Stress-activated leukocyte 12/15-lipoxygenase metabolite enhances struggle behaviour and tocotrienols relieve stress-induced behaviour alteration.

Stress induces emotional arousal causing anxiety, irritability, exaggerated startle behaviour, and hypervigilance observed in patients with trauma and stress-related mental disorders, including acute stress disorder and post-traumatic stress disorder. Central norepinephrine release promotes stress-induced emotional arousal. However, the regulator of emotional arousal remains unknown. Here, we show that the arachidonate-derived metabolite produced by stress-activated leukocyte 12/15-lipoxygenase is remarkably elevated in the plasma and upregulates the central norepinephrine release, resulting in the enhancement of the struggle behaviour (= escape behaviour) in the tail suspension test. Struggle behaviour is mimicking a symptom of emotional arousal. This stress-induced struggle behaviour was absent in 12/15-lipoxygenase deficient mice; however, intravenous administration of a 12/15-lipoxygenase metabolite to these mice after stress exposure rekindled the struggle behaviour. Furthermore, tocotrienols and geranylgeraniol reduced stress-induced 12/15-lipoxygenase metabolite production and suppressed the struggle behaviour. Our findings indicate that arachidonate-derived 12/15-lipoxygenase metabolite is involved in the regulation of stress-enhanced central norepinephrine release and struggle behaviour. In addition, we propose 12/15-lipoxygenase as a potential therapeutic target for the treatment of emotional arousal observed in stress-related mental disorders.

δ-Tocopherol promotes thermogenic gene expression via PGC-1α upregulation in 3T3-L1 cells.

Activation of thermogenic adipocytes (brown and beige) has been considered an attractive target for weight loss and treatment of metabolic disease. Peroxisome proliferator-activated receptor γ co-activator-1 α (PGC1-α) is a master regulator of thermogenic gene expression in thermogenic adipocytes. We previously reported that α-tocopherol upregulated PGC-1α gene expression and promoted thermogenic adipocyte differentiation in mammalian adipocytes. In this study, we investigated the effects of the vitamin E analogs (α-, γ- and δ-tocopherol) on PGC-1α and uncoupling protein 1 (UCP1) gene expression in 3T3-L1 cells. The expression of PGC-1α and UCP1 increased significantly with the addition of δ-tocopherol. In δ-tocopherol-treated cells, nuclear translocation of PGC-1α increased, as did p38 mitogen-activated protein kinase (MAPK) expression and phosphorylation. Our results suggest that p38 MAPK activation by δ-tocopherol contributes to PGC-1α activation and UCP1 induction.

Evaluation of probiotic and prebiotic-like effects of Bacillus subtilis BN on growth of lactobacilli.

The aim of this study was to determine the probiotic and the prebiotic-like properties of Bacillus subtilis BN, a spore-forming bacterium, also known as "natto-kin", which is used for making the Japanese fermented food, natto. We used the spores and vegetative cells of this strain and compared their effects on the growth of lactobacilli. Culture supernatant from B. subtilis BN was added to a glucose-free MRS medium used to culture lactobacilli. When lactobacilli were cultured in the supernatant-containing medium, growth was improved. This effect resulted from the digestion of starch by amylase, which was secreted by B. subtilis. Moreover, improved amylase-independent growth was also observed. Co-culture with B. subtilis improved the growth of lactobacilli, and this effect was only observed with vegetative cells; spores did not improve the growth of lactobacilli. This effect on growth was lost upon heat treatment of the vegetative cells. These results suggest that the surface protein of B. subtilis BN vegetative cells participates in the improved growth effect of lactobacilli. It is possible that B. subtilis BN could improve the intestinal flora. In addition, B. subtilis BN inhibited the growth of Salmonella enterica. Thus, it was shown that B. subtilis BN has both a probiotic and prebiotic potential. This is the first study demonstrating the selective growth improvement of indigenous intestinal lactobacilli using B. subtilis BN.

Oleuropein improves insulin resistance in skeletal muscle by promoting the translocation of GLUT4.

