Blueberry stem extract and stem active components prevent blue light-emitting diode light-induced retinal photoreceptor cell damage in vitro.

Blue light causes retinal damage that can lead to ocular diseases such as age-related macular degeneration. In this study, we determined the protective effect of blueberry stem extract (BStEx) and active components on blue light-emitting diode (LED) light-induced retinal photoreceptor cell damage in vitro. Photoreceptor cells cultured in the presence of BStEx or components were exposed to blue light to induce cell damage. BStEx, fractions of BStEx containing proanthocyanidins, chlorogenic acid, catechin, and epicatechin prevented the cell damage and/or inhibited the generation of reactive oxygen species (ROS). Furthermore, BStEx reduced apoptosis and cell death, and inhibited the phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase leading to cellular apoptosis induced by blue light exposure. These findings suggest that BStEx and components exert a protective effect against blue light-induced photoreceptor cell damage through the inhibition of MAPK phosphorylation and ROS production.

Onion (Allium cepa L.)-Derived Nanoparticles Inhibited LPS-Induced Nitrate Production, However, Their Intracellular Incorporation by Endocytosis Was Not Involved in This Effect on RAW264 Cells.

The aim of this study was to evaluate the involvement of nanoparticles prepared from Allium cepa L. as anti-inflammatory agents. In the present study, we identified nanoparticles from Allium cepa L. using the ultracentrifugation exosome purification method. The nanoparticles were referred to as 17,000× g and 200,000× g precipitates, and they contained quercetins, proteins, lipids, and small-sized RNA. The nanoparticles inhibited nitric oxide production from lipopolysaccharide (LPS)-stimulated RAW264 cells without cytotoxic properties. Cellular incorporation was confirmed by laser microscopic observation after PKH26 staining. The inhibition of caveolae-dependent endocytosis and macropinocytosis significantly prevented the incorporation of the nanoparticles but had no effect on the inhibition of nitric oxide in RAW264 cells. Collectively, the identified nanoparticles were capable of inhibiting the LPS response via extracellular mechanisms. Taken together, the way of consuming Allium cepa L. without collapsing the nanoparticles is expected to provide an efficient anti-inflammatory effect.

Suppressing effect of cordycepin on the lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells.

In this study, we focused on the anti-inflammatory effect of cordycepin, 3'-deoxyadenosine. Cordycepin potently suppressed nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 cells in an adenosine receptor-independent manner. In addition, inhibitors for adenosine kinase and nucleoside transporter abrogated the action of cordycepin. Thus, we considered that intracellular metabolism cordycepin is important for the anti-inflammatory effect of cordycepin.

The importance of 1,2-dithiolane structure in α-lipoic acid for the downregulation of cell surface ß1-integrin expression of human bladder cancer cells.

Here, we show that cell surface ß1-integrin expression, cell adhesion to fibronectin, migration, and invasion were all significantly inhibited by α-lipoic acid. These effects were not observed when cells were treated with dihydrolipoic acid or caprylic acid. These data reveal that the 1,2-dithiolane structure plays an important role in the action of α-lipoic acid.

α-Lipoic acid suppresses migration and invasion via downregulation of cell surface ß1-integrin expression in bladder cancer cells.

Our previous study showed α-lipoic acid (LA) downregulated cell surface ß1-integrin expression of v-H-ras-transformed derivative of rat fibroblast with amelioration of their malignant phenotype. Here, we evaluated the ameliorating effect of LA on the malignant characters in H-ras-transformed bladder cancer cells. H-ras mutated bladder cancer line, T24 cells were incubated with LA to evaluate the inhibitory effect on proliferation, migration, invasion and ß1-integrin expression. Fluorescence staining of F-actin and western blotting analyses of the related signaling pathways were also performed. LA inhibited the proliferation of T24 cells. Cell adhesion to collagen IV and fibronectin was strikingly inhibited by LA treatment accompanied by downregulation of cell surface but not whole cell ß1-integrin expression. LA clearly inhibited cell migration and invasion of T24 cells, which were mimicked by extracellular signal-regulated kinase (ERK) and Akt pathway inhibition. Actually, LA significantly downregulated the phosphorylated ERK and Akt levels. Moreover, LA downregulated phosphorylated focal adhesion kinase level with disappearance of stress fiber formation. Finally, although LA induced the internalization of cell surface ß1-integrin, disruption of the raft did not affect the action of LA. Taken together, LA is a promising agent to improve malignant character of bladder cancer cells through regulation of cellular ß1-integrin localization.

