Myricetin and myricitrin indirectly and directly increases uncoupling protein-1 mRNA expression in C3H10T1/2 beige adipocytes.

In thermogenic brown and beige adipocytes, the proton gradient formed by energy derived from nutrients such as lipids and carbohydrates is consumed by uncoupling protein-1 (UCP-1), resulting in thermogenesis without ATP production in the mitochondria. Accordingly, increased UCP-1 expression represents a crucial aspect of dietary management for individuals with overweight and obesity. Myricetin and its glycoside, myricitrin, are food-derived flavonoids that possess various beneficial effects. This is the first study to examine the effects of myricetin and myricitrin on the inflammation-inhibited expression of Ucp-1 using a modified cell-based assay with conditioned medium (CM). The CM derived from lipopolysaccharide (LPS)-activated RAW264.7 macrophages was observed to inhibit the Ucp-1 expression induced by adrenergic stimulation in 10T1/2 adipocytes. Conversely, the CM derived from activated macrophages treated with myricetin or myricitrin reversed this inhibition of Ucp-1 expression. Subsequently, the direct effects of both the compounds on basal and adrenaline-induced expression of Ucp-1 were investigated. In contrast to a previous report, myricetin and myricitrin did not increase the basal Ucp-1 mRNA expression in 10T1/2 adipocytes when treated during the differentiation-promoting period. However, we have found for the first time that both compounds enhanced the adrenergic sensitivity of 10T1/2 adipocytes when treated during the differentiation-inducing period. These results indicate that myricetin and myricitrin have indirect effects on inflammation-induced suppression and direct effects on adrenergic sensitivity, suggesting a novel mechanism that both compounds increase Ucp-1 expression in vivo by both indirect and direct effects, rather than by affecting basal expression.

A modified system using macrophage-conditioned medium revealed that the indirect effects of anti-inflammatory food-derived compounds improve inflammation-induced suppression of UCP-1 mRNA expression in 10T1/2 adipocytes.

Recently, it has been suggested that brown and beige adipocytes may ameliorate obesity because these adipocytes express uncoupling protein-1 (UCP-1), which generates heat by consuming lipid. However, obesity-induced inflammation suppresses the expression of UCP-1. To improve such conditions, food components with anti-inflammatory properties are attracting attention. In this study, we developed a modified system to evaluate only the indirect effects of anti-inflammatory food-derived compounds by optimizing the conventional experimental system using conditioned medium. We validated this new system using 6-shogaol and 6-gingerol, which have been reported to show the anti-inflammatory effects and to increase the basal expression of UCP-1 mRNA. In addition, we found that the acetone extract of Sarcodon aspratus, an edible mushroom, showed anti-inflammatory effects and rescued the inflammation-induced suppression of UCP-1 mRNA expression. These findings indicate that the system with conditioned medium is valuable for evaluation of food-derived compounds with anti-inflammatory effects on the inflammation-induced thermogenic adipocyte dysfunction.

Microbiota in human breast milk: Noninfectious mastitis versus without mastitis.

BACKGROUND: Long-term breastfeeding is beneficial for both mothers and infants and mastitis is associated with the premature interruption of breastfeeding. Mastitis can be infectious or noninfectious. However, the effect of noninfectious mastitis on milk microbiota is not well-understood. In this study, we aimed to clarify the relationship between noninfectious mastitis and the microbiota by conducting breast milk culture tests. METHODS: We compared the milk microbiota between women with noninfectious mastitis and without mastitis. Bacterial cultures were compared in 143 milk samples from January to November 2022, and bacterial diversity was evaluated based on the total number of bacterial species and bacterial species found per specimen. RESULTS: Women with noninfectious mastitis provided samples at a significantly later stage postpartum (p < 0.01). The total bacterial count was significantly lower in samples from participants with noninfectious mastitis (p < 0.01). The bacterial diversity of milk from participants with noninfectious mastitis was lower than that without mastitis: nine bacterial species identified in the former and 21 in the latter. The number of Rothia spp. was significantly higher, whereas the number of Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas fluorescens was significantly lower in samples from women with mastitis. There was no correlation between postpartum week and the number of bacterial species or presence of Rothia spp. CONCLUSIONS: Noninfectious mastitis is associated with a decrease in the diversity of human milk microbiota, indicating impaired immune, metabolic, and neuroendocrine development functions in infants. Rothia spp. may also be associated with noninfectious mastitis, suggesting a possible target for future research.