As the beneficial effects of the Mediterranean diet on human health are well established, the phenolic compounds in olive oil have been gaining interest. Oleuropein, a major phenolic compound in olives, is known to reduce the blood glucose levels in alloxan-induced diabetic rats and rabbits, however, its effect on type 2 diabetes caused by obesity is not clear. The purpose of this study is clarifying the effect of oleuropein on the glucose tolerance in skeletal muscle under the condition of lipotoxicity caused by type 2 diabetes. Oleuropein enhanced glucose uptake in C2C12 cells without insulin. Translocation of glucose transporter 4 (GLUT4) into the cell membrane was promoted by activation of adenosine monophosphate-activated protein kinase (AMPK) but not protein kinase B (Akt). Physiological concentration of oleuropein (10 µM) was sufficient to express beneficial effects on C2C12 cells. Oleuropein prevented palmitic acid-induced myocellular insulin resistance. Furthermore, in gastrocnemius muscles of mice fed a high fat diet, oleuropein also induced the GLUT4 localization into cell membrane. These results suggest the possibility of oleuropein to be effective for type 2 diabetes by reducing insulin resistance in skeletal muscles.

Excessive Vitamin E Intake Does Not Cause Bone Loss in Male or Ovariectomized Female Mice Fed Normal or High-Fat Diets.

Background: Animal studies on the effects of vitamin E on bone health have yielded conflicting and inconclusive results, and to our knowledge, no studies have addressed the effect of vitamin E on bone in animals consuming a high-fat diet (HFD).Objective: This study aimed to evaluate the effect of excessive vitamin E on bone metabolism in normal male mice and ovariectomized female mice fed a normal diet (ND) or HFD.Methods: In the first 2 experiments, 7-wk-old male mice were fed an ND (16% energy from fat) containing 75 (control), 0 (vitamin E-free), or 1000 (high vitamin E) mg vitamin E/kg (experiment 1) or an HFD (46% energy from fat) containing 0, 200, 500, or 1000 mg vitamin E/kg (experiment 2) for 18 wk. In the third experiment, 7-wk-old sham-operated or ovariectomized female mice were fed the ND (75 mg vitamin E/kg) or HFD containing 0 or 1000 mg vitamin E/kg for 8 wk. At the end of the feeding period, blood and femurs were collected to measure bone turnover markers and analyze histology and microcomputed tomography.Results: In experiments 1 and 2, vitamin E intake had no effect on plasma alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP) activity, or bone formation, resorption, or volume in femurs in mice fed the ND or HFDs. In experiment 3, bone volume was significantly reduced (85%) in ovariectomized mice compared with that in sham-operated mice (P < 0.05), but it did not differ among mice fed the 3 diets. Plasma ALP and TRAP activities and bone formation and resorption in femur were similar among ovariectomized mice fed the HFD containing 0 or 1000 mg vitamin E/kg.Conclusions: The results suggest that excess vitamin E intake does not cause bone loss in normal male mice or in ovariectomized or sham-operated female mice, regardless of dietary fat content.

Comparative analysis of the intestinal flora in type 2 diabetes and nondiabetic mice.

A relationship between type 2 diabetes mellitus (T2DM) and intestinal flora has been suggested since development of analysis technology for intestinal flora. An animal model of T2DM is important for investigation of T2DM. Although there are some animal models of T2DM, a comparison of the intestinal flora of healthy animals with that of T2DM animals has not yet been reported. The intestinal flora of Tsumura Suzuki Obese Diabetes (TSOD) mice was compared with that of Tsumura, Suzuki, Non Obesity (TSNO) mice in the present study. The TSOD mice showed typical type 2 diabetes symptoms, which were high-fat diet-independent. The TSOD and the TSNO mouse models were derived from the same strain, ddY. In this study, we compared the intestinal flora of TSOD mice with that if TSNO mice at 5 and 12 weeks of age. We determined that that the number of operational taxonomic units (OTUs) was significantly higher in the cecum of TSOD mice than in that of TSNO mice. The intestinal flora of the cecum and that of the feces were similar between the TSNO and the TSOD strains. The dominant bacteria in the cecum and feces were of the phyla Firmicutes and Bacteroidetes. However, the content of some bacterial species varied between the two strains. The percentage of Lactobacillus spp. within the general intestinal flora was higher in TSOD mice than in TSNO mice. In contrast, the percentages of order Bacteroidales and family Lachnospiraceae were higher in TSNO mice than in TSOD mice. Some species were observed only in TSOD mice, such as genera Turicibacter and SMB53 (family Clostridiaceae), the percentage of which were 3.8% and 2.0%, respectively. Although further analysis of the metabolism of the individual bacteria in the intestinal flora is essential, genera Turicibacter and SMB53 may be important for the abnormal metabolism of type 2 diabetes.

