1期比例(P<0.05),提高细胞内 β-catenin、c-Myc和Cyclin D1基因mRNA的转录水平(P<0.05),降低 Axin2、GSK-3β 基因mRNA的转录水平(P<0.05)。综上,婴儿源双歧杆菌可在mRNA转录水平上对人胎结肠上皮细胞有增殖促进作用,具有促进新生儿肠道发育的潜在应用价值。     

Anti‐inflammatory Potential of Quercetin‐3‐O‐β‐D‐(“2”‐galloyl)‐glucopyranoside and Quercetin Isolated from Diospyros kaki calyx via Suppression of MAP Signaling Molecules in LPS‐induced RAW 264.7 Macrophages

Diospyros kaki (DK) contains an abundance of flavonoids and has been used in folk medicine in Korea for centuries. Here, we report for the first time the anti‐inflammatory activities of Quercetin (QCT) and Quercetin 3‐O‐β‐(“2”‐galloyl)‐glucopyranoside (Q32G) isolated from DK. We have determine the no cytotoxicity of Q32G and QCT against RAW 264.7 cells up to concentration of 50 μM. QCT and Q32G demonstrated potent anti‐inflammatory activities by reducing expression of nitric oxide (NO), tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6 inducible NO synthase (iNOS), cyclooxygenase (COX)‐2, and mitogen‐activated protein kinase (MAPKs) in mouse RAW 264.7 macrophages activated with lipopolysaccharide (LPS). Both QCT or Q32G could decrease cellular protein levels of COX‐2 and iNOS as well as secreted protein levels of NO, PGE₂, and cytokines (TNF‐α, IL‐1β, and IL‐6) in culture medium of LPS‐stimulated RAW 264.7 macrophages. Immunoblot analysis showed that QCT and Q32G suppressed LPS‐induced MAP kinase pathway proteins p‐p38, ERK, and JNK. This study revealed that QCT and Q32G have anti‐inflammatory potential, however Q32G possess comparable activity as that of QCT and could be use as adjuvant to treat inflammatory diseases.     

Phenethyl isothiocyanate suppresses nitric oxide production via inhibition of phosphoinositide 3‐kinase/Akt‐induced IFN‐γ secretion in LPS‐activated peritoneal macrophages

Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, is well known to have versatile physiological activities, including chemopreventive effects. On the other hand, its anti‐inflammatory effects are poorly reported. Nitric oxide (NO) is associated with a wide variety of inflammatory diseases. In this study, we investigated the effects of PEITC on NO production in LPS‐activated peritoneal macrophages from ICR mice. The signaling pathway of LPS‐induced NO production was examined using neutralizing antibodies [anti‐interferon (IFN)‐γ and anti‐interleukin (IL‐12)] and specific protein kinase inhibitors, as well as others. The activity of PEITC toward NOx production was assessed in mice that received LPS via intraperitoneal administration. The neutralizing antibody of anti‐IFN‐γ, but not anti‐IL‐12, suppressed LPS‐induced NO production by 90%. LY294002, a specific inhibitor of phosphoinositide‐3‐kinase, suppressed Akt and IFN‐γ mRNA expression up‐regulated by LPS, whereas PEITC exhibited a similar inhibition profile. Furthermore, oral administration of PEITC significantly suppressed the serum concentration of NOx in ICR mice. Our results suggest that PEITC suppresses LPS‐induced NO production via inhibition of Akt activation and the resultant decrease in expression of IFN‐γ. This is one of the first reports to demonstrate a marked anti‐inflammatory effect of PEITC following its oral administration.     

Enhanced Anti‐Inflammatory Activities by the Combination of Luteolin and Tangeretin

Dietary components in combination may act synergistically and produce enhanced biological activities. Herein, we investigated the anti‐inflammatory effects of 2 flavonoids, that is luteolin (LUT) and tangeretin (TAN) in combination. Lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophages were treated with noncytotoxic concentrations of LUT, TAN, and their combinations. The results showed that LUT/TAN in combination produced synergistic inhibitory effects on LPS‐stimulated production of nitric oxide (NO). ELISA results demonstrated that LUT/TAN in combination caused stronger suppression on the LPS‐induced overexpression of proinflammatory mediators, such as prostaglandin E₂ (PGE₂), interleukin (IL)‐1β, and IL‐6 than LUT or TAN alone. Immunoblotting and Real‐Time PCR analyses showed that LUT/TAN combination significantly decreased LPS‐induced protein and mRNA expression of inducible nitric oxide synthase and cyclooxygenase‐2. These inhibitory effects of the combination treatment were stronger than those produced by LUT or TAN alone. Overall, our results demonstrated for the first time that combination of LUT and TAN produced synergistic anti‐inflammatory effects in LPS‐stimulated RAW 264.7 macrophages.     