(S)-Equol Is More Effective than (R)-Equol in Inhibiting Osteoclast Formation and Enhancing Osteoclast Apoptosis, and Reduces Estrogen Deficiency-Induced Bone Loss in Mice.

BACKGROUND: Equol, a metabolite of daidzein, binds to the estrogen receptor with greater affinity than daidzein and exhibits various biological properties. It exists as an enantiomer, either (S)-equol or (R)-equol. OBJECTIVES: We have previously shown that the inhibitory effect of (S)-equol on bone fragility is stronger than that of racemic equol in ovariectomized (OVX) mice; however, the effect of (R)-equol has not been elucidated. The aim of this study was to compare the activities of equol enantiomers on bone metabolism in vitro and in vivo. METHODS: Bone marrow cells (BMCs) and RAW 264.7 cells were treated with equol enantiomers. The number of osteoclasts and caspase-3/7 activity were measured. We examined the effect of equol enantiomers on osteoblast differentiation in MC3T3-E1 cells. In vivo, 8-wk-old female ddY mice were assigned to 4 groups: sham-operated (sham), OVX, OVX + 0.5 mg/d of (S)-equol (S-eq), and OVX + 0.5 mg/d of (R)-equol (R-eq). Four weeks after the intervention, femoral bone mineral density (BMD) and osteoclastic gene expression were analyzed, along with concentrations of equol enantiomers in the serum and tissues. RESULTS: (S)-equol and (R)-equol inhibited osteoclast differentiation in BMCs (97% and 60%, P < 0.05) and RAW 264.7 cells (83% and 68%, P < 0.05). (S)-equol promoted apoptosis of mature osteoclasts by inducing caspase-3/7 activity (29%, P < 0.05) and enhanced osteoblast differentiation (29%, P < 0.05). In OVX mice, BMD was ameliorated in (S)-equol-treated mice (11%, P < 0.05), but not in (R)-equol-treated mice. The concentrations of (S)-equol were greater than those of (R)-equol in the serum, tibia, liver, and kidney (by 148%, 80%, 22%, and 139%, respectively). CONCLUSIONS: These results suggest that (S)-equol is more effective than (R)-equol in inhibiting osteoclast formation and enhancing osteoclast apoptosis in vitro, supporting the beneficial effect of (S)-equol to reduce estrogen deficiency-induced bone loss in OVX mice.

Upregulation and stabilization of senescence marker protein-30 by epigallocatechin gallate against tert-butyl hydroperoxide-induced liver injury in vitro and in vivo.

Senescence marker protein-30 (SMP30), a novel ageing marker, suppresses oxidative stress in the liver. However, studies on phytochemical-mediated regulation of SMP30 expression are lacking. Here, we showed that epigallocatechin gallate (EGCg), a polyphenol abundant in green tea, positively regulates SMP30 expression in the rat hepatoma-derived Fao cells. EGCg maintained SMP30 expression even in the presence of cycloheximide, a protein synthesis inhibitor. Furthermore, treatment of cells with tert-butyl hydroperoxide (tert-BHP), an oxidative promoter, decreased SMP30 expression and ERK1/2 phosphorylation, while EGCg treatment inhibited these effects. Male mice (7-week-old) were divided into 4 groups-Control (saline), tert-BHP (1.5 mmol/kg tert-BHP), EGCg + tert-BHP (30 mg/kg/day of EGCg and 1.5 mmol/kg tert-BHP), and EGCg (30 mg/kg/day). After oral EGCg administration for 6 consecutive days, EGCg + tert-BHP group mice were administered tert-BHP. The tert-BHP-administered mice showed decreased SMP30 expression in the liver and increased aspartate aminotransferase and alanine transaminase (hepatic injury marker enzymes) activities; however, EGCg treatment attenuated these changes. Thus, EGCg-induced SMP30 upregulation may alleviate tert-BHP-induced liver injury. The findings of this study offer new perspectives of the anti-ageing properties of EGCg.

New latex agglutination assay for the determination of lactoferrin in human milk.

