Heat-killed Lactiplantibacillus plantarum Shinshu N-07 exerts antiobesity effects in western diet-induced obese mice.

AIMS: The aim of this study was to evaluate the antiobesity effects of heat-killed Lactiplantibacillus plantarum Shinshu N-07 (N-07) isolated from fermented Brassica rapa L. METHODS AND RESULTS: Male mice were divided into three groups (n = 10/group); normal diet, western diet (WD), or WD + N-07 (N-07) group and administered each diet for 56 days. The N-07 group showed significant suppression of body weight gain and epididymal fat, perirenal fat, and liver weights compared with the WD group. Higher levels of fecal total cholesterol, triglyceride (TG), and free fatty acid (FFA) were observed in the N-07 group than in the WD group. The mRNA expression of the cholesterol transporter ATP-binding cassette transporter G5 (ABCG5) was significantly increased in the small intestine of N-07-fed mice compared with WD-fed mice. Moreover, N-07 supplementation significantly increased the mRNA expression of ABCG5 and ABCG8 in Caco-2 cells. Furthermore, the TG- and FFA-removal ability of N-07 was confirmed to evaluate its soybean oil- and oleic acid-binding capacities in in vitro experiments. CONCLUSIONS: The antiobesity effects of N-07 might be due to its ability to promote lipid excretion by regulating cholesterol transporter expression and lipid-binding ability.

Lactobacillus crispatus Strain KT-11 S-Layer Protein Inhibits Rotavirus Infection.

S-layer proteins (SLPs), which are present in the external layer of certain strains of lactic acid bacteria isolated from the intestinal tract, are known to recognize and bind to specific proteins and glycan structures and contribute to adsorption to the host intestinal mucosa. The binding properties of certain SLPs are considered to exert a competitive inhibitory effect on infection because similar properties are involved in the infection mechanisms of several viruses. However, little is known regarding whether SLPs directly inhibit viral infection. In the present study, we investigated the effect of an SLP of the Lactobacillus crispatus KT-11 strain, a probiotic strain isolated from a healthy human infant, on human rotavirus infection. The impact of KT-11 lithium chloride extract (KT-11 LE), which contains SLP, on the infection of the P[4] genotype human rotavirus strain DS-1 was evaluated by monitoring the amplification of viral protein 6 (VP6) expression in human intestinal epithelial Caco-2 cells by quantitative reverse transcription-polymerase chain reaction assay after infection. KT-11 LE showed a significant suppressive effect on DS-1 infection in a dose-dependent manner with pre-infection treatment, whereas post-infection treatment was not effective. A 45 KDa protein isolated from KT-11 LE was investigated for homology using the BLAST database and was found to be a novel SLP. KT-11 SLP concentrate (KT-11 SLP) significantly inhibited the proliferative process of the DS-1 strain but not that of the P[8] genotype human rotavirus strain Wa. KT-11 SLP exerted significant inhibitory effect on DS-1 infection by pre-infection treatment even after digestion with gastric juice up to 2 h. Our results provided crucial evidence that SLPs from certain Lactobacillus strains can inhibit human rotavirus infection of intestinal epithelial cells.

Inhibitory effect of strawberry geranium (Saxifraga stolonifera) on Toll-like receptor 2-mediated inflammatory response in human skin keratinocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Strawberry geranium (Saxifraga stolonifera [L.] Meeb) has traditionally been used as a drug to treat skin disorders in Japan. However, little is known about its physiological effects on skin keratinocytes. AIM OF THE STUDY: We investigated the anti-inflammatory effects of a strawberry geranium extract (SGE) on human skin keratinocytes. MATERIALS AND METHODS: The human keratinocyte cell line, HaCaT, was treated with SGE, and then stimulated with tumor necrosis factor (TNF)-α. The expression of 207 genes related to the innate immune system was analyzed using DNA microarrays. The effect of SGE on the target proteins in primary human epidermal keratinocytes was confirmed by quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The mechanisms of action and active components involved in the suppressive effect of SGE were evaluated by fractionation and a transcription assay. RESULTS: The microarray analysis revealed that SGE primarily suppressed Toll-like receptor (TLR)2 expression through procyanidin B2 3,3'-di-O-gallate, without TLR2 downregulation, in TNF-α-stimulated HaCaT cells. SGE suppressed TLR2 expression and interleukin (IL)-8 production induced by TLR2 ligands in primary human epidermal keratinocytes and HaCaT cells. Multiple components downregulating TLR2 expression suppressed the Sp1 activity. CONCLUSIONS: We identified a novel physiological function of SGE, which suppresses TLR2 expression and TLR2-mediated inflammation in human skin keratinocytes. This study provides significant insights into the anti-inflammatory effect of SGE in human skin.