Koji Mold-derived Lipids Disrupt the Intracellular Redox State by Decreasing the GPx4 and Intracellular Glutathione Levels, Promoting Membrane Lipid Peroxidation, and Inducing Ferroptosis in HL-60 Cells.

In this study, we evaluated the cancer cell killing activity of koji mold-derived extracts using several solvents. The koji mold lipid extract (KML) exhibited potent cytotoxicity against a human leukemia cell line. Fractionation of the KML via silica gel chromatography revealed the presence of active components in fraction (Fr.) 6. Cytotoxic effects of Fr. 6 were inhibited by the ferroptosis inhibitors, ferrostatin-1 and SRS11-92, and the iron chelator, deferoxamine. Interestingly, ferroptosis inhibitors failed to prevent the KML-induced cell death. Fr. 6 decreased the expression of glutathione peroxidase 4 (GPx4) and increased the level of peroxidized plasma membrane lipids. Furthermore, Fr. 6 decreased the intracellular glutathione levels. Overall, our results suggest that Fr. 6 included in KML induces ferroptosis in HL-60 cells.

Lactiplantibacillus plantarum 06CC2 Enhanced the Expression of Intestinal Uric Acid Excretion Transporter in Mice.

ATP-binding cassette transporter subfamily G member 2 (ABCG2) is responsible for the excretion of foreign substances, such as uric acid (UA) and indoxyl sulfate (IS), from the body. Given the importance of increased ABCG2 expression in UA excretion, we investigated the enhancement of intestinal ABCG2 expression using Lactiplantibacillus plantarum 06CC2 (LP06CC2). Mice were reared on a potassium oxonate-induced high-purine model at doses of 0.02% or 0.1% LP06CC2 for three weeks. Results showed that LP06CC2 feeding resulted in increased ABCG2 expression in the small intestine. The expression level of large intestinal ABCG2 also showed a tendency to increase, suggesting upregulation of the intestinal excretion transporter ABCG2 by LP06CC2. Overall, LP06CC2 treatment increased fecal UA excretion and showed a trend towards increased fecal excretion of IS, suggesting that LP06CC2 treatment enhanced the expression of intestinal ABCG2, thereby promoting the excretion of UA and other substances from the intestinal tract.

Lactiplantibacillus plantarum 06CC2 upregulates intestinal ZO-1 protein and bile acid metabolism in Balb/c mice fed high-fat diet.

The effects of Lactiplantibacillus plantarum 06CC2 (LP06CC2), which was isolated from a Mongolian dairy product, on lipid metabolism and intestinal tight junction-related proteins in Balb/c mice fed a high-fat diet (HFD) were evaluated. The mice were fed the HFD for eight weeks, and the plasma and hepatic lipid parameters, as well as the intestinal tight junction-related factors, were evaluated. LP06CC2 slightly reduced the adipose tissue mass. Further, it dose-dependently decreased plasma total cholesterol (TC). The HFD tended to increase the plasma level of endotoxin and suppressed intestinal ZO-1 expression, whereas a low LP06CC2 dose increased ZO-1 expression and tended to reduce the plasma lipopolysaccharide level. Furthermore, a low LP06CC2 dose facilitated a moderate accumulation of Lactobacillales, a significant decrease in Clostridium cluster IV, and an increase in Clostridium cluster XVIII. The results obtained from analyzing the bile acids (BAs) in feces and cecum contents exhibited a decreasing trend for secondary and conjugated BAs in the low LP06CC2-dose group. Moreover, a high LP06CC2 dose caused excess accumulation of Lactobacillales and failed to increase intestinal ZO-1 and occludin expression, while the fecal butyrate level increased dose dependently in the LP06CC2-fed mice. Finally, an appropriate LP06CC2 dose protected the intestinal barrier function from the HFD and modulated BA metabolism.

Jacaric acid, a linolenic acid isomer with a conjugated triene system, has a strong antitumor effect in vitro and in vivo.

In this study, we compared the cytotoxic effects of natural conjugated linolenic acids (CLnAs) on human adenocarcinoma cells (DLD-1) in vitro, with the goal of finding CLnA isomers with strong cytotoxic effects. The antitumor effect of the CLnA with the strongest cytotoxic effect was then examined in mice. The results showed that all CLnA isomers have strong cytotoxic effects on DLD-1 cells, with jacaric acid (JA) having the strongest effect. Examination of the mechanism of cell death showed that CLnAs induce apoptosis in DLD-1 cells via lipid peroxidation. The intracellular levels of incorporated CLnAs were measured to examine the reason for differences in cytotoxic effects. These results showed that JA was taken into cells efficiently. Collectively, these results suggest that the cytotoxic effect of CLnAs is dependent on intracellular incorporation and induction of apoptosis via lipid peroxidation. JA also had a strong preventive antitumor effect in vivo in nude mice into which DLD-1 cells were transplanted. These results suggest that JA can be used as a dietary constituent for prevention of cancer.