BACKGROUND: Lactoferrin (LF) in human milk has various biological properties and contributes to the prevention of preterm birth complications. Enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used methods to measure LF in human milk, but this method is time-consuming and laborious. In Japanese human milk banks, the concentration of LF in donor human milk (DHM) is measured routinely. Here, we reported a rapid, simple, and accurate method for determining LF in human milk using a new reagent based on a latex agglutination assay. METHODS: We obtained 208 human milk pools from 148 mothers, and samples were collected before and after Holder pasteurization. Milk samples were diluted 100- or 200-fold and LF concentrations were measured by a latex agglutination assay using an automated analyzer. The reagent was validated in terms of repeatability, linearity, detection limit, recovery, and comparison with ELISA. RESULTS: The coefficient of variation (CV) for intra-assay precision ranged from 0.6 to 5.0% in human milk with high, medium, and low LF concentrations. The linearity was also tested by serial sample dilution and was confirmed up to 16 µg/mL with a detection limit of 0.2 µg/mL. The recovery rates in a spiked recovery test were ranged from 90 to 120% at high, medium, and low concentrations of LF. Furthermore, a strong correlation was observed between LF levels determined by the latex agglutination assay and ELISA (r = 0.978, p < 0.001, n = 255). The regression equation was y = 0.991x + 0.545 (r2 = 0.974, p < 0.001). Compared with ELISA, the latex agglutination assay reduces the measurement time by 160 min and the cost by 55%. CONCLUSIONS: The latex agglutination assay used to determine LF in human milk is rapid, simple, and accurate enough to be used routinely. Its use may contribute to the quick and easy provision of appropriate DHM to preterm infants.

Induced lactation in a transgender woman: case report.

BACKGROUND: Breastfeeding offers significant health benefits, but its practice and success can vary. While research on induced lactation in cisgender women has been documented, there is limited research on lactation induction in transgender women. CASE PRESENTATION: A 50-year-old transgender woman undergoing hormone therapy and living with a pregnant partner sought to co-feed using induced lactation. After approval by the hospital ethics committee, a regimen of estradiol, progesterone, and domperidone was initiated, accompanied by nipple stimulation. Lactation was successfully induced and maintained, with milk composition analysis indicating high levels of protein and other key nutrients. This case, the seventh reported, highlights the complexity of lactation induction in transgender women, considering factors such as age, obesity, and insulin resistance. The nutrient profile of the milk suggests its suitability for infant feeding, despite some differences from typical human milk. CONCLUSIONS: Induced lactation is feasible in transgender women, expanding the understanding of non-puerperal lactation and its potential in diverse family structures. Further research is warranted to optimize lactation induction protocols in transgender women.

AMPK negatively regulates RANKL-induced osteoclast differentiation by controlling oxidative stress.

AMP-activated protein kinase (AMPK) is a crucial energy sensor of cellular metabolism under various metabolic stresses, such as oxidative stress and inflammation. AMPK deficiency increases osteoclast numbers and reduces bone mass; however, the precise mechanisms remain unclear. This study aimed to clarify the mechanistic connection between AMPK and osteoclast differentiation, and the potential role of AMPK in the anti-resorptive effects of several phytochemicals. We found that receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL)-induced osteoclast differentiation, osteoclastic gene expression, and activation of mitogen-activated protein kinase (MAPK) and NF-κB were promoted in cells transfected with AMPK siRNA. AMPK knockdown led to defective synthesis of heme oxygenase-1, an antioxidant enzyme, and the upstream mediator, nuclear factor erythroid-2-related factor 2. Furthermore, treatment with N-acetyl-l-cysteine, an antioxidant, abolished osteoclast differentiation and MAPK/NF-κB activation induced by AMPK knockdown. AMPK activators, hesperetin, gallic acid, resveratrol, and curcumin, suppressed osteoclast differentiation via the activation of AMPK. These results suggest that AMPK inhibits RANKL-induced osteoclast differentiation by enhancing antioxidant defense system and regulating oxidative stress. AMPK activation by dietary-derived phytochemicals may be effective for the treatment of bone diseases.

Protein and Immune Component Content of Donor Human Milk in Japan: Variation with Gestational and Postpartum Age.

