Suppression of MyD88- and TRIF-dependent signaling pathways of Toll-like receptor by (-)-epigallocatechin-3-gallate, a polyphenol component of green tea

Toll-like receptors (TLRs) play an important role in recognition of microbial components and induction of innate immunity. The microbial components trigger the activation of two downstream signaling pathways of TLRs; MyD88- and/or TRIF-dependent pathways leading to activation of NF-kappaB. (-)-Epigallocatechin-3-gallate (EGCG), a flavonoid found in green tea, is known to inhibit NF-kappaB activation induced by many pro-inflammatory stimuli. EGCG was shown to inhibit the activity of IKKbeta which is the key kinase in the canonical pathway for NF-kappaB activation in MyD88-dependent pathway of TLRs. However, it is not known whether EGCG inhibits TRIF-dependent pathway through which more than 70% of lipopolysaccharide (LPS)-induced genes are regulated. Therefore, we attempted to identify the molecular target of EGCG in TRIF-dependent pathways of TLR3 and TLR4. EGCG inhibited the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C], or the overexpression of TRIF. The inhibition of IRF3 activation by EGCG was mediated through the suppression of the kinase activity of TBK1. However, EGCG did not inhibit activation of IRF3 induced by overexpression of constitutively active IRF3. These results suggest that the molecular target of EGCG is TBK1 in TRIF-dependent signaling pathways of TLR3 and TLR4. Therefore, our results suggest that green tea flavonoids can modulate both MyD88- and TRIF-dependent signaling pathways of TLRs and subsequent inflammatory target gene expression.     

Transdermal delivery of (-)-epigallocatechin-3-gallate, a green tea polyphenol, in mice

Epigallocatechin-3-gallate (EGCG) is the most studied catechin in green tea (Camellia sinensis). EGCG and green tea are cancer preventive in many animal models, and numerous mechanisms have been proposed in cell lines. EGCG is poorly bioavailable in man and rodents. We hypothesized that transdermal delivery of EGCG could result in improved bioavailability. Following application of EGCG transdermal gel (50 mg kg(-1), t.d.) to SKH-1 mice, EGCG was observed in the epidermis (1365.7-121.0 ng g(-1)) and dermis (411.2-42.6 ng g(-1)). The maximum plasma concentration (Cmax) of EGCG was 44.5 ng mL(-1). The t(1/2) (94.4 h) and AUC(0-->24 h) (881.5 ng mL(-1) h) of EGCG were greater than values previously reported for oral EGCG. The t(1/2) and area under the concentration-time curve up to 24 h (AUC(0-->24 h)) in the liver, small intestine and colon were 21.3-74.6 h and 715-2802 ng g(-1)h, respectively. Stability studies showed that the transdermal formulation was stable at 4 degrees C and had a half-life (t(1/2)) of 47.1 and 20.2 h at 25 degrees C and 37 degrees C, respectively. These data indicate that transdermal EGCG is useful for delivering prolonged levels of EGCG to plasma and tissues, and may provide an alternative to tea consumption as a dosage form of EGCG.     