Metabolomic evaluation of different starter culture effects on water-soluble and volatile compound profiles in nozawana pickle fermentation.

Metabolomic characterization of a lactic-fermented pickle of nozawana (Brassica rapa L. var. hakabura) was conducted to evaluate the effects of different starter culture strains on the chemical profiles. We compared the profiles of water-soluble and volatile compounds obtained by non-targeted nuclear magnetic resonance and solid-phase microextraction gas chromatography/mass spectrometry analyses. Principal component analyses indicated that the fermented samples differed significantly in terms of the levels of various compounds, including taste- and aroma-active components, such as water-soluble residual sugars, organic acids, mannitol, ethanol, dihydroxyacetone, ornithine, γ-aminobutyric acid, choline, volatile isothiocyanates, 3,4-epithiobutyl cyanide, 2,3-butanedione, acetoin, ethyl acetate, dimethyl trisulfide, and S-methyl thioacetate. Fermentation with a Latilactobacillus curvatus culture was associated with a unique metabolite profile characterized by higher levels of isothiocyanates and hexanoic acid and lower levels of lactic acid, acetic acid, acetoin, and 2,3-butanedione. These variations in the chemical profile might be associated with different qualities in fermented nozawana pickle products.

Suppressive Effects of Hot-water Extract of Magnolia obovata on Clostridium perfringens Enterotoxin-induced Cytotoxicity in Human Intestinal Caco-2 Cells.

The physiological functions of the leaves of Japanese big-leaf magnolia (Magnolia obovata) against enterotoxins produced by foodborne pathogens remain unclear. In this study, we evaluated the protective effects of M. obovata leaf extract (MLE) against the cytotoxicity of Clostridium perfringens enterotoxin (CPE), which causes the symptoms of C. perfringens type A food poisoning. The protective effects of MLE against CPE-induced cytotoxicity were evaluated in human intestinal epithelial Caco-2 cells. Pre-treatment with MLE significantly suppressed the cytotoxicity induced by CPE in undifferentiated and differentiated human intestinal Caco-2 cells at a pH range of 4.0 - 7.0. This CPE-suppressive effect was due to a hydrophilic sugar-containing compound without phenolic and protein structures but not the hydrophobic biologically active neolignans, honokiol and magnolol. MLE had a protective effect against cytotoxicity caused by type A C. perfringens. Our results provide novel insight regarding the usage of M. obovata in managing food poisoning.

Characterization of Microbiota that Influence Immunomodulatory Effects of Fermented Brassica rapa L.

