The rising prevalence of lifestyle diseases, such as type 2 diabetes, cardiovascular diseases, and metabolic syndrome, has increased the need for effective dietary interventions. This study aimed to evaluate the effects of heat-moisture-treated high-amylose rice (HA-HMT) on body weight, lipid metabolism, and gut microbiome composition in a rat model of obesity. Starch digestibility-specifically, resistant starch-has been shown to provide various health benefits, including improved metabolic health and gut microbiome composition. We employed a sequential approach: firstly, utilizing diet-induced obesity rat models fed with HMT-processed and HMT-non-processed low- or high-amylose rice to investigate the potential of amylose content or HMT to alter phenotypic characteristics and lipid metabolism; and secondly, using the optimal rice flour identified in the previous step to explore the underlying mechanisms. Our findings indicate that heat-moisture treatment, rather than the level of the amylose content of the rice, contributes to the observed anti-obesity and cholesterol-lowering effects. We identified candidate genes contributing to the cholesterol-regulating potential and demonstrated that HMT rice flour could influence the gut microbiome, particularly the Ruminococcus taxa. This study provides valuable insights into the health benefits of HA-HMT rice and supports its potential as a functional food ingredient in the management of obesity and cholesterol-related disorders.

Sulforaphane has several effects on the human body, including anti-inflammation, antioxidation, antimicrobial and anti-obesity effects. In this study, we examined the effect of sulforaphane on several neutrophil functions: reactive oxygen species (ROS) production, degranulation, phagocytosis, and neutrophil extracellular trap (NET) formation. We also examined the direct antioxidant effect of sulforaphane. First, we measured neutrophil ROS production induced by zymosan in whole blood in the presence of 0 to 560 µM sulforaphane. Second, we examined the direct antioxidant activity of sulforaphane using a HOCl removal test. In addition, inflammation-related proteins, including an azurophilic granule component, were measured by collecting supernatants following ROS measurements. Finally, neutrophils were isolated from blood, and phagocytosis and NET formation were measured. Sulforaphane reduced neutrophil ROS production in a concentration-dependent manner. The ability of sulforaphane to remove HOCl is stronger than that of ascorbic acid. Sulforaphane at 280 µM significantly reduced the release of myeloperoxidase from azurophilic granules, as well as that of the inflammatory cytokines TNF-α and IL-6. Sulforaphane also suppressed phagocytosis but did not affect NET formation. These results suggest that sulforaphane attenuates neutrophil ROS production, degranulation, and phagocytosis, but does not affect NET formation. Moreover, sulforaphane directly removes ROS, including HOCl.

Abstract

Background

A ketogenic diet (KD) induces nutritional ketosis (NS), benefits fatty acid oxidation (FAO), and favors moderate-intensity exercise capacity. The status that the body accommodates to produce and utilize ketone bodies (KB) and fatty acids as primary fuel sources is termed keto-adaptation. However, keto-adaptation requires time, while long-term KD also involves unfavored adverse effects. Exogenous ketone body (EKBs) administration has been introduced to elicit the advantages of NS. However, the direct use of EKBs fails to bring desired outcomes. We hypothesized that EKBs might only be effective during keto-adaptation.

Methods

Male C57BL/6J mice (n = 24) were divided into three groups: a control diet (Con, n = 8), a ketogenic diet (KD, n = 8), and a KD plus a ketone body (DL-β-Hydroxybutyric acid sodium salt, BHB) administration (KD+BHB, n = 8). After six weeks of KD administration, mice in the KD+BHB group receive BHB added into water bottles for another six weeks. Blood KB concentration is monitored throughout the experiment, while liver, gastrocnemius, and soleus mRNA are analyzed using RT-PCR.

Results

Both KD and KD+BHB induced and sustained NS and enhanced hepatic and muscular key genes regulating FAO. In addition, BHB administration upon keto-adaptation further increased circulating KB concentration and enhanced expressional levels of FAO-mediating genes (ACO, HADH, ACADM, andMLYCDin the gastrocnemius muscle;ACO, HADH, andMLYCDin the soleus muscle), and energy-regulating genes (PPARAandPPARG) in the liver and skeletal muscle compared to a KD.

Conclusion

Compared to KD alone, chronic administration of KBs upon keto-adaptation increased the expression of key genes that favor FAO or maintain energy homeostasis in the liver and skeletal muscle. Instead of directly using EKBs in non-keto-adapted individuals, it is encouraged to use EKBs upon keto-adaptation status to elicit their energy-utilizing effects.

Highlights

This is the first report to evaluate the metabolic effects using exogenous ketone bodies on keto-adapted individuals.

Administration of exogenous ketone body upon keto-adaptation furtherly increased circulating ketone bodies.

Administration of exogenous ketone body upon keto-adaptation individuals enhanced expression of genes related to fatty acid oxidation and energy hemostasis.

Citrus nobiletin (NOB) and tangeretin (TAN) show protective effects against disease-related bone destruction. We achieved demethylation of NOB and TAN into 4′-demethylnobiletin (4′-DN) and 4′-demethyltangeretin (4′-DT) using enzyme-manufacturing methods. In this study, we examined the effects of 4′-DN and 4′-DT on in vitro osteoclast differentiation, and on in vivo osteoporotic bone loss in ovariectomized (OVX) mice. 4′-DN and 4′-DT clearly suppressed the osteoclast differentiation induced by interleukin IL-1 or RANKL treatment. 4′-DN and 4′-DT treatments resulted in higher inhibitory activity in osteoclasts in comparison to NOB or TAN treatments. RANKL induced the increased expression of its marker genes and the degradation of IκBα in osteoclasts, while these were perfectly attenuated by the treatment with 4′-MIX: a mixture of 4′-DN and 4′-DT. In an in silico docking analysis, 4′-DN and 4′-DT directly bound to the ATP-binding pocket of IKKβ for functional inhibition. Finally, the intraperitoneal administration of 4′-MIX significantly protected against bone loss in OVX mice. In conclusion, 4′-DN, 4′-DT and 4′-MIX inhibited the differentiation and function of bone-resorbing osteoclasts via suppression of the NF-κB pathway. Novel 4′-DN, 4′-DT and 4′-MIX are candidates for maintaining bone health, which may be applied in the prevention of metabolic bone diseases, such as osteoporosis.

