Hydroxytyrosol induces proliferation and cytoprotection against oxidative injury in vascular endothelial cells: role of Nrf2 activation and HO-1 induction

Hydroxytyrosol (HT), a phenolic compound in olive oil and leaves, has been reported to prevent various human pathologies including cardiovascular diseases. This study investigated the effects of HT on proliferation and protection against oxidative stress-induced damage in vascular endothelial cells (VECs) and the molecular mechanism(s) involved. Treatment of VECs with HT increased cell proliferation, promoted wound repair, and protected cells against H(2)O(2) cytotoxicity through the activation of Akt and ERK1/2, but not p38 MAPK. HT increased the expression and nuclear translocation of nuclear factor-E2-related factor-2 (Nrf2). Nrf2 expression was attenuated by LY294002 and U0126, inhibitors of phosphatidylinositol-3-kinase and MEK1/2, respectively. Nrf2 siRNA decreased both proliferative and cytoprotective effects of HT and abrogated HO-1 induction. Moreover, HO-1 inhibition with HO-1 siRNA or zinc protoporphyrin IX significantly prevented HT-induced cell proliferation, cytoprotection, and reduction in intracellular reactive oxygen species (ROS), suggesting that HO-1 is involved in these HT functions. The findings demonstrate that HT positively regulates the antioxidant defense system in VECs through the activation of Nrf2 followed by cell proliferation and resistance to vascular injury. The present study provides a molecular basis for the contribution of HT in the Mediterranean diet to the prevention of cardiovascular diseases.     

Pterostilbene is more potent than resveratrol in preventing azoxymethane (AOM)-induced colon tumorigenesis via activation of the NF-E2-related factor 2 (Nrf2)-mediated antioxidant signaling pathway

Inflammatory bowel diseases have been a risk factor of colorectal cancer (CRC). The reactive oxygen species (ROS) generated by inflammatory cells create oxidative stress and contribute to neoplastic transformation, proliferation, and even metastasis. Previously, resveratrol (RS) and pterostilbene (PS) had been reported to prevent chemical-induced colon carcinogenesis by anti-inflammatory and pro-apoptotic properties. In this study, we investigated whether RS and PS could prevent the azoxymethane (AOM)-induced colon tumorigenesis via antioxidant action and to explore possible molecular mechanisms. Male BALB/c mice were injected with AOM (5 mg/kg of body weight) with or without RS or PS, and at the end of the protocol, all of the mice were euthanized and colons were analyzed. Administrations of PS can be more effective than RS in reducing AOM-induced formation of aberrant crypt foci (ACF), lymphoid nodules (LNs), and tumors. We also find that PS is functioning more effectively than RS to reduce nuclear factor-κB (NF-κB) activation by inhibiting the phosphorylation of protein kinase C-β2 (PKC-β2) and decreasing downstream target gene expression, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and aldose reductase (AR) in mouse colon stimulated by AOM. Moreover, administration of RS and PS for 6 weeks significantly enhanced expression of antioxidant enzymes, such as heme oxygenase-1 (HO-1) and glutathione reductase (GR), via activation of NF-E2-related factor 2 (Nrf2) signaling. When the above findings are taken together, they suggest that both stilbenes block cellular inflammation and oxidative stress through induction of HO-1 and GR, thereby preventing AOM-induced colon carcinogenesis. In comparison, PS was a more potent chemopreventive agent than RS for the prevention of colon cancer. This is also the first study to demonstrate that PS is a Nrf2 inducer and AR inhibitor in the AOM-treated colon carcinogenesis model.     