Donor human milk (DHM) is the second-best nutrition for preterm infants when their own mother's milk is unavailable. The nutrient content of human milk is influenced by various factors, including gestational and postpartum age, but there are no data regarding DHM composition in Japan. The aim of this study was to determine the protein and immune component content of DHM in Japan and to elucidate the effects of gestational and postpartum age on nutrient composition. From September 2021 to May 2022, 134 DHM samples were collected from 92 mothers of preterm and term infants. Protein concentrations in preterm DHM (n = 41) and term DHM (n = 93) were analyzed using a Miris Human Milk Analyzer. The concentrations of secretory immunoglobulin A (sIgA) and lactoferrin, major immune components, were measured using enzyme-linked immunosorbent assays. Preterm DHM exhibited higher protein content than term DHM (1.2 g/dL and 1.0 g/dL, respectively, p < 0.001), whereas sIgA content was higher in term DHM than in preterm DHM (110 µg/mL and 68.4 µg/mL, respectively, p < 0.001). Gestational age was negatively correlated with protein levels and positively correlated with sIgA and lactoferrin levels. Furthermore, a negative correlation was found between postpartum week and protein, sIgA, and lactoferrin concentrations. Our data suggest that gestational and postpartum age affects protein, sIgA, and lactoferrin concentrations in DHM. These results indicate the importance of nutritional analysis for the appropriate use of DHM in preterm infants.

Comparison of bacterial profiles in human milk from mothers of term and preterm infants.

BACKGROUND: Reducing the disposal of donated human milk (HM) is important for efficient management of human milk banks (HMBs). The presence of bacteria growth is the main factor that contributes to the disposal of donated HM. The bacterial profile in HM is suspected to differ between term and preterm mothers, with HM from preterm mothers containing more bacteria. Thus, elucidation of the causes of bacterial growth in preterm and term HM may help to reduce the disposal of donated preterm HM. This study compared the bacterial profiles of HM between mothers of term infants and mothers of preterm infants. METHODS: This pilot study was conducted in the first Japanese HMB, which was initiated in 2017. This study analyzed 214 human milk samples (term: 75, preterm: 139) donated by 47 registered donors (term: 31, preterm: 16) from January to November 2021. Bacterial culture results in term and preterm HM were retrospectively reviewed in May 2022. Differences in total bacterial count and bacterial species count per batch were analyzed using the Mann-Whitney U test. Bacterial loads were analyzed using the Chi-square test or Fisher's exact test. RESULTS: The disposal rate did not significantly differ between term and preterm groups (p = 0.77), but the total amount of disposal was greater in the preterm group (p < 0.01). Coagulase-negative Staphylococci, Staphylococcus aureus, and Pseudomonas fluorescens were frequently found in both types of HM. Serratia liquefaciens (p < 0.001) and two other bacteria were present in term HM; a total of five types of bacteria, including Enterococcus faecalis and Enterobacter aerogenes (p < 0.001) were present in preterm HM. The median (interquartile range) total bacterial counts were 3,930 (435-23,365) colony-forming units (CFU)/mL for term HM and 26,700 (4,050-334,650) CFU/mL for preterm HM (p < 0.001). CONCLUSIONS: This study revealed that HM from preterm mothers had a higher total bacterial count and different types of bacteria than HM from term mothers. Additionally, preterm infants can receive nosocomial-infection-causing bacteria in the NICU through their mother's milk. Enhanced hygiene instructions for preterm mothers may reduce the disposal of valuable preterm human milk, along with the risk of HM pathogen transmission to infants in NICUs.

Resveratrol Upregulates Senescence Marker Protein 30 by Activating AMPK/Sirt1-Foxo1 Signals and Attenuating H2O2-Induced Damage in FAO Rat Liver Cells.

Resveratrol (RSV) is a polyphenol with numerous biological functions, including anti-inflammatory, antioxidant, and anti-aging activities. The novel senescence marker protein-30 (SMP30) indicates aging, and it suppresses hepatic oxidative stress. However, the effects of RSV on SMP30 expression regulation remain unclear. We observed that RSV positively regulates SMP30 expression in rat hepatoma-derived FAO cells. However, this was abolished by Compound C and EX-527 that specifically inhibit AMP-activated protein kinase (AMPK) and Silent Information Regulator T1 (Sirt1), respectively. We predicted binding sites for AMPK, forkhead box protein O1 (Foxo1), and Sirt1 downstream molecules as possible SMP30 promoters using the JASPAR and UniProtKB databases. We identified a Foxo1 binding site in the promoter region of SMP30. Inhibiting Foxo1 with AS1842527 also decreased the RSV-induced upregulation of SMP30 expression. Moreover, RSV suppressed the substantial downregulation of SMP30 expression caused by oxidative stress and hydrogen peroxide (H2O2) and released accumulated lactate dehydrogenase. These results demonstrate that, as a novel food factor, RSV-induced upregulation of SMP30 by activating AMPK/Sirt1-Foxo1 signaling and may attenuates H2O2-induced oxidative damage. The findings of this study offer new perspectives of the anti-ageing properties of RSV.