Maternal malnutrition hampers the offspring health by manipulating the epigenome. Recent studies indicate that the changes in DNA methylation could be reversed by afterbirth nutrition supplementation. In this study, we used DNA methylation arrays to comprehensively investigate the DNA methylation status of the renal promoter regions and the effects of postnatal protein intake on DNA methylation. We fed stroke-prone spontaneously hypertensive (SHRSP) rat dams a normal diet or a low-protein diet during pregnancy, and their 4-week-old male offspring were fed a normal diet or a high-/low-protein diet for 2 weeks. We found that the methylation status of 2,395 differentially methylated DNA regions was reprogrammed, and 34 genes were reset by different levels of postnatal protein intake in the offspring. Among these genes, Adora2b, Trpc5, Ar, Xrcc2, and Atp1b1 are involved in renal disease and blood pressure regulation. Our findings indicate that postnatal nutritional interventions can potentially reprogram epigenetic changes, providing novel therapeutic and preventive epigenetic targets for salt-sensitive hypertension.

Skeletal muscle is programmable, and early-life nutritional stimuli may form epigenetic memory in the skeletal muscle, thus impacting adult muscle function, aging, and longevity. In the present study, we designed a one-month protein restriction model using post-weaning rats, followed by a two-month rebound feeding, to investigate how early-life protein restriction affects overall body growth and muscle development and whether these influences could be corrected by rebound feeding. We observed comprehensive alterations immediately after protein restriction, including retarded growth, altered biochemical indices, and disturbed hormone secretion. Transcriptome profiling of the gastrocnemius muscle followed by gene ontology analyses revealed that “myogenic differentiation functions” were upregulated, while “protein catabolism” was downregulated as a compensatory mechanism, with enhanced endoplasmic reticulum stress and undesired apoptosis. Furthermore, methylome profiling of the gastrocnemius muscle showed that protein restriction altered the methylation of apoptotic and hormone secretion-related genes. Although most of the alterations were reversed after rebound feeding, 17 genes, most of which play roles during muscle development, remained altered at the transcriptional level. In summary, early-life protein restriction may undermine muscle function in the long term and affect skeletal muscle development at the both transcriptional and methylation levels, which may hazard future muscle health.

Exercise-induced fatigue is a multi-origin physical and mental phenomenon. Efforts to diminish the above predisposition may contribute to endurance, along with athletic well-being, while development of nutritional strategies to optimize condition and exercise performance are essential issues for athletes and trainers. Dietary amino acids are being discussed for their specific health-promoting properties beyond their role as building blocks of proteins. Glutamine, along with cysteine, are two kinds of amino acids that are reported extensively for their anti-oxidation, anti-inflammation, and immune-regulation properties, and are promising in sport applications. In the present study, we designed a randomized, placebo-controlled, crossover trial to examine effects of 7-day supplementation of cystine/glutamine mixture (Cys2/Gln) on self-reporting fatigue index (ratings of perceived exertion, RPE), energy metabolism, and inflammation. We also employed a C2C12 myotube model to examine the capacity of cystine for fatty acid utilization. Cys2/Gln supplementation alleviated fatigue by decreasing RPE and enhanced fatty acid oxidation during a 60 min endurance exercise in human trials, while cystine increased fatty acid utilization in C2C12 myotubes by enhancing mitochondrial respiration. In summary, Cys2/Gln supplementation exerts positive effects on ameliorating exercise-induced fatigue, mechanisms of which can be attributed to enhancement of fatty acid utilization.

Black ginger (Kaempferia parviflora) extract (KPE), extracted from KP, a member of the ginger family that grows in Thailand, has a good promotion effect on cellular energy metabolism and therefore has been used to enhance exercise performance and treatment of obesity in previous studies. However, the effect of single-dose administration of KPE on endurance capacity has not been thoroughly studied, and whether the positive effect of KPE on cellular energy metabolism can have a positive effect on exercise capacity in a single dose is unknown. In the present study, we used a mouse model to study the effects of acute KPE administration 1 h before exercise on endurance capacity and the underlying mechanisms. The purpose of our study was to determine whether a single administration of KPE could affect endurance performance in mice and whether the effect was produced through a pro-cellular energy metabolic pathway. We found that a single administration of KPE (62.5 mg/kg·bodyweight) can significantly prolong the exercise time to exhaustion. By measuring the mRNA expression of Hk2, Slc2a4 (Glut4), Mct1, Ldh, Cd36, Cpt1β, Cpt2, Lpl, Pnpla2 (Atgl), Aco, Acadm (Mcad), Hadh, Acacb (Acc2), Mlycd (Mcd), Pparg, Ppargc1a (Pgc-1α), Tfam, Gp, Gs, Pfkm, Pck1 (Pepck), G6pc (G6pase), Cs, and Pfkl in skeletal muscle and liver, we found that acute high-concentration KPE administration significantly changed the soleus muscle gene expression levels (p < 0.05) related to lipid, lactate, and glycogen metabolism and mitochondrial function. In gastrocnemius muscle and liver, glycogen metabolism-related gene expression is significantly changed by a single-dose administration of KPE. These results suggest that KPE has the potential to improve endurance capacity by enhancing energy metabolism and substrate utilization in muscles and liver.

The mechanistic target of rapamycin complex 1 (mTORC1) is involved in nutrient-induced signaling and is a master regulator of cell growth and metabolism. Amino acid-deficient conditions affect mTORC1 activity; however, its upstream regulators warrant further investigation. MicroRNAs are key regulators of nutrient-related responses; therefore, the present study aimed to assess the leucine starvation-induced microRNA profile and its impact on mTORC1 activity. Transcriptome analysis of human hepatocellular carcinoma cells (HepG2) under leucine deprivation revealed that hsa-miR-663a and hsa-miR-1469 were altered in a transcription factor 4-dependent manner. Overexpression of these microRNAs induced phosphorylation of the ribosomal protein S6 kinase beta-1, a mTORC1 downstream target. Furthermore, hsa-miR-663a downregulated proline-rich Akt1 substrate of 40 kDa (PRAS40), one of the mTORC1 components. In summary, this study provides new insights into the regulatory role of microRNAs in amino acid metabolism and demonstrates alterations in microRNA profile under leucine deprivation in human hepatocytes.