Genistein induces apoptotic cell death associated with inhibition of the NF-κB pathway in adult T-cell leukemia cells.

We have shown that genistein inhibits the growth of adult T-cell leukemia (ATL) cells in vitro and in vivo, and this leads to pronounced G2/M arrest. This report shows that genistein induces apoptotic death in ATL cells. Although the pan-caspase inhibitor, Z-VAD-fmk, did not inhibit genistein-induced apoptosis, release of apoptosis-inducing factor (AIF) into the cytosol occurred. Poly-ADP ribose polymerase inhibition also abrogated genistein-induced apoptosis. Genistein decreased nuclear p65 translocation and IκBα phosphorylation, and downregulated the anti-apoptotic proteins, XIAP, cIAP and survivin, NF-κB-responsive gene products. Thus, genistein is a promising agent for ATL that induces caspase-independent apoptosis through inhibition of the NF-κB pathway.

Blueberry Leaf Extract Prevents Lacrimal Hyposecretion in Sjögren's Syndrome-like Model of Non-obese Diabetic Mice.

BACKGROUND/AIM: This study evaluated the effect of blueberry leaf hot water extract (BLEx) on Sjögren's syndrome (SS)-like lacrimal hyposecretion in male non-obese diabetic (NOD) mice. MATERIALS AND METHODS: NOD or BALB/c mice were fed 1% BLEx or control (AIN-93G) for 2 weeks from the age of 4 to 6 weeks. Pilocarpine-induced tear volume was measured using a phenol red-impregnated thread. The lacrimal glands were evaluated histologically by H&E staining. The IL-1ß and TNF-α levels in the lacrimal gland tissue were measured by ELISA. The mRNA expression levels of secretion-related proteins were measured by real-time PCR. LC3 I/II and arginase 1 expression levels were measured by western blot. RESULTS: After feeding with BLEx, pilocarpine-induced tear secretion in NOD mice was increased. In contrast, the mRNA expression levels of the cholinergic muscarinic M3 receptor, aquaporin 5, and ion channels related to lacrimal secretion were not changed by BLEx administration. In addition, the protein expression of arginase 1, which was recently reported to be involved in tear hyposecretion in NOD mice, was also not improved by BLEx administration. Although infiltration in the lacrimal gland of NOD mice was not decreased, the levels of TNF-α and the autophagy-related protein LC3 were significantly suppressed by BLEx treatment. CONCLUSION: BLEx treatment may ameliorate lacrimal hyposecretion in NOD mice by delaying the progression of autoimmune disease by suppressing autophagy in lacrimal glands.

Blueberry Stem Extract Prevents Lacrimal Hyposecretion in Non-obese Diabetic Mice via Activation of AMPK.

BACKGROUND/AIM: Tears secreted from the lacrimal gland are essential for preserving the ocular surface. Thus, dysfunction of the lacrimal gland in Sjögren's syndrome (SS) can lead to dry eye, resulting in a reduced quality of life. We previously reported that blueberry 'leaf' water extract prevents lacrimal hyposecretion in male non-obese diabetic (NOD) mice in a SS-like model. In this study, we investigated the effect of blueberry 'stem' water extract (BStEx) on lacrimal hyposecretion in NOD mice. MATERIALS AND METHODS: Male NOD mice were fed 1% BStEx or control (AIN-93G) for 2, 4, or 6 weeks from 4 weeks of age. Pilocarpine-induced tear secretion was measured using a phenol red-impregnated thread. The lacrimal glands were histologically evaluated by HE staining. Inflammatory cytokine levels in the lacrimal glands were measured using ELISA. Immunostaining was performed to examine aquaporin 5 (AQP5) localization. The expression levels of autophagy-related proteins, AQP5, and phosphorylated AMPK were measured using western blotting. RESULTS: After feeding BStEx to mice for 4 or 6 weeks, tear volume was observed to have increased in the BStEx group compared with that in the control group. There were no significant differences in inflammatory cell infiltration, autophagy-related protein expression, or the localization and expression of AQP5 in the lacrimal glands between the two groups. In contrast, AMPK phosphorylation increased in the BStEx group. CONCLUSION: BStEx prevented lacrimal hyposecretion in the SS-like model of male NOD mice, probably by opening tight junctions via the activation of AMPK in lacrimal acinar cells.