表没食子儿茶素没食子酸酯对脂肪肝细胞甲基化转移酶的调控作用

目的 探讨表没食子儿茶素没食子酸酯(EGCG)降低脂肪肝细胞脂肪含量的DNA甲基化调控机制,为应用EG CG防治脂肪肝提供实验依据.方法 利用游离脂肪酸棕榈酸处理大鼠正常肝细胞株BRL,建立大鼠脂肪肝细胞模型.用EGCG干预细胞72 h,检测处理前后脂肪肝细胞的脂肪含量及葡萄糖消耗量,用实时PCR方法检测EGCG作用前后DNA甲基化转移酶DNMT1、DNMT3a和DNMT3b的转录水平.结果 浓度为0.1 mmol/L的棕榈酸处理大鼠肝细胞BRL后,细胞的脂肪含量明显增加,吸光度(A)值从(0.22±0.04)增加到(0.31±0.06),差异具有统计学意义(P＜0.05),葡萄糖消耗量从(2.09±0.09)下降到(0.77±0.04),差异具有统计学意义(P＜0.01),即成功建立了脂肪肝细胞模型.EGCG干预脂肪肝细胞后,细胞的脂质含量明显减少,A值下降到(0.24±0.07) mM(P＜0.05),葡萄糖消耗量增加到(1.67 ±0.07)mM(P＜0.05),与DNA甲基化转移酶抑制剂5-Aza-2'-deoxycytidine(5-Aza-CdR)具有相同的效应.DNA甲基化转移酶DNMT1和DNMT3a基因在大鼠脂肪肝细胞中的mRNA表达水平分别增加了1.5和6倍(P＜0.05),EGCG和5-Aza-CdR均能够明显降低这两个基因的表达.DNMT3b在各处理组中没有明显差异.结论 EGCG可能通过抑制DNA甲基转移酶DNMT1和DNMT3a的表达,对脂肪和葡萄糖代谢中的关键基因去甲基化并使这些基因的表达上调,从而改善细胞内脂质沉积和胰岛素抵抗的状态.     

Green tea polyphenol, (−)-epigallocatechin-3-gallate,induces toxicity in human skin cancer cells by targeting β-catenin signaling

The green tea polyphenol, (−)-epigallocatechin-3-gallate (EGCG), has been shown to have anti-carcinogenic effects in several skin tumor models, and efforts are continued to investigate the molecular targets responsible for its cytotoxic effects to cancer cells. Our recent observation that β-catenin is upregulated in skin tumors suggested the possibility that the anti-skin carcinogenic effects of EGCG are mediated, at least in part, through its effects on β-catenin signaling. We have found that treatment of the A431 and SCC13 human skin cancer cell lines with EGCG resulted in reduced cell viability and increased cell death and that these cytotoxic effects were associated with inactivation of β-catenin signaling. Evidence of EGCG-induced inactivation of β-catenin included: (i) reduced accumulation of nuclear β-catenin; (ii) enhanced levels of casein kinase1α, reduced phosphorylation of glycogen synthase kinase-3β, and increased phosphorylation of β-catenin on critical serine^45,33/37 residues; and (iii) reduced levels of matrix metalloproteinase (MMP)-2 and MMP-9, which are down-stream targets of β-catenin. Treatment of cells with prostaglandin E2 (PGE₂) enhanced the accumulation of β-catenin and enhanced β-catenin signaling. Treatment with either EGCG or an EP2 antagonist (AH6809) reduced the PGE₂-enhanced levels of cAMP, an upstream regulator of β-catenin. Inactivation of β-catenin by EGCG resulted in suppression of cell survival signaling proteins. siRNA knockdown of β-catenin in A431 and SCC13 cells reduced cell viability. Collectively, these data suggest that induction of cytotoxicity in skin cancer cells by EGCG is mediated by targeting of β-catenin signaling and that the β-catenin signaling is upregulated by inflammatory mediators.     

The green tea polyphenol (−)-epigallocatechin-3-gallate inhibits leukocyte activation by bacterial formylpeptide through the receptor FPR

Although green tea polyphenol catechin is considered as a potential anti-inflammatory agent, its effect on bacterial component-induced inflammation has been poorly investigated. We examined the capacity of epigallocatechin gallate (EGCG) to regulate leukocyte responses to bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLF), which is recognized by a human G protein-coupled receptor FPR on phagocytic leukocytes. Pretreatment of human monocytic cells or FPR-transfected rat basophilic leukemia cells (ETFR cells) with EGCG significantly inhibited fMLF-induced chemotaxis. Intraperitoneal administration of EGCG in mice suppressed fMLF-induced leukocyte infiltration into the air pouch created in the skin. Mechanistic studies revealed that EGCG dose-dependently suppressed fMLF-induced calcium flux in monocytic cells and ETFR cells. fMLF-induced ETFR cell migration was significantly inhibited by a specific MEK1/2 inhibitor, PD98059, which was associated with reduction in fMLF-induced ERK1/2 phosphorylation. These results suggest that EGCG inhibits FPR-mediated leukocyte activation thus is a promising anti-inflammatory compound.     