Background and objectives: The purpose of this study was to investigate the influences of oral high-dose genistein (GE) administration on exercise-induced oxidative stress, inflammatory response and tissue damage. Materials and Methods: Thirty-two mice were randomly divided into control group (Con; sedentary/0.5% CMC-Na), GE administrated group (GE; sedentary/GE dosed), exercise group (Ex; exercise/0.5% CMC-Na), or GE administrated plus exercise group (GE + Ex; exercise/GE dosed), mice in the GE and GE + Ex group were given GE orally at the dose of 200 mg/kg weight. Results: Plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT) levels, liver interleukin (IL)-6, IL-1β, superoxide dismutase 1 (SOD1), catalase (CAT), hemeoxygenase-1 (HO-1) gene expression levels and skeletal muscle IL-6, nuclear factor erythroid 2-related factor (Nrf2), and HO-1 gene expression levels increased immediately after exhaustive exercise. GE supplementation increased liver protein carbonyl concentrations. On the other hand, GE supplementation significantly decreased SOD1, CAT gene expression levels in the liver and Nrf2, and HO-1 gene expression levels in the skeletal muscles. Conclusions: Acute exercise induced organ damage, inflammation, and oxidative stress in skeletal muscles and the liver. However, a single dose of GE supplementation before exercise did not lead to favorable antioxidant and anti-inflammatory effects in this study.

Strenuous exercise induces organ damage, inflammation and oxidative stress. To prevent exercise-induced organ damage, inflammation and oxidative stress, rehydrating may be an effective strategy. In the present study, we aimed to examine whether beverage intake after exhaustive exercise to recover from dehydration prevents such disorders. Thirteen male volunteers performed incremental cycling exercise until exhaustion. Immediately after exercise, the subjects drank an electrolyte containing water (rehydrate trial: REH) or did not drink any beverage (control trial: CON). Blood samples were collected before (Pre), immediately (Post), 1 h and 2 h after exercise. Urine samples were also collected before (Pre) and 2 h after exercise. We measured biomarkers of organ damage, inflammation and oxidative stress in blood and urine. Biomarkers of muscle, renal and intestinal damage and inflammation increased in the blood and urine after exercise. However, changes in biomarkers of organ damage and inflammation did not differ between trials (p > 0.05). The biomarker of oxidative stress, thiobarbituric acid reactive substances (TBARS), in plasma, showed different changes between trials (p = 0.027). One hour after exercise, plasma TBARS concentration in REH had a higher trend than that in CON (p = 0.052), but there were no significant differences between Pre and the other time points in each trial. These results suggest that beverage intake after exercise does not attenuate exercise-induced organ damage, inflammation or oxidative stress in healthy males. However, rehydration restores exercise-induced oxidative stress more quickly.

Obesity has become a worldwide health problem over the past three decades. During obesity, metabolic dysfunction of white adipose tissue (WAT) is a key factor increasing the risk of type 2 diabetes. A variety of diet approaches have been proposed for the prevention and treatment of obesity. The low-protein high-fat diet (LPHF) is a special kind of high-fat diet, characterized by the intake of a low amount of protein, while compared to typical high-fat diet, may induce weight loss and browning of WAT. Physical activity is another effective intervention to treat obesity by reducing WAT mass, inducing browning of WAT. In order to determine whether an LPHF, along with exercise enhanced body weight loss and body fat loss as well as the synergistic effect of an LPHF and exercise on energy expenditure in a mice model, we combined a 10-week LPHF with an 8-week forced treadmill training. Meanwhile, a traditional high-fat diet (HPHF) containing the same fat and relatively more protein was introduced as a comparison. In the current study, we further analyzed energy metabolism-related gene expression, plasma biomarkers, and related physiological changes. When comparing to HPHF, which induced a dramatic increase in body weight and WAT weight, the LPHF led to considerable loss of body weight and WAT, without muscle mass and strength decline, while it exhibited a risk of liver and pancreas damage. The mechanism underlying the LPHF-induced loss of body weight and WAT may be attributed to the synergistically upregulated expression of Ucp1 in WAT and Fgf21 in the liver, which may enhance energy expenditure. The 8-week training did not further enhance weight loss and increased plasma biomarkers of muscle damage when combined with LPHF. Furthermore, LPHF reduced the expression of fatty acid oxidation-related genes in adipose tissues, muscle tissues, and liver. Our results indicated that an LPHF has potential for obesity treatment, while the physiological condition should be monitored during application.

The low-carbohydrate ketogenic diet (LCKD) is a dietary approach characterized by the intake of high amounts of fat, a balanced amount of protein, and low carbohydrates, which is insufficient for metabolic demands. Previous studies have shown that an LCKD alone may contribute to fatty acid oxidation capacity, along with endurance. In the present study, we combined a 10-week LCKD with an 8-week forced treadmill running program to determine whether training in conjunction with LCKD enhanced fatty acid oxidation capacity, as well as whether the maximal exercise capacity would be affected by an LCKD or training in a mice model. We found that the lipid pool and fatty acid oxidation capacity were both enhanced following the 10-week LCKD. Further, key fatty acid oxidation related genes were upregulated. In contrast, the 8-week training regimen had no effect on fatty acid and ketone body oxidation. Key genes involved in carbohydrate utilization were downregulated in the LCKD groups. However, the improved fatty acid oxidation capacity did not translate into an enhanced maximal exercise capacity. In summary, while favoring the fatty acid oxidation system, an LCKD, alone or combined with training, had no beneficial effects in our intensive exercise-evaluation model. Therefore, an LCKD may be promising to improve endurance in low- to moderate-intensity exercise, and may not be an optimal choice for those partaking in high-intensity exercise.