Promoting Effect of α-Tocopherol on Beige Adipocyte Differentiation in 3T3-L1 Cells and Rat White Adipose Tissue.

Thermogenic adipocytes that are distinct from classical brown adipocytes (beige adipocytes) were identified in 2012. Beige adipocytes are also called inducible brown adipocytes because their differentiation is induced by a number of physiological stimuli, including adrenaline or myokines. PPARγ is the master regulator of adipogenesis and promotes thermogenic adipocyte differentiation. A PPARγ agonist also promotes thermogenic adipocyte differentiation in mouse white adipose tissues. The vitamin E analog α-tocopherol promotes PPARγ expression and induces mRNA expression of target genes. This study investigated the effects of vitamin E analogs on thermogenic adipocyte differentiation in mouse preadipocytes and rat white adipose tissues. We determined the effects of vitamin E analogs (α-tocopherol and γ-tocopherol) on PPARγ, PGC-1α, and uncoupling protein 1 (UCP1) gene expression in 3T3-L1 cells. UCP1 expression and the mitochondrial contents were confirmed in the cells using immunofluorescence. In an in vivo study, male SD-IGS rats were fed a high-fat diet (HFD), α-tocopherol-enriched HFD, or γ-tocopherol-enriched HFD for 8 weeks before the analysis of PPARγ, PGC-1α, UCP1, and CD137 gene expression, and pathological examinations of white adipose tissues. The expression of PPARγ, PGC-1α, and UCP1 increased in 3T3-L1 cells following α-tocopherol treatment in a concentration-dependent manner. UCP1 expression and mitochondrial content also increased in α-tocopherol-treated cells. According to the histopathological examinations of rat white adipose tissues, multilocular cells were observed in the α-tocopherol intake group. Furthermore, the gene expression levels of PGC-1α, UCP1, and CD137 increased in the α-tocopherol intake group. Our results suggest that α-tocopherol promotes thermogenic adipocyte differentiation in mammalian white adipose tissues.

Rice Bran Dietary Supplementation Improves Neurological Symptoms and Loss of Purkinje Cells in Vitamin E-Deficient Mice.

BACKGROUND: Vitamin E (VE, α-tocopherol) is a fat-soluble vitamin and is well known as an antioxidant. A deficiency in VE induces oxidative stress in the brain and causes motor and memory dysfunction. The consumption of a VE-rich diet has been given much attention in recent years, in regards to anti-aging and the prevention of age-related neuronal disorders. METHODS: A VE-deficient mouse model was prepared by feeding the animals a diet lacking VE. In addition, to evaluate the effect of VE-containing rice bran (RB) on VE deficiency, a diet including RB was also provided. VE levels in the brain tissue, as well as in the RB, were measured using an HPLC system. Behavioral tests, including rotarod, wheel running activity, Y-maze, and elevated plus maze were performed. To clarify the effect of VE deficiency and RB, we investigated the induction of heme oxygenase-1 (HO-1). Histological studies were performed using HE staining and immunohistochemical studies were performed using antibodies against glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1). RESULTS: VE in the mouse brain under a VE-deficient diet was decreased, and recovered α-tocopherol levels were observed in the brain of mice fed an RB diet. Motor behavioral scores were decreased in VE-deficient conditions, while the supplementation of RB improved motor function. HO-1, a marker of oxidative stress, was upregulated in the mouse brain under VE deficiency, however, RB supplementation inhibited the increase of HO-1. Histological analyses showed neuronal degeneration of Purkinje cells and decreased GFAP-immunoreactivity of Bergmann glia in the cerebellum. In addition, activated astrocytes and microglia were observed in mice fed the VE-deficient diet. Mice fed the RB diet showed improvement in these histological abnormalities. CONCLUSION: A VE-deficient diet induced motor dysfunction in mice due to the degeneration of Purkinje cells in the cerebellum. Oral supplementation of RB increases VE in the brain and improved the motor dysfunction caused by VE deficiency. Thus, RB or unpolished rice may be a promising VE supplement.