Release of SMP30 in Extracellular Vesicles under Conditions of Ascorbic Acid Deficiency Is Involved with Acute Phase Response in ODS Rat.

Senescence marker protein-30 (SMP30) is a senescence marker molecule that exhibits lactonase activity in the ascorbic acid (AsA) biosynthesis pathway, except in primate mammals, including humans. Although numerous studies have shown that hepatic AsA deficiency causes acute-phase responses, details of the relationship between SMP30 expression and acute-phase responses in AsA-deficient conditions remain to be elucidated. Here, we investigated the effects of AsA deficiency on the relationship between SMP30 and acute liver injury in osteogenic disorder Shionogi (ODS) rats, which have a hereditary defect in AsA biosynthesis. Male-ODS rats (4 wk old) were pair-fed an AsA-free diet with distilled or 0.1% AsA-dissolved water for 14 d. Under AsA-deficient conditions, hepatic SMP30 protein level was decreased and liver injury markers, the serum aspartate aminotransferase/alanine transaminase ratio and cytokine-induced neutrophil chemoattractant-1 (CINC-1) concentration, were elevated. In contrast, SMP30 protein level in extracellular vesicles (EVs) was significantly increased in addition to the positive acute proteins haptoglobin and asialoglycoprotein receptor 1 (ASGPR1), hepatic-derived specific markers expression under AsA-deficient conditions. AsA deficiency also activated signal transducer and activator of transcription 3 (STAT3) which is linked to EVs release in the liver. These results suggest that the release of SMP30 in EVs by AsA deficiency is involved with acute-phase responses.

Gallic acid regulates adipocyte hypertrophy and suppresses inflammatory gene expression induced by the paracrine interaction between adipocytes and macrophages in vitro and in vivo.

Obesity-induced chronic inflammation in adipose tissue plays a critical role in the development of insulin resistance and various lifestyle-related diseases. Although gallic acid (GA) is known to exert protective effects on obesity-related complications, its function in adipose tissue inflammation has not been elucidated. Recently, we reported that GA exerts protective effects against inflammation. To test our hypothesis that the anti-inflammatory effect of GA partially contributes to the improvement of metabolic diseases, we examined the effect of GA on inflammation caused by adipocyte-macrophage crosstalk in obesity. We showed that GA enhanced adipocyte differentiation in 3 T3-L1 adipocytes. Consistent with the enhancement of adipogenesis, GA decreased the gene expression of monocyte chemoattractant protein-1 and increased that of adiponectin and the upstream mediator peroxisome proliferator-activated receptor gamma. GA also reduced inflammatory mediator expression induced by the co-culture of 3 T3-L1 adipocytes with RAW 264 macrophages. Diet-induced obese mice treated with GA showed decreased serum cholesterol levels and adipocyte size, and improved insulin sensitivity without changes in body weight. Moreover, GA-treated mice had decreased expression of interleukin-6, inducible nitric oxide synthase, cyclooxygenase-2, F4/80, and sterol regulatory element binding transcription factor-1 in their adipose tissue. These results indicate that GA suppresses adipocyte hypertrophy and inflammation caused by the interaction between adipocytes and macrophages, thereby improving metabolic disorders such as insulin resistance and dyslipidemia.

Gallic Acid Inhibits Lipid Accumulation via AMPK Pathway and Suppresses Apoptosis and Macrophage-Mediated Inflammation in Hepatocytes.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease, sometimes ranges from simple steatosis to nonalcoholic steatohepatitis (NASH). Various hits including excessive hepatic steatosis, oxidative stress, apoptosis, and inflammation, contribute to NASH development. Gallic acid (GA), a natural polyphenol, was reported to exert a protective effect on hepatic steatosis in animal models, but the precise molecular mechanisms remain unclear. Here, we examined the effect of GA on hepatic lipid accumulation, apoptosis, and inflammatory response caused by hepatocyte-macrophage crosstalk. We demonstrated that GA attenuated palmitic acid (PA)-induced fat accumulation via the activation of AMP-activated protein kinase (AMPK) in HepG2 cells. GA also ameliorated cell viability and suppressed apoptosis-related gene expression and caspase 3/7 activity induced by PA and H2O2. In a co-culture of lipid-laden Hepa 1-6 hepatocytes and RAW 264 macrophages, GA reduced inflammatory mediator expression and induced antioxidant enzyme expression. These results indicate that GA suppresses hepatic lipid accumulation, apoptosis, and inflammation caused by the interaction between hepatocytes and macrophages. The potential effects of GA observed in our study could be effective in preventing NASH and its complications.