Strenuous exercise induces organ damage, inflammation, and oxidative stress. Currently, to monitor or investigate physiological conditions, blood biomarkers are frequently used. However, blood sampling is perceived to be an invasive method and may induce stress. Therefore, it is necessary to establish a non-invasive assessment method that reflects physiological conditions. In the present study, we aimed to search for useful biomarkers of organ damage, inflammation, oxidative stress, and bone turnover in urine following exercise. Ten male runners participated in this study and performed a 3000-m time trial. We measured biomarkers in urine collected before and immediately after exercise. Renal damage markers such as urea protein, albumin, N-acetyl-β-D-glucosaminidase (NAG), and liver-fatty acid binding protein (L-FABP), and an intestinal damage marker, intestine-fatty acid binding protein (I-FABP), increased following exercise (p < 0.05). However, a muscle damage marker, titin N-terminal fragments, did not change (p > 0.05). Inflammation-related factors (IRFs), such as interleukin (IL)-1β, IL-1 receptor antagonist (IL-1ra), IL-6, complement (C) 5a, myeloperoxidase (MPO), calprotectin, monocyte chemoattractant protein (MCP)-1, and macrophage colony-stimulating factor (M-CSF), increased whereas IRFs such as IL-4 and IL-10 decreased following exercise (p < 0.05). IRFs such as tumor necrosis factor (TNF)-α, IL-2, IL-8, IL-12p40, and interferon (IFN)-γ did not change (p > 0.05). Oxidative stress markers, such as thiobarbituric acid reactive substances (TBARS) and nitrotyrosine, did not change following exercise (p > 0.05) whereas 8-hydroxy-2'-deoxyguanosine (8-OHdG) decreased (p < 0.05). Bone resorption markers, such as cross-linked N-telopeptide of type I collagen (NTX) and deoxypyridinoline (DPD), did not change following exercise (p > 0.05). These results suggest that organ damage markers and IRFs in urine have the potential to act as non-invasive indicators to evaluate the effects of exercise on organ functions.

Previous studies have not investigated the determinants of resting oxidative stress, including physical fitness, as it relates to redox regulation. The present study therefore was aimed at identifying lifestyle and biological factors that determine resting oxidative stress, including objectively measured physical fitness. In 873 middle-aged and elderly men and women, age and anthropometric parameters, lifestyle-related parameters, medication and supplementation status, physical fitness, biochemical parameters, and nutritional intake status, as well as three plasma oxidative stress markers: protein carbonyl (PC), F2-isoprostane (F2-IsoP), and thiobarbituric acid reactive substances (TBARS), were surveyed and measured. The determinants of PC, F2-IsoP, and TBARS in all participants were investigated using stepwise multiple regression analysis. In PC, age (β = -0.11, P = 0.002), leg extension power (β = -0.12, P = 0.008), BMI (β = 0.12, P = 0.004), and HDL-C (β = 0.08, P = 0.040) were included in the regression model (adjusted R 2 = 0.018). In the F2-IsoP, smoking status (β = 0.07, P = 0.060), BMI (β = 0.07, P = 0.054), and HbA1c (β = -0.06, P = 0.089) were included in the regression model (adjusted R 2 = 0.006). In TBARS, glucose (β = 0.18, P < 0.001), CRF (β = 0.16, P < 0.001), age (β = 0.15, P < 0.001), TG (β = 0.11, P = 0.001), antioxidant supplementation (β = 0.10, P = 0.002), and HbA1c (β = -0.13, P = 0.004) were included in the regression model (adjusted R 2 = 0.071). In conclusion, the present study showed that age, anthropometric index, lifestyle-related parameters, medication and supplementation status, objectively measured physical fitness, biochemical parameters, and nutritional intake status explain less than 10% of oxidative stress at rest.

Exhaustive exercise induces systemic inflammatory responses, which are associated with exercise-induced tissue/organ damage, but the sources and triggers are not fully understood. Herein, the basics of inflammatory mediator cytokines and research findings on the effects of exercise on systemic inflammation are introduced. Subsequently, the association between inflammatory responses and tissue damage is examined in exercised and overloaded skeletal muscle and other internal organs. Furthermore, an overview of the interactions between oxidative stress and inflammatory mediator cytokines is provided. Particularly, the transcriptional regulation of redox signaling and pro-inflammatory cytokines is described, as the activation of the master regulatory factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is involved directly or indirectly in controlling pro-inflammatory genes and antioxidant enzymes expression, whilst nuclear factor-kappa B (NF-κB) regulates the pro-inflammatory gene expression. Additionally, preventive countermeasures against the pathogenesis along with the possibility of interventions such as direct and indirect antioxidants and anti-inflammatory agents are described. The aim of this review is to give an overview of studies on the systematic inflammatory responses to exercise, including our own group as well as others. Moreover, the challenges and future directions in understanding the role of exercise and functional foods in relation to inflammation and oxidative stress are discussed.

Prolonged strenuous exercise may induce inflammation, cause changes in gastrointestinal permeability, and lead to other unfavorable biological changes and diseases. Nutritional approaches have been used to prevent exercise-induced inflammatory responses and gastrointestinal disorders. Hyperimmunized milk, obtained by immunizing cows against specific antigens, promotes the development of immunity against pathogens, promotes anti-inflammatory effects, and protects intestinal function. Immune protein (IMP) is a concentrated product of hyperimmunized milk and is a more promising means of supplementation to protect against acute infections and inflammation. To determine whether IMP has protective properties against exercise-induced gastrointestinal dysfunction and inflammation, we examined biochemical markers, intestinal damage markers, and pro-/anti-inflammatory profiles of young male runners using a randomized, placebo controlled, cross-over design. Urine samples were collected and used for measurements of creatinine, N-acetyl-β-d-glucosaminidase, osmotic pressure, and specific gravity. Titin was measured as a muscle damage marker. Further, urine concentrations of complement 5a, calprotectin, fractalkine, myeloperoxidase, macrophage colony-stimulating factor, monocyte chemotactic protein-1, intestinal fatty acid binding protein (I-FABP), interferon (IFN)-γ, interleukin (IL)-1β, IL-1 receptor antagonist, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, and tumor necrosis factor (TNF)-α were measured by enzyme-linked immunosorbent assays. We demonstrated that urine osmotic pressure, urine specific gravity, I-FABP, IFN-γ, IL-1β, and TNF-α were reduced by 8 weeks of IMP supplementation, indicating that IMP may have potential in preventing strenuous exercise-induced renal dysfunction, increased intestinal permeability, and inflammation. Thus, IMP supplementation may be a feasible nutritional approach for the prevention of unfavorable exercise-induced symptoms.