Tocotrienol improves learning and memory deficit of aged rats.

To define whether tocotrienol (T-3) improves cognitive deficit during aging, effect of T-3 on learning and memory functions of aged rats was assessed. It was found that T-3 markedly counteracts the decline in learning and memory function in aged rats. Quantitative analysis of T-3 content in the rat brain showed that the aged rats fed T-3 mixture-supplemented diet revealed the transport of α- and γ-T-3 to the brain. In contrast, normal young rats fed the same diet did not exhibit brain localization. Furthermore, the T-3 inhibited age-related decreases in the expression of certain blood brain barrier (BBB) proteins, including caludin-5, occludin and junctional adhesion molecule (JAM). It was found that the activation of the cellular proto-oncogene c-Src and extracellular signal-regulated protein kinase (ERK), in the mitogen-activated protein kinase (MAPK) cell signaling pathway for neuronal cell death, was markedly inhibited by T-3. These results may reveal that aging induces partial BBB disruption caused by oxidative stress, thereby enabling the transport of T-3 through the BBB to the central nervous system, whereupon neuronal protection may be mediated by inhibition of c-Src and/or ERK activation, resulting in an improvement in age-related cognitive deficits.

Estrogen receptor-mediated effect of δ-tocotrienol prevents neurotoxicity and motor deficit in the MPTP mouse model of Parkinson's disease.

Neuroprotection following signal transduction has been investigated recently as a strategy for Parkinson's disease (PD) therapy. While oxidative stress is important in the pathogenesis of PD, neuroprotection using antioxidants such as α-tocopherol have not been successful. δ-tocotrienol (δT3), a member of the vitamin E family, has received attention because of activities other than its antioxidative effects. In the present study, we examined the estrogen receptor-ß (ERß)-mediated neuroprotective effects of δT3 in a mouse model of PD. ERß is expressed in neuronal cells, including dopaminergic neurons in the substantia nigra. Daily forced oral administration of δT3 inhibited the loss of dopaminergic neurons in the substantia nigra. In addition, the ER inhibitor tamoxifen canceled the neuroprotective effects of δT3. Moreover, δT3 administration improved the performance of the PD mice in the wheel running activity, while tamoxifen inhibited this improved performance. These results suggest that the oral administration of δT3 may be useful in the treatment of PD patients, and ERß may be a candidate target for the neuroprotection activity of δT3.

Oleuropein-Rich Diet Attenuates Hyperglycemia and Impaired Glucose Tolerance in Type 2 Diabetes Model Mouse.

Oleuropein, a phenolic compound found in abundance in olive leaves, has beneficial effects on various diseases. However, it is unknown whether an oleuropein-rich diet is efficacious against type 2 diabetic phenotypes. In this study, we investigated the effects of the oleuropein-containing supplement OPIACE, whose oleuropein content exceeds 35% (w/w), on the diabetic phenotypes in type 2 diabetes model Tsumura Suzuki Obese Diabetes (TSOD) mouse. TSOD mice were fed OPIACE at 4 weeks of age, i.e., before the TSOD mice exhibited diabetic phenotypes. We revealed that OPIACE attenuated hyperglycemia and impaired glucose tolerance in TSOD mice over the long-term (from 10 to 24 weeks of age) but had no effect on obesity. Furthermore, we demonstrated that OPIACE mildly reduced oxidative stress in TSOD mice by 26.2% and attenuated anxiety-like behavioral abnormality in aged TSOD mice. The results suggest that oleuropein suppresses the progression of type 2 diabetes and diabetes-related behavioral abnormality over the long-term.

Radical-scavenging Activity and Antioxidative Effects of Olive Leaf Components Oleuropein and Hydroxytyrosol in Comparison with Homovanillic Alcohol.