Fucoxanthin enhances HO-1 and NQO1 expression in murine hepatic BNL CL.2 cells through activation of the Nrf2/ARE system partially by its pro-oxidant activity

To determine whether fucoxanthin, a major carotenoid in brown sea algae, may activate cellular antioxidant enzymes via up-regulation of the Nrf2/antioxidant-response element (ARE) pathway, we incubated mouse hepatic BNL CL.2 cells with fucoxanthin (0.5-20 μM) for 0-24 h. We found that fucoxanthin (≥5 μM) significantly increased cellular reactive oxygen species (ROS) at 6 h of incubation, whereas preincubation with α-d-tocopherol (30 μM) significantly attenuated the increase of ROS, indicating the pro-oxidant nature of fucoxanthin. Fucoxanthin significantly increased the phosphorylation of ERK and p38 and markedly increased nuclear Nrf2 protein accumulation after incubation for 12 h. Moreover, fucoxanthin significantly enhanced binding activities of nuclear Nrf2 with ARE and increased mRNA and protein expression of HO-1 and NQO1 after incubation for 12 h. siRNA inhibition of Nrf2 led to markedly decreased HO-1 and NQO1 protein expression. Thus, fucoxanthin may exert its antioxidant activity, at least partly, through its pro-oxidant actions.     

Suppression of free fatty acid-induced insulin resistance by phytopolyphenols in C2C12 mouse skeletal muscle cells

It was reported that increased plasma levels of free fatty acids (FFAs) are associated with profound insulin resistance in skeletal muscle and may also play a critical role in the insulin resistance of obesity and type 2 diabetes mellitus. Skeletal muscle is the major site for insulin-stimulated glucose uptake and is involved in energy regulation and homeostasis. In this study, we used 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C (PKC) activator, and palmitate to induce insulin resistance in C2C12 mouse skeletal muscle cells. Our data show that epigallocatechin gallate (EGCG) and curcumin treatment reduce insulin receptor substrate-1 (IRS-1) Ser307 phosphorylation, and curcumin is more potent to increase Akt phosphorylation in TPA induction. Moreover, we found that after 5 h of palmitate incubation, epicatechin gallate (ECG) can suppress IRS-1 Ser307 phosphorylation and significantly promote Akt, ERK1/2, p38 MAPK, and AMP-activated protein kinase activation. With a longer incubation with palmitate, IRS-1 exhibited a dramatic depletion, and treatment with EGCG, ECG, and curcumin could reverse IRS-1 expression, Akt phosphorylation, and MAPK signaling cascade activation and improve glucose uptake in C2C12 skeletal muscle cells, especially ECG and curcumin. In addition, treatment with these polyphenols can suppress acetyl-CoA carboxylase activation, but only EGCG could inhibit lipid accumulation in the intracellular site. These findings may suggest that curcumin shows the best capacity to improve FFA-induced insulin resistance than the other two, and ECG was more effective than EGCG in attenuating insulin resistance.     

Neuroprotective effects of dehydroglyasperin C through activation of heme oxygenase-1 in mouse hippocampal cells

Licorice, the root of the Glycyrrhiza species ( Glycyrrhiza uralensis Fisher), is known to have antioxidant, anti-inflammatory, antiviral, and antitumor properties. The objective of this study is to explore the neuroprotective effect of dehydroglyasperin C (DGC) against glutamate-induced oxidative stress in mouse hippocampal HT22 cells. DGC significantly reduced cytotoxicity and reactive oxygen species (ROS) generation induced by glutamate in HT22 cells, whereas DGC did not restore glutathione depletion caused by glutamate. In addition, it was further investigated whether DGC affected the expression of heme oxygenase (HO)-1, one of the major cellular antioxidant defense systems, and it was found that DGC dose-dependently increased HO-1 expression. DGC-mediated cytoprotection of HT22 neuronal cells from glutamate insult was abrogated by either HO-1 inhibitor (Tin protoporphyrin, SnPP) or AKT inhibitor (LY294002). In conclusion, the present results demonstrate for the first time that DGC protects neuronal cells against glutamate-induced oxidative injury through the induction of HO-1 expression, which is, in turn, activated maybe through Nrf2-Keap1 and PI3K/AKT signaling pathways.     