Regular exercise is beneficial to maintain a healthy lifestyle, but the beneficial effects are lost in the case of acute exhaustive exercise; this causes significant inflammation, oxidative stress along with organ damage. Recently, sulforaphane (SFN), an indirect antioxidant, has drawn special attention for its potential protective effect against inflammation and oxidative stress. However, no studies have been performed regarding acute exhaustive exercise-induced organ damage in association with SFN administration. Therefore, the aim of this study was to investigate the effects of SFN on acute exhaustive exercise-induced organ damage and the mechanisms involved. To perform the study, we divided mice into four groups: Control, SFN, exercise, and SFN plus exercise. The SFN group was administered orally (50 mg/kg body wt) 2 h before the running test. We measured plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), and acute exhaustive exercise significantly increased these biomarkers. In addition, the mRNA expression of pro-inflammatory cytokines, IL-6, IL-1β, and TNF-α, were significantly increased in the liver of exercise group. However, the SFN plus exercise group showed a significant reduction in the expression of cytokines and blood biomarkers of tissue damage or cell death. Furthermore, we measured mRNA expression of Nrf2, heme oxygenase (HO)-1, and antioxidant defense enzymes expression, i.e., superoxide dismutase (SOD1), catalase (CAT), and glutathione peroxidase (GPx1) in the liver. The expression of all these biomarkers was significantly upregulated in the SFN plus exercise group. Collectively, SFN may protect the liver from exhaustive exercise-induced inflammation via inducing antioxidant defense response through the activation of Nrf2/HO-1 signal transduction pathway.

Phytonutrients are widely recognized for providing protective human health benefits. Among the phytonutrients, epidemiological and experimental studies show that dietary organosulfur compounds (OSC) play a significant role in preventing various human pathological progressions, including chronic inflammation, by decreasing inflammatory mediators such as nitric oxide (NO), prostaglandin (PG)E2, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and IL-17, which are all typical hallmarks of inflammation. Evidence supports OSC in reducing the expression of these markers, thereby attenuating chronic inflammatory processes. Nuclear factor-kappa B (NF-κB) is a key regulating factor during inflammation, and novel evidence shows that OSC downregulates this transcriptional factor, thus contributing to the anti-inflammatory response. In vitro and in vivo studies show that inflammation is mechanistically linked with acute and chronic pathological conditions including cancer, diabetes, obesity, neural dysfunction, etc. Furthermore, a considerable number of experiments have demonstrated that the anti-inflammatory properties of OSC occur in a dose-dependent manner. These experiments also highlight indirect mechanisms as well as potent co-functions for protective roles as antioxidants, and in providing chemoprotection and neuroprotection. In this brief review, we provided an overview of the anti-inflammatory effects of OSC and elucidated probable mechanisms that are associated with inflammation and chronic disorders.

Postpubescent females may have negative mood or premenstrual syndrome during the menstrual cycle; with the emotional and physical symptoms interfering with their quality of life. Little is known about the relationship of dietary behaviors and dietary antioxidant intake with negative mood or premenstrual syndrome in university students in China; so we explored the relationship between negative mood and dietary behavior in female university students during the three menstrual cycle phases. Random sampling was used to enroll 88 individuals from a university in Guangzhou; China in the study. Data were collected using self-administered questionnaires. Descriptive statistics and multiple logistic regression analyses were performed. During the menstrual phase, tea, black coffee and carbonated beverage intake was higher in the group with a high negative affect scale score than in the low score group (p < 0.05). Likewise; during the premenstrual phase, fresh fruit (banana and red Chinese dates) intake was higher in the group with a high negative affect scale score than in the low-score group (p < 0.05). The logistic regression analysis results showed that negative mood was positively associated with tea, coffee, and carbonated beverage intake during the menstrual phase (β = 0.21, p = 0.0453, odds ratio = 1.23), and negative mood was positively associated with banana and red Chinese dates intake during the premenstrual phase (β = 0.59, p = 0.0172, odds ratio = 1.81). Our results suggest that negative mood may be associated with diet and specific food in university postpubescent females.

Inflammation is an essential part for the general or innate immune defenses to defend against tissue damage and accelerate the curing process by providing protection against pathogens. Sulforaphane (SFN) is a natural isothiocyanate that has potential properties against inflammation, along with other protective functions. The purpose of this study was to examine the mechanism of its protective effect on lipopolysaccharide (LPS)-induced inflammation in Raw 264.7 macrophages. Here, we compared LPS-challenged macrophages with or without SFN pretreatment. Macrophages were pre-incubated for 6 h with a wide range of concentrations of SFN (0 to 50 µM), and then treated with LPS for 24 h. Nitric oxide (NO) concentration and gene expression of different inflammatory mediators, i.e., interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β, were measured. SFN neither directly reacted with cytokines, nor with NO. To understand the mechanisms, we performed analyses of the expression of regulatory enzyme inducible nitic oxide synthase (iNOS), the transcription factor NF-E2-related factor 2 (Nrf2), and its enzyme heme-oxygenase (HO)-1. Our results revealed that LPS increased significantly the expression of inflammatory cytokines and concentration of NO in non-treated cells. SFN was able to prevent the expression of NO and cytokines through regulating inflammatory enzyme iNOS and activation of Nrf2/HO-1 signal transduction pathway.

Background and objectives: Aroma therapy is a complementary therapy using essential oils diluted with carrier oils. Jojoba oils have been widely used as carrier oils. However, limited information is available regarding their effects on blood biochemical parameters. This study aimed to investigate the effect of transdermal administration of jojoba oil on blood biochemical parameters in mice. Materials and Methods: Eight-week-old male hairless mice were randomly divided into naïve control and treatment groups. In the treatment group, mice were topically administered 4 μL of jojoba oil, per gram of body weight, on the dorsa 30 min before euthanasia. Thereafter, serum biochemical parameters were assayed, and gene expression was analyzed in various tissues via a real-time polymerase chain reaction. Results: Serum non-esterified fatty acid (NEFA) levels increased significantly 30 min after topical application of jojoba oil (p < 0.05). Atgl was significantly upregulated in the liver (p < 0.05), and Atgl upregulation in the liver was positively correlated with serum NEFA levels (r = 0.592, p < 0.05). Furthermore, a trend of decreasing fatty acid trafficking-related gene (FABPpm, FATP-1, FATP-3, and FATP-4) expression in the skin after topical application of jojoba oil (p = 0.067, 0.074, 0.076, and 0.082, respectively) was observed. Conclusions: Serum NEFA levels were elevated 30 min after transdermal administration of jojoba oil. The mechanisms of elevated serum NEFA levels might be related to both enhanced lipolysis in the liver and reduced fatty acid trafficking in the skin.