Lactic acid bacteria (LAB) exert beneficial health effects by regulating immune responses. Brassica rapa L., known as Nozawana, is commonly consumed as a lactic acid-fermented food called nozawana-zuke. Few studies have investigated changes in the bacterial community and cytokine production activities during the fermentation of B. rapa L. In order to obtain more detail information, we herein conducted a study on fresh B. rapa L. fermented for 28 d. An amplicon analysis of the 16S rRNA gene revealed that Lactobacillales predominated during fermentation, and the microbiota became less diverse on day 7 or later. Fermented B. rapa L. promoted the production of interferon (IFN)-γ and interleukin (IL)-10 by mouse spleen cells more than non-fermented vegetables. Lactobacillus curvatus was the predominant species during fermentation, followed by L. plantarum and L. brevis. L. sakei was occasionally detected. A correlation analysis showed that IFN-γ concentrations positively correlated with the numbers of L. curvatus and L. plantarum, while those of IL-10 correlated with the numbers of L. sakei in addition to these 2 species. Significantly higher levels of IFN-γ and IL-10 were induced by fermented B. rapa L. when isolated Lactobacillus strains were added as starter cultures. These results suggest that the Lactobacillus species present in fermented B. rapa L. are beneficial for manufacturing vegetables with immunomodulatory effects.

Effect of the topical application of an ethanol extract of quince seeds on the development of atopic dermatitis-like symptoms in NC/Nga mice.

BACKGROUND: Quince (Cydonia oblonga Miller) is a deciduous shrub belonging to the Rosaceae family. Quince seed extract has long been used as a cosmetic ingredient for its moisturizing effect. However, little is known about whether quince seed extract has therapeutic effects on keratinocyte-associated skin inflammation. METHODS: In the present study, we investigated the effect of the topical application of ethanol extract of quince seeds (QSEtE) on atopic dermatitis (AD) symptoms in NC/Nga mice. The direct effect of QSEtE on keratinocytes was evaluated using the human keratinocyte cell line HaCaT. RESULTS: The preliminary application of QSEtE markedly reduced house dust mite allergen-induced skin lesions. The expression of thymus- and activation-regulated chemokine (TARC) in dorsal skin was downregulated. QSEtE directly suppressed the expression and production of TARC in HaCaT cells. CONCLUSIONS: The results suggest that the topical application of QSEtE is effective in preventing the onset of and ameliorating the atopic symptoms of keratinocyte-associated skin inflammation by suppressing TARC production in keratinocytes.

Inhibitory Effect of a Hot-Water Extract of Leaves of Japanese Big-Leaf Magnolia (Magnolia obovata) on Rotavirus-Induced Diarrhea in Mouse Pups.

The leaf of Japanese big-leaf magnolia (Magnolia obovata Thunb.) has long been used as a natural packaging material for traditional foods in Japan. However, many of the physiological functions of the leaves against oral infection and resultant illness remain unclear. The aim of the present study was to investigate the effects of a hot-water extract of the leaves of Magnolia obovata on diarrhea induced by rotavirus (RV), a major cause of acute diarrhea. RV strain SA11 was mixed with the M. obovata leaf extract and inoculated orally to neonatal BALB/c mouse pups. Simultaneous inoculation of SA11 with the extract significantly decreased the incidence of diarrhea. In addition, the extract significantly inhibited cytopathic effects and mRNA expression of viral proteins in SA11-infected MA104 cells. Two flavonoid glycosides, quercitrin and rutin, were strongly suggested to be major anti-RV agents in the extract by serial solvent extraction and reversed-phase HPLC-ESI-MS analysis. Our results suggest that the hot-water extract of M. obovata leaves can be used as a medicine or food additive to prevent and ameliorate RV-induced diarrhea in individuals that may have difficulty in benefitting from the RV vaccines.

Establishment and characterization of mouse bone marrow-derived mast cell hybridomas.