Isoobtusilactone A sensitizes human hepatoma Hep G2 cells to TRAIL-induced apoptosis via ROS and CHOP-mediated up-regulation of DR5

Hepatoma cells are relatively resistant to TRAIL. We have previously shown that isoobtusilactone A (IOA), a potent anticancer agent isolated from Cinnamomum kotoense, induced mitochondria-mediated apoptosis in hepatoma cells. Here, we report that IOA could potentiate TRAIL-induced apoptosis in Hep G2 cells. The combined treatment with IOA and TRAIL significantly induced caspase-dependent apoptosis. This correlated with the up-regulation of C/EBP homologous protein (CHOP) and death receptor 5 (DR5) protein levels. Gene silencing of the DR5 by small interfering RNA abrogated the apoptosis induced by the combined regimen of IOA and TRAIL, suggesting that the sensitization to TRAIL was mediated through DR5. By analyzing the DR5 promoter, we found that IOA induced a CHOP-dependent DR5 transactivation. DR5 expression after IOA treatment was accompanied by provoking intracellular reactive oxygen species (ROS) generation. Pretreatment with N-acetyl-L-cysteine (NAC) attenuated IOA-induced CHOP and DR5 expression and inhibited TRAIL-induced apoptosis. Taken together, our data suggested that ROS-dependent and CHOP-regulated DR5 expression played a pivotal role in the synergistic enhancement of TRAIL-induced apoptosis instigated by IOA in Hep G2 cells.     

Effect of eriodictyol on glucose uptake and insulin resistance in vitro

Eriodictyol [2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-2,3-dihydrochromen-4-one] is a flavonoid with anti-inflammatory and antioxidant activities. Because inflammation and oxidative stress play critical roles in the pathogenesis of diabetes mellitus, the present study was designed to explore whether eriodictyol has therapeutic potential for the treatment of type 2 diabetes. The results show that eriodictyol increased insulin-stimulated glucose uptake in both human hepatocellular liver carcinoma cells (HepG2) and differentiated 3T3-L1 adipocytes under high-glucose conditions. Eriodictyol also up-regulated the mRNA expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) and adipocyte-specific fatty acid-binding protein (aP2) as well as the protein levels of PPARγ2 in differentiated 3T3-L1 adipocytes. Furthermore, it reactivated Akt in HepG2 cells with high-glucose-induced insulin resistance. This response was strongly inhibited by pretreatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, indicating that eriodictyol increased Akt phosphorylation by activating the PI3K/Akt pathway. These results imply that eriodictyol can increase glucose uptake and improve insulin resistance, suggesting that it may possess antidiabetic properties.     

Steam-dried ginseng berry fermented with Lactobacillus plantarum controls the increase of blood glucose and body weight in type 2 obese diabetic db/db mice

This study examined whether steam-dried ginseng berries fermented with Lactobacillus plantarum (FSGB) could improve the indices of type 2 diabetes mellitus (T2DM) in obese db/db mice. FSGB was shown to have an effect on body weight and blood glucose/serum parameters when administered at a dose of 0.5 g/kg. In the intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT), FSGB was clearly shown to improve insulin tolerance and glucose tolerance. Moreover, FSGB was shown to enhance immune activities by increasing the immune cell population, and glucose transpoter 1 (GLUT1) mRNA expression in L6 cells was up-regulated, suggesting that FSGB can increase glucose transport activity in target cells. These results indicate that steam- and dry-processed ginseng berries fermented with L. plantarum can be used to effectively control blood sugar metabolism via improving insulin and glucose tolerance and body weight gain in db/db mice.     