Selective inhibition by apocynin of the proliferation and adhesion to fibronectin of v-H-ras-transformed 3Y1 cells.

We determined the effects of apocynin, a representative inhibitor of NADPH oxidase, on the proliferative and adhesive properties of 3Y1 rat fibroblasts and the 3Y1 v-H-ras-transformed derivative, HR-3Y1-2. Apocynin inhibited the proliferation of HR-3Y1-2 but not 3Y1 cells at 10 µM and 100 µM. Apocynin also decreased the intracellular reactive oxygen species (ROS) level in HR-3Y1-2 but not 3Y1 cells. We also evaluated the effects of apocynin on cell adhesion to fibronectin and found decreased adhesion of HR-3Y1-2 cells to fibronectin-coated plates. Our results indicate that apocynin selectively down-regulated ß1-integrin cell surface expression on the HR-3Y1-2 cells. It also inhibited the migration and invasion of these cells. These data suggest that reducing the production of NADPH oxidase-mediated ROS could be an effective means for ameliorating the abnormal growth, adhesion and motility of v-H-ras-transformed cells.

Two cases of cervical esophageal perforation treated by surgery.

Cervical esophageal perforation is rare, but it is associated with high mortality. We describe two patients with cervical esophageal perforation that required surgical treatment. In both cases, good outcomes were evenly achieved, despite the presence of risk factors. A prompt diagnosis and treatment with collaboration between a surgeon and a gastroenterologist are important.

Enzymatic sulfation of tocopherols and tocopherol metabolites by human cytosolic sulfotransferases.

Tocopherols are essential micronutrients for mammals widely known as potent lipid-soluble antioxidants that are present in cell membranes. Recent studies have demonstrated that most of the carboxychromanol (CEHC), a tocopherol metabolite, in the plasma exists primarily in sulfate- and glucuronide-conjugated forms. To gain insight into the enzymatic sulfation of tocopherols and their metabolites, a systematic investigation was performed using all 14 known human cytosolic sulfotransferases (SULTs). The results showed that the members of the SULT1 family displayed stronger sulfating activities toward tocopherols and their metabolites. These enzymes showed a substrate preference for γ-tocopherol over α-tocopherol and for γ-CEHC over other CEHCs. Using A549 human lung epithelial cells in a metabolic labeling study, a similar trend in the sulfation of tocopherols and CEHCs was observed. Collectively, the results obtained indicate that SULT-mediated enzymatic sulfation of tocopherols and their metabolites is a significant pathway for regulation of the homeostasis and physiological functions of these important compounds.

Genetic and transcriptomic analyses in a rare case of human papillomavirus-related oropharyngeal squamous-cell carcinoma combined with small-cell carcinoma.

Human papillomavirus (HPV)-related oropharyngeal small-cell carcinoma (OPSmCC) is a rare malignancy with aggressive behavior, whereas HPV-related oropharyngeal squamous-cell carcinoma (OPSqCC) displays a favorable prognosis. Notably, these two malignancies occasionally arise in an identical tumor. In this case study, we explored the molecular characteristics that distinguishes these two carcinomas using a rare case of HPV-related oropharyngeal carcinoma (OPC) with the combined histology of SmCC and SqCC. Immunohistochemical analysis and HPV-RNA in situ hybridization (ISH) suggested that both SmCC and SqCC were HPV-related malignancies. Targeted exome sequencing revealed that SmCC and SqCC had no significant difference in mutations of known driver genes. In contrast, RNA sequencing followed by bioinformatic analyses suggested that aberrant transcriptional programs may be responsible for the neuroendocrine differentiation of HPV-related OPC. Compared to SqCC, genes up-regulated in SmCC were functionally enriched in inflammatory and immune responses (e.g., arachidonic acid metabolism). We then developed a SmCC-like gene module (top 10 up-regulated genes) and found that OPC patients with high module activity showed poor prognosis in The Cancer Genome Atlas (TCGA) and GSE65858 cohort. Gene set enrichment analysis of the SmCC-like gene module suggested its link to MYC proto-oncogene in the TCGA data set. Taken together, these findings suggest that the SmCC-like gene module may contribute to acquisition of aggressive phenotypes and tumor heterogeneity of HPV-related OPC. The present case study is the first report of genetic and transcriptomic aberrations in HPV-related OPSmCC combined with SqCC.