Olive leaf has great potential as a natural antioxidant, and one of its major phenolic components is oleuropein. In this study, the antioxidant activity of oleuropein against oxygen-centered radicals was measured by examining its sparing effects on the peroxyl radical-induced decay of fluorescein and pyrogallol red, in comparison with related compounds. The antioxidant capacity of oleuropein against lipid peroxidation was also assessed through its effect on the free radical-induced oxidation of methyl linoleate in a micelle system. On a molar basis, oleuropein and hydroxytyrosol inhibited the decay of fluorescein for longer than both homovanillic alcohol and the vitamin-E mimic 2-carboxy-2,5,7,8-tetramethyl-6-chromanol (Trolox), but did not suppress pyrogallol red decay in a concentration-dependent manner. Measurement of the fluorescein decay period revealed that the stoichiometric number of oleuropein and hydroxytyrosol against peroxyl radicals was twice that of Trolox, which is substantially higher than expectations based on chemical structure. Oleuropein and hydroxytyrosol were also more effective than Trolox and homovanillic alcohol at suppressing the oxidation of methyl linoleate in the micelle system. Thus, both oleuropein and hydroxytyrosol exhibit high antioxidative activity against lipid peroxidation induced by oxygen-centered radicals, but the high reactivity of phenolic/catecholic radicals makes their mechanism of action complex.

Kinetic study of the quenching reaction of singlet oxygen by α-, ß-, γ-, δ-tocotrienols, and palm oil and soybean extracts in solution.

Measurements of the singlet oxygen ((1)O2) quenching rates (kQ (S)) and the relative singlet oxygen absorption capacity (SOAC) values were performed for 11 antioxidants (AOs) (eight vitamin E homologues (α-, ß-, γ-, and δ-tocopherols and -tocotrienols (-Tocs and -Toc-3s)), two vitamin E metabolites (α- and γ-carboxyethyl-6-hydroxychroman), and trolox) in ethanol/chloroform/D2O (50:50:1, v/v/v) and ethanol solutions at 35 °C. Similar measurements were performed for five palm oil extracts 1-5 and one soybean extract 6, which included different concentrations of Tocs, Toc-3s, and carotenoids. Furthermore, the concentrations (wt%) of Tocs, Toc-3s, and carotenoids included in extracts 1-6 were determined. From the results, it has been clarified that the (1)O2-quenching rates (kQ (S)) (that is, the relative SOAC value) obtained for extracts 1-6 may be explained as the sum of the product {Σ kQ(AO-i) (S) [AO-i]/100} of the rate constant (kQ(AO-i) (S)) and the concentration ([AO-i]/100) of AO-i (Tocs, Toc-3s, and carotenoid) included.

Finding of synergistic and cancel effects on the aroxyl radical-scavenging rate and suppression of prooxidant effect for coexistence of α-tocopherol with ß-, γ-, and δ-tocopherols (or -tocotrienols).

Measurements of aroxyl radical (ArO•)-scavenging rate constants (k(s)(AOH)) of antioxidants (AOHs) [α-, ß-, γ-, and δ-tocopherols (TocHs) and -tocotrienols (Toc-3Hs)] were performed in ethanol solution via stopped-flow spectrophotometry. k(s)(AOH) values of α-, ß-, γ-, and δ-Toc-3Hs showed good agreement with those of the corresponding α-, ß-, γ-, and δ- TocHs. k(s)(AOH) values were measured not only for each antioxidant but also for mixtures of two antioxidants: (i) α-TocH with ß-, γ-, or δ-TocH and (ii) α-TocH with α-, ß-, γ-, or δ-Toc-3H. A synergistic effect in which the k(s)(AOH) value increases by 12% for γ-TocH (or by 12% for γ-Toc-3H) was observed for solutions including α-TocH and γ-TocH (or γ-Toc-3H). On the other hand, a cancel effect in which the k(s)(AOH) value decreases (a) by 7% for ß-TocH (or 11% for ß-Toc-3H) and (b) by 24% for δ-TocH (or 25% for δ-Toc-3H) was observed for solutions including two kinds of antioxidants. However, only a synergistic effect may function in edible oils, because contents of ß- and δ-TocHs (and ß- and δ-Toc-3Hs) are much less than those of α- and γ-TocHs (and α- and γ-Toc-3Hs) in many edible oils. UV-vis absorption of α-Toc•, which was produced by reaction of α-TocH with ArO•, decreased remarkably for coexistence of α-TocH with ß-, γ-, or δ-TocH (or ß-, γ-, or δ-Toc-3H), indicating that the prooxidant effect of α-Toc• is suppressed by the coexistence of other TocHs and Toc-3Hs.