Interleukin (IL)-3-dependent mouse bone marrow-derived mast cells (BMMCs) are an important model for studying the function of mucosal-type mast cells. In the present study, BMMCs were successfully immortalized by cell fusion using a hypoxanthine-aminopterin-thymidine medium-sensitive variant of P815 mouse mastocytoma (P815-6TgR) as a partner cell line. The established mouse mast cell hybridomas (MMCHs) expressed α, ß, and γ subunits of high-affinity immunoglobulin E (IgE) receptor (FcεRI) and possessed cytoplasmic granules devoid of or partially filled with electron-dense material. Four independent MMCH clones continuously proliferated without supplemental exogenous IL-3 and showed a degranulation response on stimulation with IgE+antigen. Furthermore, histamine synthesis and release by degranulation were confirmed in MMCH-D5, a MMCH clone that showed the strongest degranulation response. MMCH-D5 exhibited elevated levels of IL-3, IL-4, IL-13, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-α, and cyclooxygenase 2, and production of prostaglandin D(2) and leukotriene C(4) in response to IgE-induced stimulation. MMCH clones also expressed Toll-like receptors (TLRs) 1, 2, 4, and 6 and showed elevated levels of TNF-α expression in response to stimulation with TLR2 and TLR4 ligands. The MMCHs established using this method should be suitable for studies on FcεRI- and TLR-mediated effector functions of mast cells.

Inhibitory effect of hot-water extract of quince (Cydonia oblonga) on immunoglobulin E-dependent late-phase immune reactions of mast cells.

We evaluated the effect of a crude hot-water extract (HW) of quince (Cydonia oblonga Miller) fruit on immunoglobulin E (IgE)-dependent late-phase immune reactions of mast cells using in vitro system. Mast cell-like RBL-2H3 cells were treated with quince HW and late-phase reaction was then induced by stimulation with IgE + Antigen. Quince HW reduced the elevation of interleukin-13 and tumor necrosis factor-α expression level. Furthermore, quince HW suppressed these cytokine expressions of mouse bone marrow-derived mast cells (BMMCs), a normal mast cell model. Leukotriene C(4) and prostaglandin D(2) production in BMMCs after 1 and 6 h of stimulation, respectively, were also reduced by treating the cells with quince HW. We found that the induction of intracellular cyclooxygenase (COX)-2 expression but not COX-1 expression in BMMCs was reduced by quince HW. These results suggest that quince HW has an inhibitory effect on broad range of the late-phase immune reactions of mast cells.

Inhibitory effect of heat-killed Lactobacillus strain on immunoglobulin E-mediated degranulation and late-phase immune reactions of mouse bone marrow-derived mast cells.

This study investigated the in vitro effect of Lactobacillus strains, a major group of probiotic lactic acid bacteria, on immunoglobulin E (IgE)- and antigen-induced mast cell degranulation and subsequent gene expression. Bone marrow-derived mast cells (BMMCs) from DBA/2 mice were cultured with heat-killed Lactobacillus strains for 24 h. Some strains significantly inhibited IgE- and antigen-induced ß-hexosaminidase release from BMMCs. Furthermore, Lactobacillus reuteri NBRC 15892, which exhibited the strongest inhibitory activity, significantly reduced the elevated interleukin (IL)-4, IL-13, tumor necrosis factor-α, and cyclooxygenase-2 expression levels that was induced by 1-2 h of stimulation with IgE and antigens. The suppressive effect of NBRC 15892 strain on BMMC degranulation was significantly reduced in the presence of a toll-like receptor (TLR)2-neutralizing antibody. In addition, downregulation of cell surface FcεRIα expression was observed after 6 h of NBRC 15892 treatment. These results suggest that some Lactobacillus strains inhibited IgE-mediated mast cell degranulation and subsequent late-phase reactions involving mast cells via a TLR2-dependent mechanism with FcεRIα downregulation.

Anti-allergic effect of a hot-water extract of quince (Cydonia oblonga).

We examined the effect of a crude hot-water extract (HW) of quince (Cydonia oblonga Miller) fruit on type I allergy in vivo and in vitro. The oral administration of the quince HW-added diet to NC/Nga mice for 63 d showed a significant decrease in the development of atopic dermatitis-like skin lesions under conventional conditions. The concentration of IgE in the serum collected from mice fed with quince HW was also lowered in a dose-dependent manner. Moreover, we found that quince HW inhibited the release of beta-hexosaminidase from rat basophilic leukemia cell line RBL-2H3 after a 24-h treatment. The quince HW fraction of less than 3 kDa reduced the mRNA expression of the high-affinity IgE receptor (FcepsilonRI) gamma subunit. These results suggest that quince HW had an inhibitory effect on type I allergy by suppressing IgE production and IgE-mediated degranulation.