The main catechin of green tea, (-)-epigallocatechin-3-gallate (EGCG), reduces bleomycin-induced DNA damage in human leucocytes.

Interest in the beneficial effects of green tea has led to investigations on activities by the main catechin (-)-epigallocatechin-3-gallate (EGCG). This antioxidative compound could contribute to cancer chemoprevention by acting antigenotoxic. To further explore this hypothesis we investigated antigenotoxic potentials of low EGCG concentrations in human peripheral leucocytes. Leucocytes isolated from whole blood were (1) stimulated with phytohaemagglutinin, (2) damaged with genotoxic bleomycin, and (3) post-incubated to allow DNA repair. After each phase DNA integrity was measured with the comet assay. EGCG (2, 20, 100 microM) was added either during phases 1, 2 or 3 or during the whole process (1-3), to delineate mechanisms of antigenotoxicity reflecting induction of detoxification (phase 1), scavenging of radicals (phase 2), stimulation of repair (phase 3), respectively. Bleomycin induced breaks and endonuclease III specific damage, but EGCG did not affect damage or repair of these lesions when added during phases 1, 2 or 3. However, the application of EGCG during phases 1 and 2 significantly reduced both bleomycin-induced breaks and endonuclease III sensitive sites. EGCG added during all phases impaired persistence of damage. Our studies show that the continuous presence of EGCG can reduce radical-induced DNA damage in primary leucocytes, possibly due to a combination of different mechanisms. Together the findings support the hypotheses that EGCG acts protective in human cells.     

Inhibition of influenza virus internalization by (−)-epigallocatechin-3-gallate

(−)-Epigallocatechin-3-gallate (EGCG), one of the major flavonoid components of green tea, is known to have a broad antiviral activity against several enveloped viruses, including the influenza virus. However, its mode of action and the mechanism that allows it to target influenza virus molecules have not been fully elucidated. Thus, this study investigated the molecular mechanism by which EGCG suppresses influenza virus infections. EGCG was found to block an early step in the influenza viral life cycle, but it did not affect viral adsorption to target cells or viral RNA replication. However, EGCG inhibited hemifusion events between virus particles and the cellular membrane by reducing the viral membrane integrity, thereby resulting in the loss of the cell penetration capacity of the influenza virus. EGCG also marginally suppressed the viral and nonviral neuraminidase (NA) activity in an enzyme-based assay system. In conclusion, it is suggested that the anti-influenza viral efficacy of EGCG is attributable to damage to the physical properties of the viral envelope and partial inhibition of the NA surface glycoprotein. These results may facilitate future investigations of the antiviral activity of EGCG against other enveloped viruses as well as influenza virus.     

Tea polyphenol (-)-epigallocatechin-3-gallate: a new trapping agent of reactive dicarbonyl species

Previous studies have demonstrated that reactive dicarbonyl compounds [e.g., methylglyoxal (MGO) and glyoxal (GO)] irreversibly and progressively modify proteins over time and yield advanced glycation end products (AGEs), which are thought to contribute to the development of diabetes mellitus and its subsequent complications. Thus, decreasing the levels of MGO and GO will be an effective approach to reduce the formation of AGEs and the development of diabetic complications. In our studies to find nontoxic trapping agents of reactive dicarbonyl species from dietary sources, we found that (-)-epigallocatechin-3-gallate (EGCG), the major bioactive green tea polyphenol, could efficiently trap reactive dicarbonyl compounds (MGO or GO) to form mono- and di-MGO or GO adducts under physiological conditions (pH 7.4, 37 degrees C). The products formed from EGCG and MGO (or GO), combined at different ratios, were analyzed using LC/MS. We also developed a method to purify the two major mono-MGO adducts of EGCG without derivatization, and their structures were identified as stereoisomers of mono-MGO adducts of EGCG based on their 1D and 2D NMR spectra. Our LC/MS and NMR data showed that positions 6 and 8 of the EGCG A-ring were the major active sites for trapping reactive dicarbonyl compounds. We also found that EGCG lost its trapping efficacy under acidic conditions (pH相似文献   