Tocotrienol prevents AAPH-induced neurite degeneration in neuro2a cells.

OBJECTIVES: Reactive oxygen species induce neurite degeneration before inducing cell death. However, the degenerative mechanisms have not yet been elucidated. While tocotrienols have a known neuroprotective function, the underlying mechanism remains unclear and may or may not involve antioxidant action. In this study, we hypothesize that free radical-derived membrane injury is one possible mechanism for inducing neurite degeneration. Therefore, we examined the potential neuroprotective effect of tocotrienols mediated through its antioxidant activity. METHODS: Mouse neuroblastoma neuro2a cells were used to examine the effect of the water-soluble free radical generator 2,2'-azobis(2-methylpropionamide) dihydrochloride (AAPH) on neurite dynamics. After 24 hours of AAPH treatment, cell viability, neurite number, and the number of altered neurites were measured in the presence or absence of α-tocotrienol. RESULTS: Treatment of neuro2a cells with a low concentration of AAPH induces neurite degeneration, but not cell death. Treatment with 5 µM α-tocotrienol significantly inhibited neurite degeneration in AAPH-treated neuro2a cells. Furthermore, morphological changes in AAPH-treated neuro2a cells were similar to those observed with colchicine treatment. CONCLUSIONS: α-Tocotrienol may scavenge AAPH-derived free radicals and alkoxyl radicals that are generated from AAPH-derived peroxyl radicals on cell membranes. Therefore, α-tocotrienol may have a neuroprotective effect mediated by its antioxidant activity.

Vitamin E prevents hyperoxia-induced loss of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins in the rat neuronal cytoplasm.

This study examines the ability of vitamin E to inhibit hyperoxia-induced loss of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins in the neuronal cytoplasm. Here, the effects of vitamin E on hyperoxia-induced changes in the expressions of N-ethylmaleimide-sensitive factor (NSF) and soluble NSF-attachment protein α (α-SNAP) in the rat brain were analyzed. When rats were subjected to hyperoxia, the expression of both SNARE proteins was markedly decreased compared to normal rats. Vitamin E significantly inhibited the decrease in the expression of NSF in rats subjected to hyperoxia. Rats showed the tendency to improve the loss of α-SNAP by vitamin E-supplementation, although it was not statistically significant. On the other hand, vitamin E deficient rats showed marked loss of these proteins in the brain in the absence of oxidative stress. These results suggest that hyperoxia induces a loss of SNARE proteins, which are involved in membrane docking between synaptic vesicles and pre-synaptic membranes, and that vitamin E prevents the oxidative damage of SNARE proteins. Consequently, it is implied that vitamin E inhibits impaired neurotransmission caused by oxidative stress through the prevention of oxidative damage to SNARE proteins by probably its antioxidant effect.

Double-blind placebo-controlled trial of hydroxytyrosol of Olea europaea on pain in gonarthrosis.

Hydroxytyrosol is mainly found in olive leaves after hydrolysis of oleuropein and has anti-oxidant, anti-bacterial, and anti-inflammatory properties. The aim of this study was to investigate the effect of hydroxytyrosol for alleviating the pain in patients with gonarthrosis. We conducted a double-blind clinical trial in which hydroxytyrosol or placebo was administered to adult patients with gonarthrosis for 4 weeks. The group administered hydroxytyrosol showed significant improvement in the Japanese Orthopedic Association score (pain measurement index) and the visual analog scale score compared to the placebo group.

Effects of tocotrienol on tumor necrosis factor-α/d-galactosamine-induced steatohepatitis in rats.