Inhibitory effect of electrolyzed reduced water on tumor angiogenesis.

Vascular endothelial growth factor (VEGF) is a key mediator of tumor angiogenesis. Tumor cells are exposed to higher oxidative stress compared to normal cells. Numerous reports have demonstrated that the intracellular redox (oxidation/reduction) state is closely associated with the pattern of VEGF expression. Electrolyzed reduced water (ERW) produced near the cathode during the electrolysis of water scavenged intracellular H(2)O(2) and decreased the release of H(2)O(2) from a human lung adenocarcinoma cell line, A549, and down-regulated both VEGF transcription and protein secretion in a time-dependent manner. To investigate the signal transduction pathway involved in regulating VEGF expression, mitogen-activated kinase (MAPK) specific inhibitors, SB203580 (p38 MAPK inhibitor), PD98059 (ERK1/2 inhibitor) and JNKi (c-Jun N-terminal protein kinase inhibitor) were applied. The results showed that only PD98059 blocks VEGF expression, suggesting an important role for ERK1/2 in regulating VEGF expression in A549 cells. As well, ERW inhibited the activation of extracellular signal-regulated kinase (ERK) in a time-dependent manner. Co-culture experiments to analyze in vitro tubule formation assay revealed that A549 cell-derived conditioned medium significantly stimulated the formation of vascular tubules in all analyzed parameters; tubule total area, tubule junction, number of tubules, and total tubule length. ERW counteracted the effect of A549 cell-conditioned medium and decreased total tube length (p<0.01). The present study demonstrated that ERW down-regulated VEGF gene transcription and protein secretion through inactivation of ERK.

Oral ingestion of phosphorylated dextrin stimulates antibody responses in mice.

Corn dextrin with an average degradation degree of 16.7, or molecular mass of 2,700, was covalently introduced with 4.5 mol of phosphoric acid per mole by dry-heating with sodium phosphate. The effect on immunoglobulin production of the phosphorylated dextrin in mice that orally ingested lipopolysaccharide (LPS) from Salmonella typhimurium was investigated. No significant difference in body weight gain was observed between mice fed a phosphorylated dextrin-containing diet and those fed a control (dextrin-containing) diet. Fecal and intestinal anti-LPS immunoglobulin (Ig) A, intestinal and serum anti-LPS IgG, and fecal and intestinal total IgA levels were significantly higher in the mice given the phosphorylated dextrin. In contrast, serum and intestinal levels of IgM specific to the LPS were similar between the two groups. Moreover, spleen cells from mice fed the phosphorylated dextrin-added diet had significantly higher levels of anti-LPS IgG and IgA than those from mice on the control diet. These results suggest that dietary phosphorylated dextrin protects against local and systemic invasions of pathogenic microorganisms in mice.

[A case of adenosquamous gastric carcinoma successfully treated with TS-1, low-dose CDDP and docetaxel as neoadjuvant chemotherapy].

The patient was a 66-year-old male with extremely advanced gastric cancer type 3 and diagnosed with adenocarcinoma by endoscopic biopsies specimens. Combined chemotherapy of TS-1, CDDP and docetaxel was prescribed in order for tumor reduction and downstaging. TS-1 (80 mg/m(2)) was administered 28 days followed by 14 days rest as one course. CDDP (8 mg/m(2)) was administered on days 1, 2, 14 and 15 and docetaxel (40 mg/m(2)) was administered on day 1 and 14, followed by 4 weeks rest as one course. After 2 courses of treatment, a CT scan revealed a minor response of tumor reduction. Therefore, total gastrectomy, partial pancreas body and tail resection, and D 2 lymph node dissection were performed. The patient had undergone adjuvant chemotherapy of TS-1 and biweekly docetaxel after surgery with no recurrence for 13 months. Adverse reactions were grade 3 neutropenia and grade 2 diarrhea. Combined chemotherapy of TS-1, low-dose CDDP and docetaxel were intensive and required constant patient monitoring. However, it proved effective and feasible as a neoadjuvant chemotherapy regimen for advanced gastric cancer.

