Yuko Takano-Ishikawa

Inhibitory Effect of Some Flavonoids on Tumor Necrosis Factor-α Production in Lipopolysaccharide-Stimulated Mouse Macrophage Cell Line J774.1

Certain naturally occurring flavonoids affect immunoregulatory activities in vitro and in vivo against cytokine production. Since tumor necrosis factor (TNF)-alpha is one of the major inflammatory cytokines, the effects of various dietary flavonoids on TNF-alpha production in lipopolysaccharide (LPS)-stimulated J774.1 cells were evaluated in vitro. Flavones, flavonols, and chalcone are the most potent inhibitors of production of TNF-alpha. Flavanone, naringenin, anthocyanidin, pelargodinin, and cyanidin exhibit moderate inhibitory activity. In contrast, genistein isoflavone displays weak inhibition, while eriodictyol flavanone is inactive. It is clear that the double bond between carbons 2 and 3 and the ketone group at position 4 of flavonoids are necessary for potent inhibitory effect. The difference in inhibitory action appears to depend on the categorized subclass of flavonoids.

Orally Administered Bisphenol A Disturbed Antigen Specific Immunoresponses in the Naïve Condition

Bisphenol A [2,2-bis(4-hydoxyphenyl)propane; BPA] is an endocrine disrupter widely used in polycarbonate plastics and epoxy resins. We investigated the effects of orally administered BPA on antigen-specific responses of the naïve immune system. BPA was orally administered to T cell receptor transgenic mice, and the antigen-specific responses of immune cells were investigated. Administered BPA moderately reduced interleukin (IL)-2, 4, and interferon (IFN)-γ secretion and increases in IgA and IgG2a production. Additionally, it was found that orally administered BPA increased antigen-specific IFN-γ production of T cells and modified whole antigen presenting cells (APCs) to suppress antigen-specific cytokine production from T cells. These findings suggest that BPA can augment the Th1-type responses of naïve immune systems, though the bioavailability of orally administered BPA was low in our experiments.

Wheat Alkylresorcinols Suppress High-Fat, High-Sucrose Diet-Induced Obesity and Glucose Intolerance by Increasing Insulin Sensitivity and Cholesterol Excretion in Male Mice

BACKGROUND Epidemiologic studies have shown that the consumption of whole grains can reduce the risk of type 2 diabetes mellitus, cardiovascular disease, and all-cause mortality. However, the underlying mechanisms remain a matter of debate. OBJECTIVE We aimed to determine the effects of wheat bran-derived alkylresorcinols on diet-induced metabolic disorders in mice. METHODS We fed C57BL/6J mice a normal refined diet or a high-fat, high-sucrose diet [29.1% fat, 20.7% protein, 34.0% carbohydrates containing 20.0% sucrose (w/w)] alone (FS) or containing 0.4% (wt:wt) alkylresorcinols (FS-AR) for 10 wk. RESULTS The alkylresorcinols suppressed FS-induced increases in body weight by 31.0% as well as FS-induced hepatic triglyceride accumulation (means ± SEMs: 29.6 ± 3.18 and 19.8 ± 2.42 mg/g tissue in the FS and FS-AR groups, respectively), without affecting energy intake. We measured circadian changes in blood metabolic hormones and found that FS-induced hyperinsulinemia (5.1 and 2.1 μg/L at night in the FS and FS-AR groups, respectively) and hyperleptinemia (21.6 and 10.8 μg/L at night in the FS and FS-AR groups, respectively) were suppressed by alkylresorcinols. Glucose and insulin tolerance tests showed that alkylresorcinols significantly reduced fasting blood glucose concentrations (190 ± 3.62 and 160 ± 8.98 mg/dL in the FS and FS-AR groups, respectively) and suppressed glucose intolerance as well as insulin resistance induced by the FS diet. Furthermore, alkylresorcinols significantly increased insulin-stimulated hepatic serine/threonine protein kinase B phosphorylation compared to the FS diet (+81.3% and +57.4% for Ser473 and Thr308, respectively). On the other hand, pyruvate and starch tolerance tests suggested that alkylresorcinols did not affect gluconeogenesis and carbohydrate digestion, respectively. Alkylresorcinols significantly increased fecal cholesterol excretion by 39.6% and reduced blood cholesterol concentrations by 30.4%, while upregulating the expression of hepatic cholesterol synthetic genes such as sterol regulatory element binding protein 2 (Srebf2) and 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (Hmgcs1). CONCLUSIONS These findings suggest that wheat alkylresorcinols increase glucose tolerance and insulin sensitivity by suppressing hepatic lipid accumulation and intestinal cholesterol absorption, which subsequently suppresses diet-induced obesity in mice.