It has been reported that α-tocopherol (α-Toc), a vitamin E analog, is effective for treatment of non-alcoholic steatohepatitis (NASH). However, it is unknown whether or not other vitamin E analogs are effective. Therefore we designed a new rat model of steatohepatitis induced by tumor necrosis factor-α (TNF-α) stimulation, and used it to investigate the effects of vitamin E analogs. The rat liver triglyceride content increased with the dosage of TNF-α/d-galactosamine (GalN), but was suppressed by intake of both tocotrienol (T3) and α-tocopherol. Moreover, lipid peroxides (thiobarbituric acid-reactive substances) level in the liver level was also lower in both groups after tocotrienol and α-Toc intake. Intake of both tocotrienol and α-tocopherol also tended to control the increase of liver damage marker activity. In the tocotrienol and α-tocopherol groups, increases of inflammatory cytokines mRNA expression in the liver were inhibited, and these effects were considered to contribute to improvement of inflammation and fibrosis. The expression of mRNAs for inflammatory cytokines in rat primary hepatocytes was increased by TNF-α stimulation, but was inhibited by addition of α-tocotrienol and γ-tocotrienol. Transforming growth factor-ß1 mRNA expression in particular was significantly inhibited by γ-tocotrienol. These findings suggest that tocotrienol species are effective for amelioration of steatohepatitis, and that tocotrienol and α-tocopherol exert a synergistic effect.

Gamma-tocotrienol reduces the triacylglycerol level in rat primary hepatocytes through regulation of fatty acid metabolism.

The present study was carried out to investigate the effect of vitamin E analogs, especially gamma-tocotrienol (γ-T3), on hepatic TG accumulation and enzymes related to fatty acid metabolism in three types of rat primary hepatocytes: (1) normal hepatocytes, (2) hepatocytes incubated in the presence of palmitic acid (PA), and (3) hepatocytes with fat accumulation. Our results showed that γ-T3 significantly reduced the TG content of normal hepatocytes. γ-T3 also increased the expression of carnitine palmitoyltransferase 1 (CPT1A) mRNA, and tended to reduce that of sterol regulatory element binding protein 1c (SREBP-1c) mRNA. In addition, γ-T3 markedly suppressed the gene expression of both C/EBP homologous protein (CHOP) and SREBP-1c induced by PA. As these two genes are located downstream of endoplasmic reticulum (ER) stress, their suppression by γ-T3 might result from a decrease of ER stress. Moreover, γ-T3 suppressed the expression of interleukin 1ß (IL-1ß), which lies downstream of CHOP signaling. Taken together, our data suggest that γ-T3 might prevent hepatic steatosis and ameliorate ER stress and subsequent inflammation in the liver.

Interleukin-12- and interferon-gamma-mediated natural killer cell activation by Agaricus blazei Murill.

Dried fruiting bodies of Agaricus blazei Murill (A. blazei) and its extracts have generally used as complementary and alternative medicines (CAMs). Here, we report that the oral administration of A. blazei augmented cytotoxicity of natural killer (NK) cells in wild-type (WT) C57BL/6, C3H/HeJ, and BALB/c mice. Augmented cytotoxicity was demonstrated by purified NK cells from treated wild-type (WT) and RAG-2-deficient mice, but not from interferon-gamma (IFN-gamma) deficient mice. NK cell activation and IFN-gamma production was also observed in vitro when dendritic cell (DC)-rich splenocytes of WT mice were coincubation with an extract of A. blazei. Both parameters were largely inhibited by neutralizing anti-interleukin-12 (IL-12) monoclonal antibody (mAb) and completely inhibited when anti-IL-12 mAb and anti-IL-18 mAb were used in combination. An aqueous extract of the hemicellulase-digested compound of A. blazei particle; (ABPC) induced IFN-gamma production more effectively, and this was completely inhibited by anti-IL-12 mAb alone. NK cell cytotoxicty was augmented with the same extracts, again in an IL-12 and IFN-gamma-dependent manner. These results clearly demonstrated that A. blazei and ABPC augmented NK cell activation through IL-12-mediated IFN-gamma production.