Modulation of gold(III) porphyrin 1a-induced apoptosis by mitogen-activated protein kinase signaling pathways

Gold(III) porphyrin 1a is a novel gold(III) complex with selective anticancer effect in a number of human carcinoma cell lines. We have previously shown that gold(III) porphyrin 1a mediated mitochondrial transmembrane potential (DeltaPsi(m)) depletion, leading to cytochrome c release, nucleus translocation of apoptosis-inducing factor (AIF), and generation of reactive oxygen species (ROS). The current study addressed gold(III) porphyrin 1a-induced phosphoproteome alterations and modulation of cell death by the mitogen-activated protein kinase (MAPK) family proteins. ERK and p38(MAPK) were transiently activated upon gold(III) porphyrin 1a treatment. Inhibition of p38(MAPK) phosphorylation rescued gold(III) porphyrin 1a-induced cell death upstream of caspase activation. Attenuation of DeltaPsi(m) was the primary effect of gold(III) porphyrin 1a leading to p38(MAPK) phosphorylation. Further functional proteomic study suggested that differential regulation of phosphotyrosine proteins were related to p38(MAPK) activation in gold(III) porphyrin 1a-induced signal transduction cascade. In summary, p38(MAPK) modulated gold(III) porphyrin 1a-induced cell death downstream of mitochondria, and phosphorylation of multiple proteins also involved in this process. These results suggested that gold(III) porphyrin 1a is a promising anticancer agent directed toward the mitochondria.     

表没食子儿茶素没食子酸酯(EGCG)抗炎机理研究进展

EGCG是从绿茶中提取分离的茶多酚中的一种主要活性成分。基于其显著的抗氧化、抗癌以及抗炎活性,已成为近来研究的热点。其抗炎机理主要表现在:抗氧化活性介导、干预信号通路、抑制转录因子激活等方面,本文就此展开综述。     

Effects of (-)-epigallocatechin-3-gallate, the main component of green tea, on the cloned rat brain Kv1.5 potassium channels

The interaction of (-)-epigallocatechin-3-gallate (EGCG), the main component of green tea (Camellia sinensis), with rat brain Kv1.5 channels (rKv1.5) stably expressed in Chinese hamster ovary (CHO) cells was investigated using the whole-cell patch-clamp technique. EGCG inhibited rKv1.5 currents at +50 mV in a concentration-dependent manner, with an IC50 of 101.2+/-6.2 microM. Pretreatment with protein tyrosine kinase (PTK) inhibitors (10 microM genistein, 100 microM AG1296), a tyrosine phosphatase inhibitor (500 microM sodium orthovanadate), or a protein kinase C (PKC) inhibitor (10 microM chelerythrine) did not block the inhibitory effect of EGCG on rKv1.5. The inhibition of rKv1.5 by EGCG displayed voltage-independence over the full activation voltage range positive to +10 mV. EGCG had no effect on the midpoint potential or the slope factor for steady-state activation and inactivation. EGCG did not affect the ion selectivity of rKv1.5. The activation (at +50 mV) kinetics was significantly slowed by EGCG. During repolarization (at -40 mV), EGCG also slowed the deactivation of the tail currents, resulting in a crossover phenomenon. Reversal of inhibition was detected by the application of repetitive depolarizing pulses and of identical double pulses, especially during the early part of the activating pulse, in the presence of EGCG. EGCG-induced inhibition of rKv1.5 showed identical affinity between EGCG and the multiple closed states of rKv1.5. These results suggest that EGCG interacts directly with rKv1.5 channels. Furthermore, by analyzing the kinetics of the interaction between EGCG and rKv1.5, we conclude that the inhibition of rKv1.5 channels by EGCG includes at least two effects: EGCG preferentially binds to the channel in the closed state, and blocks the channel by pore occlusion while depolarization is maintained.     

