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.

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.

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.

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.

Blueberry Leaf Polyphenols Prevent Body Fat Accumulation in Mice Fed High-fat, High-sucrose Diet.

Blueberry leaf is currently a popular dietary supplement. Effects of dietary blueberry leaf and its active components on body fat accumulation were examined. C57BL/6J mice were fed high-fat, high-sucrose diet with or without 3% blueberry leaf extract (BLEx) or 3% concentrated-polyphenolic BLEx (CP BLEx) for 8 weeks. Compared to mice fed a high-fat, high-sucrose diet without blueberry leaf, BLEx and CP BLEx significantly reduced body weight and adipose tissue weight gain. Adipocytes were also smaller and and liver lipid accumulatioin was significantly inhibited in mice fed either BLEx or CP BLEx. These effects tended to be more pronounced in mice fed CP BLEx compared to in mice fed BLEx. Together, results suggest that blueberry leaf inhibits body fat accumulation typically observed in mice fed a high-fat, high-sucrose diet, and that inhibition is attributable to polyphenolic components in leaf extracts.

Carnosol, rosemary ingredient, induces apoptosis in adult T-cell leukemia/lymphoma cells via glutathione depletion: proteomic approach using fluorescent two-dimensional differential gel electrophoresis.

Adult T-cell leukemia/lymphoma (ATL) is a fatal malignancy caused by infection with human T-lymphotropic virus type-1 and there is no accepted curative therapy for ATL. We searched for biological active substances for the prevention and treatment of ATL from several species of herbs. The ATL cell growth-inhibitory activity and apoptosis assay showed that carnosol, which is an ingredient contained in rosemary (Rosmarinus officinalis), induced apoptosis in ATL cells. Next, to investigate the apoptosis-inducing mechanism of carnosol, we applied proteomic analysis using fluorescent two-dimensional differential gel electrophoresis and mass spectrometry. The proteomic analysis showed that the expression of reductases, enzymes in glycolytic pathway, and enzymes in pentose phosphate pathway was increased in carnosol-treated cells, compared with untreated cells. These results suggested that carnosol affected the redox status in the cells. Further, the quantitative analysis of glutathione, which plays the central role for the maintenance of intracellular redox status, indicated that carnosol caused the decrease of glutathione in the cells. Further, N-acetyl-L-cystein, which is precursor of glutathione, canceled the efficiency of carnosol. From these results, it was suggested that the apoptosis-inducing activity of carnosol in ATL cells was caused by the depletion of glutathione.

Induction of apoptotic cell death in HL-60 cells by jacaranda seed oil derived fatty acids.

Various fatty acids are attracting considerable interest for their anticancer effects. Among them, fatty acids containing conjugated double bonds show one of the most potent cytotoxic effects on cancer cells. Here, we focused on the cancer cell killing activity of jacaranda seed oil. The seed oil of jacaranda harvested from Miyazaki in Japan contained 30.9% cis-8, trans-10, cis-12 octadecatrienoic acid, called jacaric acid (JA). Fatty acid prepared from this oil (JFA) and JA strongly induced cell death in human leukemia HL-60 cells. On the other hand, linoleic acid and trans-10, cis-12 conjugated linoleic acid (<10 µM) did not affect cell proliferation and viability. An increase in the sub-G1 population and internucleosomal fragmentation of DNA was observed in JA- and JFA-treated cells, indicating induction of apoptotic cell death. Finally, the cytotoxic effects of JA and JFA were completely abolished by α-tocopherol. Taken together, these data suggest that jacaranda seed oil has potent apoptotic activity in HL-60 cells through induction of oxidative stress.

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.

Mustard oil in "Shibori Daikon" a variety of Japanese radish, selectively inhibits the proliferation of H-ras-transformed 3Y1 cells.

Cruciferous vegetables and their isothiocyanates are promising foods and agents for cancer prevention. We focus here on the effects of mustard oil (SMO) in a variety of the Japanese radish, Shibori Daikon (Raphanus sativus), on the proliferation of 3Y1 rat fibroblasts and the H-ras-transformed derivative, HR-3Y1-2. SMO (1.6 microg/ml) inhibited the proliferation of HR-3Y1-2, but not 3Y1 after 24 h after treatment. A cell cycle analysis showed that SMO induced G2/M arrest after 6 h, although this effect was not observed 24 h after the treatment. SMO transiently decreased the cellular reduced glutathione level accompanied with up-regulation of the intracellular reactive oxygen species 2-3 h post-treatment. Glutathione ethyl ester and N-acetyl-L-cysteine prevented the growth inhibitory effect of SMO. This mustard oil extract consisted of 95.6% 4-methylthio-3-butenyl isothiocyanate and 4.4% 4-methylthiobutyl isothiocyanate. SMO selectively inhibited H-ras-transformed 3Y1 cells associated with transient oxidative stress via reduced glutathione (GSH) depletion.

Antioxidant activity of vitamin B6 delays homocysteine-induced atherosclerosis in rats.

Elevated plasma homocysteine is a risk factor for atherosclerotic disease. In the present study, we have examined whether the oxidative stress due to a low level of vitamin B6 accelerates the development of homocysteine-induced atherosclerosis in rats. First, the effect of homocysteine thiolactone intake (50 mg/kg per d) on vascular integrity, lipid peroxide concentration, endothelial NO synthase (eNOS) expression and biochemical profiles was examined at day 1, day 21 and day 42 (five rats per group). The histochemical staining of the rat aorta showed no change at day 1 and day 21, but the subendothelial space was observed to be enlarged in rat aorta at day 42 with exposure to homocysteine thiolactone. Expression of eNOS was observed in rat aorta at day 42, but not at day 1 and day 21. Serum lipid peroxide concentration and biochemical profiles including glucose cholesterol and triacylglycerol showed no change at any day. Second, the effect of homocysteine thiolactone intake in the presence and absence of vitamin B6 on vascular integrity was examined at day 1 and day 14 (five rats per group). Aortic lesions were observed in vitamin B6-deficient rat aorta at day 14 but not in vitamin B6-supplemented rats. The expression of eNOS was also observed in vitamin B6-deficient rat aorta at day 14. Serum lipid concentrations of the vitamin B6-deficient group significantly increased compared with concentrations of the vitamin B6-supplemented group, though serum concentration of homocysteine did not change between both groups. These results suggest that the oxidative stress caused by a low level of vitamin B6 accelerates the development of homocysteine-induced atherosclerosis in rats.