Catalyser-21(TM), a mineral water derived from leaf soil, inhibits tumor cell invasion and angiogenesis.

Catalyser-21(TM) is a mineral water derived from natural leaf soil containing various organic and inorganic substances. Previous reports suggested a possibility that Catalyser-21(TM) has antioxidative potential and could inhibit angiogenesis and cancer cell invasiveness. Angiogenesis is a prerequisite for cancer cells to spread to surrounding tissues. Vascular endothelial growth factor (VEGF) is a major angiogenic factor in the formation of blood capillaries by cancer cells to supply nutrients and oxygen for their sustained growth. Matrix metalloproteinase-2 (MMP-2) is another key enzyme for cancer cell metastasis. To assess the anti-angiogenic activity of Catalyser-21(TM), we first examined cell viability using a human cervical cancer cell line, HeLa, and a fibrosarcoma cell line, HT1080. The results showed that Catalyser-21(TM) decreased the viability of both cell types in a dose-dependent manner. Flow cytometric analysis proved that Catalyser-21(TM) scavenges intracellular H(2)O(2) in both cell types. RT-PCR demonstrated that both VEGF and MMP-2 gene transcription was suppressed after Catalyser-21(TM) treatment. Both Matrigel and tubule formation experiments showed an effect of Catalyser-21(TM). These results suggest that Catalyser-21(TM) has potential as an anti-tumor agent.

Bovine beta-casein (1-28), a casein phosphopeptide, enhances proliferation and IL-6 expression of mouse CD19+ cells via Toll-like receptor 4.

This study was conducted to elucidate the target cells and receptors which participate in the mitogenic and interleukin (IL)-6-enhancing effect of bovine beta-casein (1-28), a casein phosphopeptide. When the spleen lymphocyte subset (CD4+, CD8+, and CD19+ cells) from C3H/HeN mice was cultured with the beta-casein (1-28), it exerted a dose-dependent mitogenic effect on CD19+ cells. The effect of beta-casein (1-28) was not apparent in the case of CD19+ cells from C3H/HeJ mouse. In addition, the effect was significantly inhibited by treating the C3H/HeN mouse-derived CD19+ cells with neutralizing antibody for toll-like receptor 4 (TLR4). Reverse transcription-polymerase chain reaction analysis showed that the beta-casein (1-28) exerted an IL-6-enhancing effect on the CD19+ cells. The effect was also abrogated in either C3H/HeJ mouse-derived CD19+ cell culture or the anti-TLR4 antibody-added culture. These results suggest that the beta-casein (1-28) stimulates both proliferation and IL-6 expression of CD19+ cells via TLR4.

Stimulatory effect of lactic acid bacteria from commercially available Nozawana-zuke pickle on cytokine expression by mouse spleen cells.

We investigated the effect of lactic acid bacteria (LAB) isolated from eight samples of commercially available Nozawana-zuke, a traditional Japanese pickle, on cytokine expression by mouse spleen cell cultures. The 12 isolated strains of LAB (Nz1-Nz12), which were identified as genus Lactobacillus or Leuconostoc by the API50CHL test, enhanced the expression of interferon (IFN)-gamma with 6 h of culture. Ten of these 12 LAB, particularly Nz8, enhanced interleukin (IL)-12 p40 expression. The actinase E- and Benzonase-treated or untreated cell wall fraction of Nz8 enhanced both IFN-gamma and IL-12 p40 expression, while the cell plasma fraction had little effect. In the presence of anti-toll like receptor 4 antibody, the effect of the cell wall fraction of Nz8 was significantly abrogated. These results suggest that some LAB from Nozawana-zuke have a T helper 1-type immunoenhancing effect.