Roles of mitogen-activated protein kinase pathways for mediator release from human cultured mast cells

Human cultured mast cells (HCMC) secrete histamine, sulfidoleukotrienes (LTs), and prostaglandin D(2) (PGD(2)), and produce a variety of cytokines after aggregation of high-affinity receptors for IgE (FcepsilonRI). With respect to the mitogen-activated protein kinase (MAPK) family, extracellular signal-regulated kinases (ERKs), c-Jun NH(2)-terminal kinases (JNKs), and p38 mitogen-activated protein kinase (p38 MAPK) are known. To investigate the roles of these kinase pathways for mediator release from human mast cells, we examined the participation of the activation of these kinases in mediator release, using 1,4-diamino-2, 3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), an ERK pathway inhibitor, and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imid azo le (SB203580), a p38 MAPK pathway inhibitor. U0126 inhibited ERK activation, LT and PGD(2) release, and granulocyte macrophage-colony stimulating factor (GM-CSF) production after stimulation of HCMC. SB203580, on the other hand, potentiated JNK activation and GM-CSF production. The findings of the present study demonstrated that: (i) the release of arachidonic acid metabolites is mediated by the ERK pathway; (ii) GM-CSF production may be driven by both the ERK and JNK pathways; and (iii) the p38 MAPK pathway negatively regulates the JNK pathway. This suggests that MAPK pathways play important roles in mediator release from human mast cells.     

EGCG对体外培养的胚鼠皮层神经细胞的保护作用

目的研究表没食子儿茶酚没食子酸酯(EGCG)对FeSO4和H2O2诱导神经细胞损伤的保护作用。方法采用FeSO4和H2O2作用产生自由基的方法诱导离体培养的小鼠胚鼠大脑皮层神经细胞损伤建立模型。倒置相差显微镜进行神经元形态学观察,中性红比色法和MTT法测定神经细胞的存活数量和活性,TBARS法和BCA法分别测定神经细胞中丙二醛(MDA)的水平和总蛋白合成的能力。结果与FeSO4和H2O2损伤组进行比较,EGCG增加了神经细胞的存活数量和活性,降低MDA的含量和提高了总蛋白的合成能力。EGCG显示出较强的抗氧化能力。结论EGCG对FeSO4和H2O2诱导神经细胞损伤有明显的保护作用,其作用机制可能源于EGCG清除自由基和抑制脂质过氧化过程。     

表没食子儿茶素没食子酸酯对肾缺血再灌注损伤的保护作用

目的　探讨表没食子儿茶素没食子酸酯 (EGCG)对大鼠肾缺血再灌注损伤的影响。方法　通过结扎肾动脉 60min后再灌注 ,建立大鼠肾缺血再灌注损伤模型 ,给药组于结扎前后分别静脉给予 10mg·kg-1和 40mg·kg-1EGCG。化学法观察大鼠血清肌酐 (Scr)、尿素氮 (Bun)、丙二醛(MDA)含量 ,肾组织内MDA、活性氧 (ROS)含量和超氧化物歧化酶 (SOD)、Ca2 + ATP酶活性的变化 ,并观察肾组织的病理变化。结果　与模型对照组比较 ,40mg·kg-1的EGCG组可抑制由肾缺血再灌注引起的血清Bun、Scr、MDA含量和组织MDA、ROS含量的变化 ,增强SOD和Ca2 + ATP酶活性 ,减轻肾组织病理改变。结论　EGCG有保护大鼠肾缺血再灌注损伤的作用 ,其机制可能与抗自由基损伤和减少细胞内钙有关     

Cell growth inhibition and gene expression regulation by (-)-epigallocatechin-3-gallate in human cervical cancer cells

EGCG [(-)-epigallocatechin-3-gallate] has shown its antitumor ability and perhaps a potential regimen for cancer patients. The goal of this study was to investigate the effect of EGCG on human papilloma virus (HPV) positive cervical cancer cell lines. EGCG inhibited the growth of CaSki (HPV16 positive) and HeLa (HPV18 positive) cells in a time- and concentration-dependent manner. Cell cycle arrest and apoptosis were observed in two cell lines after EGCG exposure. More importantly, we focused on EGCG regulation ability on pivotal genes involved in cervical cancer: viral oncogenes E6/E7, estrogen receptor (ER) and aromatase. Our results suggested that EGCG may be suitable for prevention and treatment of cervical cancer.     