Iron deficiency negatively regulates protein methylation via the downregulation of protein arginine methyltransferase.

Iron is an essential trace metal for all biological processes and plays a role in almost every aspect of body growth. Previously, we found that iron-depletion downregulated the expression of proteins, arginine methyltransferase-1 and 3 (PRMT1 and PRMT3), by an iron-specific chelator, deferoxamine (DFO), in rat liver FAO cell line using DNA microarray analysis (unpublished data). However, regulatory mechanisms underlying the association between iron deficiency and PRMT expression are unclear in vitro and in vivo. In the present study, we revealed that the treatment of cells with two iron-specific chelators, DFO and deferasirox (DFX), downregulated the gene and protein expression of PRMT1 and 3 as compared with the untreated cells. Subsequently, DFO and DFX treatments decreased protein methylation. Importantly, these effects were attenuated by a holo-transferrin treatment. Furthermore, weanling Wistar-strain rats were fed a control diet or an iron-deficient diet for 4 weeks. Dietary iron deficiency was found to decrease the concentration of hemoglobin and liver iron while increasing the heart weight. PRMT and protein methylation levels were also significantly reduced in the iron-deficient group as compared to the control group. To our knowledge, this is the first study to demonstrate that PRMT levels and protein methylation are reduced in iron-deficient models, in vitro and in vivo.

Estimated Dietary Polyphenol Intake and Its Seasonal Variations among Japanese University Students.

The intake of polyphenols among Japanese has been estimated in several adult populations, but there has been no information regarding their intake among young adults, especially in those in their twenties. We conducted a food frequency questionnaire (FFQ)-based dietary assessment four times a year (once in each season) among Japanese university students and evaluated the total polyphenol intake across and within seasons. Forty-nine subjects (aged 20.7±0.6 y) completed our FFQ regarding polyphenol intake in February, May, August, and November 2016. We then calculated their total polyphenol intake using our polyphenol content database. The mean intake of total polyphenol across the seasons was 567±236 mg/d, which was largely sourced from beverages (62%). No significant differences were found in the total polyphenol intake or polyphenol intake from beverages among the four seasons. By contrast, we observed significant seasonal differences in the subjects' polyphenol intake from food; the polyphenol intake from food in February (255 mg/d) was significantly higher than that in May (215 mg/d), August (187 mg/d) and November (196 mg/d) (p<0.0001). These findings should assist in future estimations of dietary polyphenol intakes that consider differences according to age and season.

Aronia berry extract inhibits TNF-α-induced vascular endothelial inflammation through the regulation of STAT3.

BACKGROUND: Inflammation in endothelial cells induces production of inflammatory cytokines and monocytes adhesion, which are crucial events in the initiation of atherosclerosis. Aronia berry (Aronia meranocalpa), also called black chokeberry, contains abundant anthocyanins that have received considerable interest for their possible relations to vascular health. OBJECTIVE: The aim of this study was to investigate whether an anthocyanin-rich extract obtained from aronia berry can attenuate inflammatory responses in vascular endothelial cells. METHODS: As a model of vascular endothelial inflammation, human umbilical vein endothelial cells (HUVECs) pretreated with aronia berry extract were stimulated with tumor necrosis factor-alpha (TNF-α). The expression levels of cytokines and adhesion molecules were analyzed. To investigate the effects of aronia berry extract on the adhesion of THP-1 monocytic cell, the static adhesion assay was carried out. The possible molecular mechanisms by which aronia berry extract regulated vascular inflammatory responses were explored. RESULTS: The mRNA expressions of interleukins (IL-1ß, IL-6, and IL-8) and monocyte chemoattractant protein-1 (MCP-1) upregulated by TNF-α were significantly suppressed by pretreatment with aronia berry extract. Aronia berry extract decreased TNF-α-induced monocyte/endothelial adhesion and suppressed vascular cell adhesion molecule-1 (VCAM-1) expression, but did not affect intercellular adhesion molecule-1 (ICAM-1) expression. Moreover, aronia berry extract decreased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the nuclear levels of STAT3 and interferon regulatory transcription factor-1 (IRF1). The nuclear translocation of nuclear factor-kappa B (NF-κB) was not inhibited by aronia berry extract. CONCLUSION: Aronia berry extract could exert anti-atherosclerotic effects on TNF-α-induced inflammation through inhibition of STAT3/IRF1 pathway in vascular endothelial cells.