Studies have shown that diet and nutrition play significant roles in the prevention of depression and its clinical treatment. The present review aims to provide a clear understanding of the associations between diet patterns, specific foods, nutrients such as antioxidants, and depression. As a result, balanced dietary patterns such as the Mediterranean diet and certain foods such as fish, fresh vegetables, and fruits have been associated with a lower risk of depression or depressive symptoms, while high-fat Western diets and sugar-sweetened beverages have been associated with higher risk of depression or depressive symptoms. Dietary antioxidants such as green tea polyphenols or isoflavonoid intake have been negatively associated with depression or depressive symptoms. It is concluded that diet patterns, specific foods, and antioxidants play important roles in the prevention and clinical treatment of depression.

BACKGROUND: Carbohydrate (CHO) supplementation during exercise attenuates exercise-induced increases in plasma Interleukin (IL)-6 concentration. However, the effects of CHO supplementation on muscle IL-6 production during endurance exercise is controversial. The purpose of this study was to investigate the effects of CHO supplementation on muscle IL-6 production during endurance exercise with a special focus on the IL-6 producing cells. METHODS: C57BL/6J mice were divided into three groups-sedentary with water ingestion group as the control (Con; n = 10), exercise with water ingestion group (Ex; n = 10), and exercise with 6% glucose ingestion group (Ex + glucose; n = 10). The Ex and Ex + glucose groups completed 3 h of treadmill running (24 m/min, 7% incline) and were sacrificed immediately after exercise. RESULTS: The exercise-induced increases of plasma IL-6 concentration and gastrocnemius IL-6 gene expression were attenuated by glucose ingestion. However, the increases of soleus IL-6 gene expression and gastrocnemius and soleus IL-6 protein expression were not attenuated by glucose ingestion. Furthermore, we observed that macrophages that infiltrated muscle produce IL-6 and glucose ingestion attenuated the infiltration of IL-6-producing macrophages. CONCLUSION: This study revealed that infiltrating macrophages may be one type of IL-6-producing cells during endurance exercise, and the infiltration of these cells in muscle was attenuated by glucose ingestion. However, the effects of glucose ingestion on muscle IL-6 production were limited.

A ketogenic diet (KD) could induce nutritional ketosis. Over time, the body will acclimate to use ketone bodies as a primary fuel to achieve keto-adaptation. Keto-adaptation may provide a consistent and fast energy supply, thus improving exercise performance and capacity. With its anti-inflammatory and anti-oxidative properties, a KD may contribute to muscle health, thus preventing exercise-induced fatigue and damage. Given the solid basis of its potential to improve exercise capacity, numerous investigations into KD and exercise have been carried out in recent years. This narrative review aims to summarize recent research about the potential of a KD as a nutritional approach during endurance exercise, focusing on endurance capacity, recovery from fatigue, and the prevention of exhaustive exercise-induced muscle and organ damage.

Beta-cryptoxanthin (β-cry) is a typical carotenoid found abundantly in fruit and vegetables such as the Japanese mandarin orange, persimmon, papaya, paprika, and carrot, and exerts various biological activities (e.g., antioxidant effects). We previously reported that β-cry suppressed lipopolysaccharide (LPS)-induced osteoclast differentiation via the inhibition of prostaglandin (PG) E₂ production in gingival fibroblasts and restored the alveolar bone loss in a mouse model for periodontitis in vivo. In this study, we investigated the molecular mechanism underlying the inhibitory effects of β-cry on osteoclast differentiation. In mouse calvarial organ cultures, LPS-induced bone resorption was suppressed by β-cry. In osteoblasts, β-cry inhibited PGE₂ production via the downregulation of the LPS-induced mRNA expression of cyclooxygenase (COX)-2 and membrane-bound PGE synthase (mPGES)-1, which are PGE synthesis-related enzymes, leading to the suppression of receptor activator of NF-κB ligand (RANKL) mRNA transcriptional activation. In an in vitro assay, β-cry directly suppressed the activity of the inhibitor of NF-κB kinase (IKK) β, and adding ATP canceled this IKKβ inhibition. Molecular docking simulation further suggested that β-cry binds to the ATP-binding pocket of IKKβ. In Raw264.7 cells, β-cry suppressed RANKL-mediated osteoclastogenesis. The molecular mechanism underlying the involvement of β-cry in LPS-induced bone resorption may involve the ATP-competing inhibition of IKK activity, resulting in the suppression of NF-κB signaling.

Adjusting dietary fat intake is reported to affect mitochondrial biogenesis and fatty acid oxidation (FAO), and thus may enhance exercise capacity. However, a high-fat diet where carbohydrate intake is not limited enough also makes it difficult for athletes to maintain weight, and may fail to force the body to utilize fat. As such, a low-carbohydrate, high-fat, ketogenic diet (KD) may be viable. We have previously reported that an eight-week KD enhances exercise capacity, and suggested the mechanism to be enhanced lipolysis and ketolysis. In the present study, we investigated how an eight-week KD alters mRNA expression during fatty acid mobilization, FAO and ketolysis. We found that an eight-week KD may remodel the lipid metabolism profile, thus contributing to influence exercise capacity. We also found that ketolysis, lipolysis and FAO adaptations may contribute to enhanced exhaustive exercise performance. Along with enhanced FAO capacity during exhaustive exercise, a KD may also alter IL-6 synthesis and secretion profile, thus contribute to fatty acid mobilization, ketolysis, lipolysis and preventing muscle damage. Both the lipid metabolism response and IL-6 secretion appeared to be muscle fiber specific. Taken together, the previous and present results reveal that an eight-week KD may enhance exercise performance by up-regulating ketolysis and FAO ability. Therefore, a KD may have the potential to prevent muscle damage by altering IL-6 secretion profile, indicating that a KD may be a promising dietary approach in endurance athletes, sports, and for injury prevention.