Effect of (-)-epigallocatechin-3-gallate on respiratory burst of rat macrophages

The toxic effects derived from overproduction of oxygen radicals [reactive oxygen species (ROS)] by immune cells can be partially abolished by the antioxidant activities of plant polyphenols. In the present study, we investigated the antioxidant action of a catechin, (-)-epigallocatechin-3-gallate (EGCG), on the respiratory-burst responses of rat peritoneal macrophages. EGCG at concentrations of 50-200 microM blocked the production of nitric oxide by macrophages stimulated in vivo with sodium thioglycollate then 5 days later in vitro with lipopolysaccharide and gamma-interferon. At 1-100 microM, EGCG also inhibited the extracellular liberation of oxygen radicals by resident peritoneal macrophages stimulated with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA). At low concentrations (1-5 microM), EGCG increased the reduction of nitro blue tetrazolium (NBT) by the superoxide anions generated in the non-enzymatic system NADH/PMS, acting as a pro-oxidant agent, while at concentrations above 10 microM, EGCG acts as a scavenger of superoxide anions. These results show that EGCG is capable of modulating ROS production during the respiratory burst of rat peritoneal macrophages by acting as a superoxide anion scavenger. EGCG may therefore be useful in the prevention and treatment of diseases due to increased free radical production.     

Inhibitory modulation of ATP-sensitive potassium channels by gallate-ester moiety of (-)-epigallocatechin-3-gallate

(-)-Epigallocatechin-3-gallate (EGCG), a major polyphenolic substance found in green tea, is well recognized to be beneficial for human health. However, it is still controversial as to what dose of this compound is indeed good for human health. Though some recent studies have interestingly reported various beneficial effects of EGCG in cell culture system, however, plasma levels of EGCG attainable by oral regular intake in humans are normally in nanomolar range. However, potential side effects of EGCG when administered parenterally at higher concentration have not been thoroughly tested. Here, we evaluated the effect of EGCG on ATP-sensitive potassium (K(ATP)) channels expressed in Xenopus oocytes. EGCG inhibited the activity of the Kir6.2/SUR1 and Kir6.2DeltaC36 channels with IC(50) of 142+/-37 and 19.9+/-1.7microM, respectively. Inhibition of EGCG was also observed in Kir6.2/SUR2A or Kir6.2/SUR2B channels. Notably, (-)-epicatechin-3-gallate (ECG), another major polyphenolic substance in green tea, was found to reduce the channel activity with greater potency than EGCG. In contrast to EGCG and ECG, which have the gallic acid-ester moiety in their own structures, (-)-epigallocatechin and (-)-epicatechin exhibited very weak inhibition of the K(ATP) channel. Collectively, these results suggest that the gallate-ester moiety of epicatechins may be critical for inhibiting the K(ATP) channel activity via the pore-forming subunit Kir6.2 and this may be a possible mechanism by which green tea extracts or EGCG may cause unexpected side effects at micromolar plasma level.     

Inhibition of bcl-x(l) phosphorylation by tea polyphenols or epigallocatechin-3-gallate is associated with prostate cancer cell apoptosis

Prostate cancer cells demonstrate slow growth kinetics and chemoresistance. Tea polyphenols have been shown to exert prostate cancer-preventative effects. Here we report that growth-arrested prostate cancer cells expressed high levels of a hyperphosphorylated Bcl-X(L) in mitochondria. Treatment with tea polyphenols or the major tea component epigallocatechin-3-gallate blocked expression of the hyper-, but not hypophosphorylated Bcl-X(L) in mitochondria, accompanied by cytochrome c release, caspase activation, and apoptosis. Studies using specific inhibitors suggest that tea inhibits p38 mitogen-activated protein kinase and the proteasome activities, leading to inhibition of Bcl-X(L) phosphorylation and induction of prostate cancer cell death.