Identification of 4-hydroxy-2-nonenal-histidine adducts that serve as ligands for human lectin-like oxidized LDL receptor-1

LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) is an endothelial scavenger receptor that is important for the uptake of OxLDL (oxidized low-density lipoprotein) and contributes to the pathogenesis of atherosclerosis. However, the precise structural motifs of OxLDL that are recognized by LOX-1 are unknown. In the present study, we have identified products of lipid peroxidation of OxLDL that serve as ligands for LOX-1. We used CHO (Chinese-hamster ovary) cells that stably express LOX-1 to evaluate the ability of BSA modified by lipid peroxidation to compete with AcLDL (acetylated low-density lipoprotein). We found that HNE (4-hydroxy-2-nonenal)-modified proteins most potently inhibited the uptake of AcLDL. On the basis of the findings that HNE-modified BSA and oxidation of LDL resulted in the formation of HNE-histidine Michael adducts, we examined whether the HNE-histidine adducts could serve as ligands for LOX-1. The authentic HNE-histidine adduct inhibited the uptake of AcLDL in a dose-dependent manner. Furthermore, we found the interaction of LOX-1 with the HNE-histidine adduct to have a dissociation constant of 1.22×10(-8) M using a surface plasmon resonance assay. Finally, we showed that the HNE-histidine adduct stimulated the formation of reactive oxygen species and activated extracellular-signal-regulated kinase 1/2 and NF-κB (nuclear factor κB) in HAECs (human aortic endothelial cells); these signals initiate endothelial dysfunction and lead to atherosclerosis. The present study provides intriguing insights into the molecular details of LOX-1 recognition of OxLDL.

Comparison of scavenging capacities of vegetables by ORAC and EPR

Reactive oxygen species (ROS) are considered to be causative agents of many health problems. In spite of this, the radical-specific scavenging capacities of food samples have not been well studied. In the present work, we have developed an electron paramagnetic resonance (EPR) spin trapping method for analysis of the scavenging capacities of food samples for multiple ROS, utilising the same photolysis procedure for generating each type of radical. The optimal conditions for effective evaluation of hydroxyl, superoxide, and alkoxyl radical scavenging capacity were determined. Quantification of radical adducts was found to be highly reproducible, with variations of less than 4%. The optimised EPR spin trapping method was used to analyse the scavenging capacities of 54 different vegetable extracts for multiple radicals, and the results were compared with oxygen radical absorption capacity values. Good correlations between the two methods were observed for superoxide and alkoxyl radicals, but not for hydroxyl.

Extraction Efficiency of Hydrophilic and Lipophilic Antioxidants from Lyophilized Foods Using Pressurized Liquid Extraction and Manual Extraction