Antidiabetic effects of rice hull smoke extract in alloxan-induced diabetic mice

This study investigated the protective effect of a liquid rice hull smoke extract (RHSE) against diabetes in alloxan-induced diabetic mice. Antidiabetic effects of RHSE were evaluated in both the rat insulinoma-1 cell line (INS-1) and diabetic ICR mice induced by intraperitoneal (ip) injection of alloxan. Alloxan treatment (10 mM) increased cellular reactive oxygen species (ROS) levels in the INS-1 cells, which were inversely related to cell viabilities. RHSE inhibited alloxan-induced nitric oxide (NO) generation through inhibition of inducible nitric oxide synthase (iNOS) gene expression and suppressed the inflammatory reaction in INS-1 cells through inhibition of expression of pro-inflammatory genes, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Dietary administration of 0.5 or 1% RHSE to alloxan-induced diabetic mice caused a decrease in blood glucose and increases in both serum insulin and hepatic glycogen levels. RHSE induced decreases in glucose-6-phosphatase (G6 Pase) and phosphoenolpyruvate carboxykinase (PEPCK) levels and an increase in the glucokinase (GCK) level. These changes resulted in restoring glucose-regulating enzyme levels to control values. Histopathology showed that alloxan also induced damage of Langerhans islet cells of the pancreas and liver necrosis associated with diabetes. Oral administration of RHSE restored the islet and liver cells to normal levels. RHSE-supplemented functional food could protect insulin-producing islet cells against damage triggered by oxidative stress and local inflammation associated with diabetes.     

Extract of lotus leaf ( Nelumbo nucifera ) and its active constituent catechin with insulin secretagogue activity

The effect of lotus leaf ( Nelumbo nucifera Gaertn.) on diabetes is unclear. We hypothesized that lotus leaf can regulate insulin secretion and blood glucose levels. The in vitro and in vivo effects of lotus leaf methanolic extract (NNE) on insulin secretion and hyperglycemia were investigated. NNE increased insulin secretion from β cells (HIT-T15) and human islets. NNE enhanced the intracellular calcium levels in β cells. NNE could also enhance phosphorylation of extracellular signal-regulated protein kinases (ERK)1/2 and protein kinase C (PKC), which could be reversed by a PKC inhibitor. The in vivo studies showed that NNE possesses the ability to regulate blood glucose levels in fasted normal mice and high-fat-diet-induced diabetic mice. Furthermore, the in vitro and in vivo effects of the active constituents of NNE, quercetin, and catechin, on glucose-induced insulin secretion and blood glucose regulation were evaluated. Quercetin did not affect insulin secretion, but catechin significantly and dose-dependently enhanced insulin secretion. Orally administered catechin significantly reversed the glucose intolerance in high-fat-diet-induced diabetic mice. These findings suggest that NNE and its active constituent catechin are useful in the control of hyperglycemia in non-insulin-dependent diabetes mellitus through their action as insulin secretagogues.     

Inhibitory effects of dioscorea polysaccharide on TNF-α-induced insulin resistance in mouse FL83B cells

Dioscorea is a traditional medicinal food in Asia. This study investigated the anti-insulin resistance of dioscorea polysaccharide (DPS) in inflammatory factor (tumor necrosis factor-α; TNF-α) induced mouse normal liver FL83B cells. Insulin resistance was induced by treating cells with TNF-α (20 ng/mL) for 5 h; subsequently, the medium was replaced with insulin and DPS for 60 min of incubation (model 1; alleviating group). In addition, cells were cotreated with TNF-α and DPS for 5 h in model 2 (preventing group). DPS effectively increased glucose uptake and glucose transporter 2 (GLUT2) expression of insulin-resistant cells. Furthermore, DPS stimulated insulin receptor substrate (IRS) tyrosyl phosphorylation and increased p-Akt level to alleviate insulin resistance in models 1 and 2. Finally, the possible mechanism of DPS promoting insulin sensitivity in TNF-α-induced FL83B cells was investigated in this study. DPS may attenuate c-Jun N-terminal kinases (JNK) and insulin resistance caused by TNF-α induction; therefore, DPS also elevated the levels of p-IRS(Tyr) and p-Akt(Ser) to improve insulin sensitivity in the TNF-α-induced FL83B cells.     