Endurance capacity is important for maintenance of quality of life as well as performance of endurance athletes. In order to improve endurance, intake of nutritional supplements as well as exercise training is also important. Indeed, polyphenolic extracts from plants are known to improve endurance capacity via increase of fatty acid utilization, mitochondrial biogenesis or inhibition of oxidative stress. Taheebo, the extract obtained from inner bark of Tabebuia avellanedae has been reported to have beneficial effects for treatment of inflammation, oxidative stress and obesity. Here, we investigated the effects and mechanisms of polyphenol fraction of taheebo (taheebo polyphenol; TP) on endurance capacity of mice. Single dose administration of TP significantly increased running time until exhaustion. Acute TP administration increased blood glucose and muscle glycogen levels (p < 0.05) through alteration on expression level of genes involved with glycogen metabolism and gluconeogenesis. Furthermore, TP administration decreased exercise-induced increase of protein carbonyls in skeletal muscle. These results suggest that TP administration improve endurance capacity via up-regulation of skeletal muscle glycogen levels and maintenance of blood glucose by acceleration of gluconeogenesis as well as inhibition of exercise-induced oxidative stress. Single administration of TP also increased phosphorylation of AMP-activated protein kinase (AMPK) and gene expression level of sirtuin 1 (SIRT1) but did not change the marker of mitochondrial biogenesis.

A low-carbohydrate, high-fat ketogenic diet (KD) is a nutritional approach ensuring that the body utilizes lipids. In our previous study, we found that an eight-week ketogenic high-fat, low-carbohydrate diet increased the capacity of endurance exercise in mice without aggravated muscle injury, despite the decrease of absolute muscle volume. The potential mechanism is most possibly to be enhanced capacity to mobilize and utilize fat. In the present study, we investigated whether a ketogenic diet influences post-exercise recovery by measuring blood biomarkers, muscle and liver oxidative state as well as fatigue recovery 24 h post exercise by employing an open-field locomotion test. Several biochemistry markers indicating exercise-induced injury after exhaustive exercise were improved by KD, followed by a 24-h rest with free feed access, including lactate. No aggravated hepatic oxidative damage was observed, whereas muscular oxidative stress was increased by KD. Accelerated recovery induced by exhaustive exercise was also observed from blood biomarkers of injury. For fatigue recovery, lactate concentration, a marker often employed as exhaustion index was lowered by KD, whereas an open field test showed that KD application contributed to increased locomotion after exhaustive exercise, followed by a 24-h rest. These results suggest that KD has the potential to be used as a fatigue-preventing and/or recovery-promoting diet approach in endurance athletes.

Current fueling tactics for endurance exercise encourage athletes to ingest a high carbohydrate diet. However, athletes are not generally encouraged to use fat, the largest energy reserve in the human body. A low carbohydrate, high fat ketogenic diet (KD) is a nutritional approach ensuring that the body utilizes lipids. Although KD has been associated with weight-loss, enhanced fat utilization in muscle and other beneficial effects, there is currently no clear proof whether it could lead to performance advantage. To evaluate the effects of KD on endurance exercise capacity, we studied the performance of mice subjected to a running model after consuming KD for eight weeks. Weight dropped dramatically in KD-feeding mice, even though they ate more calories. KD-feeding mice showed enhanced running time without aggravated muscle injury. Blood biochemistry and correlation analysis indicated the potential mechanism is likely to be a keto-adaptation enhanced capacity to transport and metabolize fat. KD also showed a potential preventive effect on organ injury caused by acute exercise, although KD failed to exert protection from muscle injury. Ultimately, KD may contribute to prolonged exercise capacity.

We aimed to examine the effects of ingestion of different amounts of carbohydrate (CHO) after endurance exercise on neutrophil count, circulating cytokine levels, and the markers of neutrophil activation and muscle damage. Nine participants completed three separate experimental trials consisting of 1 h of cycling exercise at 70% V · O₂ max, followed by ingestion of 1.2 g CHO·kg body mass-1·h-1 (HCHO trial), 0.2 g CHO·kg body mass-1·h-1 (LCHO trial), or placebo (PLA trial) during the 2 h recovery phase in random order. Circulating glucose, insulin, and cytokine levels, blood cell counts, and the markers of neutrophil activation and muscle damage were measured. The concentrations of plasma glucose and serum insulin at 1 h after exercise were higher in the HCHO trial than in the LCHO and PLA trials. Although there were significant main effects of time on several variables, including neutrophil count, cytokine levels, and the markers of neutrophil activation and muscle damage, significant time × trial interactions were not observed for any variables. These results suggest that CHO ingestion after endurance exercise does not enhance exercise-induced increase in circulating neutrophil and cytokine levels and markers of neutrophil activation and muscle damage, regardless of the amount of CHO ingested.

[This corrects the article on p. 63 in vol. 4, PMID: 29312947.].

AIM OF STUDY: Taheebo polyphenols (TP) are water extracts of Tabebuia spp. (Bignoniaceae), taken from the inner bark of the Tabebuia avellanedae tree, used extensively as folk medicine in Central and South America. Some anti-inflammatory drugs act by inhibiting both cyclooxygenase-2 (COX-2) and COX-1 enzymes. COX-2 syntheses prostaglandin (PG) E2, which is a species of endogenous pain-producing substance, whereas COX-1 acts as a house-keeping enzyme. Inhibiting both COX-1 and -2 simultaneously can have side effects such as gastrointestinal bleeding and renal dysfunction. Some polyphenols have been reported for its selective inhibiting activity toward COX-2 expression. Our study aimed to demonstrate the potential and mechanisms of TP as an anti-inflammation action without the side effects of COX-1 inhibition. MATERIALS AND METHODS: Free fatty acid-stimulated macrophage cell lines were employed to mimic macrophage behaviors during lifestyle-related diseases such as atherosclerosis and non-alcoholic steatohepatitis. Real-time polymerase chain reaction was used to detect expression of inflammatory cytokine mRNA. Griess assay was used to measure the production of nitric oxide (NO). ELISA was used to measure PG E2 production. Molecular docking was adopted to analyze the interactions between compounds from T. avellanedae and COX-2. RESULTS: TP significantly suppressed the production of NO production, blocked the mRNA expression of iNOS, and COX-2 in both cell lines, blocked the mRNA expression of TNF-α, IL-1β, IL-6, and PGE2 in the murine cell line. However, there was no inhibitory effect on COX-1. Molecular docking result indicated that the inhibitory effects of TP on COX-2 and PGE2 could be attributed to acteoside, which is the main compound of TP that could bind to the catalytic zone of COX-2. After the interaction, catalytic ability of COX-2 is possibly inhibited, followed by which PGE2 production is attenuated. COX inhibitor screening assay showed TP as a selective inhibitor of COX-2 enzyme. CONCLUSION: The anti-inflammatory effects of TP can possibly regulate macrophages due to the targeted inhibition of COX-2 activity, without affecting COX-1 activity with other anti-inflammatory effects including suppression of iNOS and inflammatory cytokines. As such, TP is potentially useful in prevention and treatment of lifestyle-related disease by attenuating inflammation caused by macrophages infiltration.