Terminalia bellirica (Gaertn.) Roxb. Extract and Gallic Acid Attenuate LPS-Induced Inflammation and Oxidative Stress via MAPK/NF-κB and Akt/AMPK/Nrf2 Pathways.

Excessive oxidative stress plays a critical role in the progression of various diseases. Recently, we showed that Terminalia bellirica (Gaertn.) Roxb. extract (TBE) inhibits inflammatory response and reactive oxygen species (ROS) production in THP-1 macrophages. However, molecular mechanisms underlying anti-inflammatory and antioxidant activities of TBE and its major polyphenolic compounds gallic acid (GA) and ellagic acid (EA) remain unclear. We found that TBE and GA attenuated LPS-induced inflammatory mediator expression, ROS production, and activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) in RAW 264 macrophages. Furthermore, TBE and GA increased antioxidant enzyme expression along with upstream mediators nuclear factor erythroid-2-related factor 2 (Nrf2), Akt, and AMP-activated protein kinase (AMPK). Importantly, knockdown of Nrf2 by siRNA and specific inhibition of Akt and AMPK significantly reduced antioxidant enzyme expression induced by TBE and GA. Finally, in vivo effects on histopathology and gene expression were assessed in tissues collected after intraperitoneal injection of LPS with or without TBE treatment. TBE enhanced antioxidant enzyme expression and improved acute kidney injury in LPS-shock model mice. In conclusion, TBE and GA exert protective effects against inflammation and oxidative stress by suppressing MAPK/NF-κB pathway and by activating Akt/AMPK/Nrf2 pathway. These results suggest that TBE and GA might be effective for the treatment of inflammation-related diseases.

Dietary Polyphenol Intake Estimated by 7-Day Dietary Records among Japanese Male Workers: Evaluation of the Within- and Between-Individual Variation.

Polyphenol intake has been estimated in some populations; however, information about day-to-day and individual differences in polyphenol intake has not been well-evaluated. In this study, we aimed to examine within- and between-individual variation in polyphenol intake in Japanese male workers. First, 56 male subjects (aged 37.9±10.4 y) completed detailed 7-d dietary records (DR). We then calculated their total polyphenol intake using our polyphenol content database and the within- and between-individual variations. We also estimated the minimum number of days of dietary assessment required both to rank individuals within a group and to assess an individual's usual polyphenol intake with acceptable accuracy. The estimated daily total polyphenol intake was 965±471 mg/d, which was largely sourced from beverages. The day-to-day variation (CVw) for polyphenol intake was 43.6%, and the variation between the individuals in the population (CVb) for polyphenol intake was 45.9%. A 4-d DR was required to rank individuals within a group with high correlation coefficients (r=0.9), and a 19-d DR was required to assess the individual's usual polyphenol intake with 20% deviation. The CVw for polyphenol intake was intermediate between those of the other nutrients, but the CVb for polyphenol intake was largest among the nutrients. These results suggest that the dietary intake of polyphenols should be carefully estimated considering its within- and between-individual variation.

Terminalia bellirica Extract Inhibits Low-Density Lipoprotein Oxidation and Macrophage Inflammatory Response in Vitro.

The deciduous tree Terminalia bellirica found in Southeast Asia is extensively used in traditional Indian Ayurvedic medicine for the treatment of hypertension, rheumatism, and diabetes. The anti-atherogenic effect of Terminalia bellirica fruit has not been fully elucidated. Here, we investigated the effect of Terminalia bellirica extract (TBE) on low-density lipoprotein (LDL) oxidation and inflammation in macrophages. TBE showed 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (EC50: 7.2 ± 1.2 µg/mL) and 15-lipoxygenase inhibitory activity. TBE also significantly inhibited free radical-induced LDL oxidation compared to the solvent control in vitro. In THP-1 macrophages, TBE treatment resulted in significant decreases of the mRNA expression of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), and lectin-like oxidized LDL receptor-1 (LOX-1). TBE also reduced matrix metalloproteinase (MMP)-9 secretion and intracellular reactive oxygen species (ROS) production in THP-1 macrophages. These results show that TBE has the inhibitory effects on LDL oxidation and macrophage inflammatory response in vitro, suggesting that its in vivo use might inhibit atherosclerosis plaque progression.