HepG2细胞胰岛素抵抗模型的建立及其应用

为建立方便、可靠、重复性好的胰岛素抵抗细胞模型,研究胰岛素受体底物-2(IRS-2)蛋白磷酸化与胰岛素抵抗的关系以及不同铬制剂对细胞葡萄糖代谢的作用。试验采用高胰岛素、高糖联合诱导Hep G2细胞建立胰岛素抵抗模型,通过葡萄糖氧化酶法及噻唑蓝比色法(MTT)分析受试物对细胞葡萄糖代谢及活性的影响,优化最佳造模条件;同时,利用蛋白免疫印迹法(WB)分析IRS-2的蛋白表达及其蛋白磷酸化,验证模型的胰岛素抵抗性。在此基础上,将模型应用于分析葡萄糖耐量因子(GTF)及其它铬制剂的降糖效果。结果表明,高胰岛素(10-6mol·L~(-1))和高糖培养基(25 mmol·L~(-1))处理细胞48h,细胞存活率达96%,与对照组相比,葡萄糖消耗量降低了5.7%,IRS-2含量减少31.02%,胰岛素抵抗效果显著,且该胰岛素抵抗模型可稳定维持48 h。相比于吡啶酸铬和三氯化铬,GTF对细胞葡萄糖代谢有显著的改善作用,其最佳作用浓度为1.0μg·m L-1。利用HepG2细胞建立体外胰岛素抵抗模型,方法简单可靠、重复性好,可广泛应用于天然活性物质的降糖功能研究。     

Hispolon induces apoptosis and cell cycle arrest of human hepatocellular carcinoma Hep3B cells by modulating ERK phosphorylation

Hispolon is an active phenolic compound of Phellinus igniarius , a mushroom that has recently been shown to have antioxidant, anti-inflammatory, and anticancer activities. This study investigated the antiproliferative effect of hispolon on human hepatocellular carcinoma Hep3B cells by using the MTT assay, DNA fragmentation, DAPI (4,6-diamidino-2-phenylindole dihydrochloride) staining, and flow cytometric analyses. Hispolon inhibited cellular growth of Hep3B cells in a time-dependent and dose-dependent manner, through the induction of cell cycle arrest at S phase measured using flow cytometric analysis and apoptotic cell death, as demonstrated by DNA laddering. Hispolon-induced S-phase arrest was associated with a marked decrease in the protein expression of cyclins A and E and cyclin-dependent kinase (CDK) 2, with concomitant induction of p21waf1/Cip1 and p27Kip1. Exposure of Hep3B cells to hispolon resulted in apoptosis as evidenced by caspase activation, PARP cleavage, and DNA fragmentation. Hispolon treatment also activated JNK, p38 MAPK, and ERK expression. Inhibitors of ERK (PB98095), but not those of JNK (SP600125) and p38 MAPK (SB203580), suppressed hispolon-induced S-phase arrest and apoptosis in Hep3B cells. These findings establish a mechanistic link between the MAPK pathway and hispolon-induced cell cycle arrest and apoptosis in Hep3B cells.     

Tangeretin reduces ultraviolet B (UVB)-induced cyclooxygenase-2 expression in mouse epidermal cells by blocking mitogen-activated protein kinase (MAPK) activation and reactive oxygen species (ROS) generation

The present study examined the effects of tangeretin, a polymethoxylated flavonone present in citrus fruits, on ultraviolet B (UVB)-induced cyclooxygenase-2 (COX-2) expression in JB6 P+ mouse skin epidermal cells. Tangeretin suppressed UVB-induced COX-2 expression and transactivation of nuclear factor-κB and activator protein-1 in JB6 P+ cells. Moreover, tangeretin blocked UVB-induced phosphorylation of Akt and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38, and attenuated the phosphorylation of MAPK kinases 1/2, 3/6, and 4. Tangeretin also limited the endogenous generation of reactive oxygen species (ROS), thereby protecting the cells against oxidative stress. However, tangeretin did not scavenge the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and influence the nicotinamide adenine dinucleotide phosphate oxidase activity. These results suggest that the anti-inflammatory effects of tangeretin stem from its modulation of cell signaling and suppression of intracellular ROS generation. Tangeretin may have a potent chemopreventive effect in skin cancer.