Absorption of anthocyanins through intestinal epithelial cells – Putative involvement of GLUT2

Anthocyanins bioavailability is a major issue regarding their biological effects and remains unclear due to few data available on this matter. This work aimed to evaluate the absorption of anthocyanins at the intestine using Caco‐2 cells. Anthocyanin extract, rich in malvidin‐3‐glucoside, was obtained from red grape skins and tested on Caco‐2 cells. The absorption of anthocyanins, in absence or presence of 1% ethanol, was detected by HPLC/DAD/LC‐MS. Our results showed that this transport was significantly increased in the presence of ethanol especially after 60 min of incubation. In addition, cells that were pretreated for 96 h with anthocyanins (200 μg/mL) showed an increase of their own transport (about 50% increase). Expression of glucose transporters sodium‐dependent glucose transporter 1, facilitative glucose transporters 5, and facilitative glucose transporters 2 was assessed by RT‐PCR. It was found that facilitative glucose transporters 2 expression was increased (60%) in Caco‐2 cells pretreated with anthocyanins, by comparison with controls. When the effect of anthocyanin extract on ³H‐2‐deoxy‐D ‐glucose uptake was tested, an inhibitory effect was observed (about 60% decrease). However, the malvidin aglycone was tested and had no effect. In conclusion, anthocyanins could be absorbed through Caco‐2 cells, and can interfere with their own transport and also with glucose intestinal uptake.     

Apple extract induces increased epithelial resistance and claudin 4 expression in Caco-2 cells

BACKGROUND: The small intestinal epithelium functions both to absorb nutrients, and to provide a barrier between the outside, luminal, world and the human body. One of the passageways across the intestinal epithelium is paracellular diffusion, which is controlled by the properties of tight junction complexes. We used a differentiated Caco‐2 monolayer as a model for small intestinal epithelium to study the effect of crude apple extracts on paracellular permeability. RESULTS: Exposure of crude apple homogenate to the differentiated Caco‐2 cells increased the paracellular resistance, determined as trans‐epithelial electrical resistance (TEER). This increase was linearly related to the concentration of apple present. The TEER‐enhancing effect of apple extract was due to factors mainly present in the cortex, and the induction was not inhibited by protein kinase inhibitors. Apple‐induced resistance was accompanied by increased expression of several tight junction related genes, including claudin 4 (CLDN4). CONCLUSION: Crude apple extract induces a higher paracellular resistance in differentiated Caco‐2 cells. Future research will determine whether these results can be extrapolated to human small intestinal epithelia. Copyright © 2011 Society of Chemical Industry     

Determination of S‐methyl‐L‐methionine (SMM) from Brassicaceae Family Vegetables and Characterization of the Intestinal Transport of SMM by Caco‐2 Cells

The objectives of the current study were to determine S‐methyl‐L‐methionine (SMM) from various Brassicaceae family vegetables by using validated analytical method and to characterize the intestinal transport mechanism of SMM by the Caco‐2 cells. The SMM is well known to provide therapeutic activity in peptic ulcers. The amount of SMM from various Brassicaceae family vegetables ranged from 89.08 ± 1.68 μg/g to 535.98 ± 4.85 μg/g of dry weight by using validated ultra‐performance liquid chromatography‐electrospray ionization‐mass spectrometry method. For elucidating intestinal transport mechanism, the cells were incubated with or without transport inhibitors, energy source, or a metabolic inhibitor. Phloridzin and verapamil as inhibitors of sodium glucose transport protein (SGLT1) and P‐glycoprotein, respectively, were not responsible for cellular uptake of SMM. Glucose and sodium azide were not affected by the cellular accumulation of SMM. The efflux ratio of SMM was 0.26, implying that it is not effluxed through Caco‐2 cells. The apparent coefficient permeability (P _app) of SMM was 4.69 × 10^?5 cm/s, indicating that it will show good oral absorption in in vivo .     

柚叶总黄酮对LPS所致Caco-2结肠上皮细胞间高通透性的保护作用

探讨柚叶总黄酮(PLTFE)对脂多糖(LPS,2 μg/mL)诱发Caco-2高通透性细胞模型的保护作用。Caco-2模型细胞以不同浓度的PLTFE(0、10、50、100、150 μg/mL)处理培养24 h进行后续实验。MTT法测定细胞生存率,细胞乳酸脱氢酶(lactate dehydrogenase,LDH)水平依说明书使用试剂盒测定。酶联法(enzyme linked immunosorbent assay,ELISA)测定白介素(interlukin-1β,IL-1β)、IL-8和肿瘤坏死因子(tumor necrosis factor-α,TNF-α)分泌水平。跨上皮细胞电阻(trans epithelial electrical resistance,TEER)值和异硫氰酸荧光素-右旋糖酐(FD40)透过度用于评估细胞通透性水平。实时定量PCR(Quantitative real-time PCR,qRT-PCR)检测细胞IL-1β、IL-8、TNF-α、闭锁蛋白(Occludin)、紧密连接蛋白-1(claudin-1)、封闭小带蛋白(ZO-1)和肌球蛋白轻链激酶(myosin light chain kinase,MLCK)的mRNA表达。结果表明,柚叶总黄酮能显著提高受损细胞生存率至86.1%,抑制受损细胞中LDH的溢出(p<0.05)。同时还能有效抑制受损细胞中炎性细胞因子(IL-1β、IL-8、TNF-α)的分泌及mRNA转录。此外,柚叶总黄酮可增强细胞紧密连接因子(Occludin、claudin-1、ZO-1)的mRNA转录,抑制MLCK的mRNA转录,改善细胞间高通透性qRT-PCR法检测细胞中相关因子的mRNA转录水平。结果提示,柚叶总黄酮具有较强的抗炎活性,能通过上调细胞内细胞紧密连接相关因子的mRNA转录显著改善LPS造成的Caco-2细胞间高通透性的发生(p<0.05)。