Vaccinium virgatum Aiton Leaves Extract Suppressed Lipid Accumulation and Uric Acid Production in 3T3-L1 Adipocytes.

Blueberry (Vaccinium virgatum Aiton; Kinisato 35 Gou) leaves have recently attracted increasing attention as a useful material for the prevention of lifestyle diseases. Here, we examined the effects of the hot water extract of blueberry leaves (BLEx) on lipogenesis and uric acid production in 3T3-L1 adipocytes. The results showed that BLEx suppressed lipid accumulation and the mRNA expression of differentiation markers in 3T3-L1 adipocytes. A fractionation study showed that the highly polymerized proanthocyanidin-rich fraction was responsible for this effect. Upon maturation to adipocytes, 3T3-L1 cells produced uric acid and tumor necrosis factor-α, and hypoxia stimulated the production of uric acid and xanthine oxidoreductase activity. BLEx suppressed the production of uric acid under these conditions. Although BLEx inhibited the enzymatic activity of xanthine oxidase, this activity was observed in several fractions containing catechin, epicatechin, chlorogenic acid, rutin, and low molecular weight proanthocyanidins. Taken together, these results indicate that BLEx contains various compounds with the ability to suppress lipid accumulation and uric acid production in adipocytes.

Novel screening method for potential skin-whitening compounds by a luciferase reporter assay.

Measurement of the melanin content by using B16 melanoma cells is generally applied to find novel skin-whitening agents. However, this measurement method using B16 melanoma cells has such disadvantages, as the time taken, its sensitivity, and troublesomeness. We therefore attempted in the present study to establish a reporter assay system by measuring the tyrosinase promoter activity to use for convenient, high-throughput screening of new melanogenesis inhibitors. We first confirmed the validity of this reporter assay system by using such known skin-whitening agents, as arbutin, sulforaphane, and theaflavin 3,3'-digallate. We then compared the effect of 56 compounds on the tyrosinase promoter activity to test this reporter assay system. Carnosol, and rottlerin strongly inhibited the tyrosinase promoter activity. Moreover, carnosol and rottlerin decreased melanin synthesis and tyrosinase expression in a dose-dependent manner when using B16 melanoma cells. These results indicate this new luciferase reported assay system to be an effective and convenient method for screening potential skin-whitening compounds.

Genistein induced apoptotic cell death in adult T-cell leukemia cells through estrogen receptors.

Adult T-cell leukemia (ATL) occurs in human T-lymphotropic virus type I-infected individuals and is endemic to the southwestern area of Kyushu in Japan. Here, we found that nM levels of genistein and 17ß-estradiol had cytotoxic effects on ATL cells and activated caspase-3. The estrogen receptor antagonist ICI182780 negated the cytotoxic effect of genistein. In addition, G protein-coupled estrogen receptor agonist G-1 also had a cytotoxic effect on ATL cells. This is the first report suggesting that estrogen receptors are a molecular target for ATL therapy.

Lactobacillus plantarum 06CC2 reduces hepatic cholesterol levels and modulates bile acid deconjugation in Balb/c mice fed a high-cholesterol diet.

Previous study suggested that dietary intake of Lactobacillus plantarum 06CC2 (LP06CC2) isolated from Mongolian dairy products showed various health beneficial effects. Here, the effect of LP06CC2 on the cholesterol metabolism in mice fed a cholesterol-loaded diet was evaluated. Cholesterol and LP06CC2 were incorporated into the AIN93G-based diet to evaluate the effect on cholesterol metabolism in Balb/c mice. Serum and liver cholesterol levels were significantly increased in mice fed a cholesterol-loaded diet whereas the LP06CC2 ingestion suppressed the increase of liver cholesterol. LP06CC2 suppressed the increase of the hepatic damage indices. The increase of the cecal content and fecal butyrate were observed in mice fed LP06CC2. The analysis of bile acids clearly showed that LP06CC2 increased their deconjugation indicating the decrease of bile acid absorption. The protein expression of hepatic Cyp7A1 was also suppressed by LP06CC2 in mice fed cholesterol. Finally, in vitro studies showed that LP06CC2 had the most potent ability to deconjugate bile acids using glycocholate among the tested probiotic lactic acid bacteria isolated from Mongolian dairy products. Taken together, LP06CC2 is a promising microorganism for the reduction of the cholesterol pool via modulation of bile acid deconjugation.