The efficient extraction of antioxidants from food samples is necessary in order to accurately measure their antioxidant capacities. α-Tocopherol and gallic acid were spiked into samples of 5 lyophilized and pulverized vegetables and fruits (onion, cabbage, Satsuma mandarin orange, pumpkin, and spinach). The lipophilic and hydrophilic antioxidants in the samples were sequentially extracted with a mixed solvent of n-hexane and dichloromethane, and then with acetic acid-acidified aqueous methanol. Duplicate samples were extracted: one set was extracted using an automated pressurized liquid extraction apparatus, and the other set was extracted manually. Spiked α-tocopherol and gallic acid were recovered almost quantitatively in the extracted lipophilic and hydrophilic fractions, respectively, especially when pressurized liquid extraction was used. The expected increase in lipophilic oxygen radical absorbance capacity (L-ORAC) due to spiking with α-tocopherol, and the expected increase in 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities and total polyphenol content due to spiking with gallic acid, were all recovered in high yield. Relatively low recoveries, as reflected in the hydrophilic ORAC (H-ORAC) value, were obtained following spiking with gallic acid, suggesting an interaction between gallic acid and endogenous antioxidants. The H-ORAC values of gallic acid-spiked samples were almost the same as those of postadded (spiked) samples. These results clearly indicate that lipophilic and hydrophilic antioxidants are effectively extracted from lyophilized food, especially when pressurized liquid extraction is used.

Improvement of the Lipophilic-Oxygen Radical Absorbance Capacity (L-ORAC) Method and Single-Laboratory Validation

We improved the procedure for lipophilic-oxygen radical absorbance capacity (L-ORAC) measurement for better repeatability and intermediate precision. A sealing film was placed on the assay plate, and glass vials and microdispensers equipped with glass capillaries were used. The antioxidant capacities of food extracts can be evaluated by this method with nearly the same precision as antioxidant solutions.

Continuous Orally Administered Coffee Enhanced the Antigen-Specific Th1 Response and Reduced Allergic Development in a TCR-Transgenic Mice Model

Coffee is a globally consumed beverage. Although recent studies have suggested that coffee reduced the risk of lifestyle-related diseases, there are few studies regarding allergic response. This study investigates the effects of orally administered coffee (91 ml/kg/d) on allergic responses using a T cell receptor (TCR)-transgenic DO11.10 mouse allergic model. Splenocytes from coffee-administered naïve mice increased antigen (Ag)-specific interleukin (IL)-12p40 secretion. When Ag sensitization and coffee administration were concurrently performed, the splenocytes from coffee-administered mice showed a decrease of IL-2 and an increase of IL-12p40 secretion. The Ag-specific cutaneous response and serum IgE level were reduced in coffee-administered mice, although, after establishing the allergy, coffee administration did not suppress the allergic reaction. These results suggest that coffee could induce a Th1-type response of the immune system and prevent an allergy developing. Further studies on the optimum dose, cultivar differences, and roasted degree need to be undertaken.

Mac1 Positive Cells Are Required for Enhancement of Splenocytes Proliferation Caused by Bisphenol A

We examined the effects of bisphenol A (BPA) on immune cells and it was shown that BPA upregulated the proliferation of murine splenocytes stimulated with Concanavalin A (ConA). The upregulating effects of BPA were removed with depleting Mac1+ cells from the splenocytes. This study provides evidence for the first time that Mac1+ cells were required for enhancement of splenocytes proliferation caused by bisphenol A.

An improved method for measuring vascular permeability in rat and mouse skin

INTRODUCTION We have improved a rodent vascular permeability measurement method employing fluorescent dye-labeled bovine serum albumin. METHODS The incubation duration for direct fluorescent detection of skin injected with an inflammatory agent was decided based on regression curve parameters with the correlation coefficient obtained from the least squares method. RESULTS A suitable incubation time was determined to be 2-6 h. The recovery of FITC-BSA from the skin sample was very good, and the correlation coefficient of the linear regression curve was .99. The linear relation between the previous dye extraction method using brilliant blue 6B and the new and improved fluorescence method was very high. In mice, histamine-induced serum exudation in the back skin increased from 0.31 to 1.25 microg/site in a dose-dependent manner and reached a plateau at 1.25-2.5 microg/site. The serum exudation caused by histamine increased to 10 microg/site and almost reached a plateau at 10-40 microg/site in rats. The time required for the measurement of fluorescence intensity was very short because a microplate reader was used as the measurement apparatus. CONCLUSION The improved method is easy to use and sensitive and does not necessitate extraction of dye from the skin.