Gasoline exhaust particles (GEP) and diesel exhaust particles (DEP) are considered to be some of the most important air pollutants. Among the many constituents in these pollutant particles, 4-pentylphenol (PP) and 3-methyl-4-nitrophenol (PNMC) are considered important phenolics in GEP and DEP, respectively. The aim of this study was to investigate the effect of in vitro exposure to commercially-supplied PP and PNMC on populations of, and production of interleukin (IL)-2, IL-4 and granzyme-B by, mouse splenic lymphocytes. After in vitro exposure to PP or PNMC for 48 h, splenocyte viability was measured, cell phenotypes, e.g. B-cell (CD19), T-cells (CD3), T-cell subsets (CD4 and CD8), were quantified by flow cytometry and production of IL-2, IL-4 and granzyme-B was assessed via ELISA. The oxidative toxicity of PP and PNMC toward the splenocytes was also evaluated using measures of hydroxyl radical and malondiadehyde production and changes in glutathione peroxidase and superoxide dismutase activities. Results showed that in vitro exposure to PP and PNMC inhibited splenic cell parameters in a dose-related manner. Exposure to PP and PNMC decreased splenic T-lymphocyte populations and splenocyte production of cytokines and granzyme B, as well as induced oxidative stress in the splenocytes. The results also showed that the percentages of CD3(+) T-cells overall and of CD4(+) and CD8(+) T-cells therein, among exposed splenocytes, were reduced; neither compound appeared to affect levels of CD19(+) B-cells. Overall, the suppressive effects of PP were stronger than PNMC. The data here provide support for the proposal that PP-/PNMC-induced toxicity in splenocytes may be due at least in part to oxidative damage and that PP and PNMC - as components of GEP and DEP - might significantly impact on splenic T-cell formation/release of cytokines/granzymes in situ.

4-pentylphenol (PP) and 3-methyl-4-nitrophenol (PNMC), two important components of vehicle emissions, have been shown to confer toxicity in splenocytes. Certain natural products, such as those derived from walnuts, exhibit a range of antioxidative, antitumor, and anti-inflammatory properties. Here, we investigated the effects of walnut polyphenol extract (WPE) on immunotoxicity induced by PP and PNMC in murine splenic lymphocytes. Treatment with WPE was shown to significantly enhance proliferation of splenocytes exposed to PP or PNMC, characterized by increases in the percentages of splenic T lymphocytes (CD3+ T cells) and T cell subsets (CD4+ and CD8+ T cells), as well as the production of T cell-related cytokines and granzymes (interleukin-2, interleukin-4, and granzyme-B) in cells exposed to PP or PNMC. These effects were associated with a decrease in oxidative stress, as evidenced by changes in OH, SOD, GSH-Px, and MDA levels. The total phenolic content of WPE was 34,800 ± 200 mg gallic acid equivalents/100 g, consisting of at least 16 unique phenols, including ellagitannins, quercetin, valoneic acid dilactone, and gallic acid. Taken together, these results suggest that walnut polyphenols significantly attenuated PP and PNMC-mediated immunotoxicity and improved immune function by inhibiting oxidative stress.

The Chinese brown frog (Rana dybowskii) has one special physiological phenomenon, which is that its oviduct goes through expansion prior to hibernation instead of during the breeding period. In this study, we investigated the localization and expression level of interleukin-1 (IL-1β) and its functional membrane receptor type I (IL1R1) proteins in the oviduct of R. dybowskii during pre-hibernation and the breeding period. There were significant differences in both oviductal weight and pipe diameter, with values markedly higher in pre-hibernation than in the breeding period. Histologically, epithelium cells, glandular cells and tubule lumen were identified in the oviduct during pre-hibernation and the breeding period, while sizes of both cell types are larger in the pre-hibernation than those of the breeding period. IL-1β was immunolocalized in the cytoplasm of epithelial and glandular cells in both periods, whereas IL-1R1 was observed in the membrane of epithelial and glandular cells in the breeding period, whereas only in epithelial cells during pre-hibernation. Consistently, the protein levels of IL-1β and IL-1R1 were higher in pre-hibernation as compared to the breeding period. These results suggested that IL-1β may play an important autocrine or paracrine role in oviductal cell proliferation and differentiation of R. dybowskii.

BACKGROUND: Walnut is unique because they have a perfect balance of n-6 and n-3 polyunsaturated fatty acids. The increasing market demand of walnut lipids results in the large amount of the oil extraction residue. The walnut residue is rich in nutritional proteins, and the uneconomic use of the by-product discouraged the development of walnut industry. Anticancer peptides have recently received attention as alternative chemotherapeutic agents that overcome the limits of current drugs. The aim of this study was to investigate whether anticancer bioactive peptide is contained in walnut. METHODS: Walnut residual protein was hydrolyzed separately by five different proteases. The sequential purification of the hydrolysates was carried out by ultra-filtration, gel filtration chromatography and RP-HPLC to obtain a cancer cell growth inhibitory peptide. Cell cycle distribution, Annexin V-FITC/PI double staining, TUNEL assay, western blot and immunofluorescence for LC3-II assay were used to detect apoptosis and autophagy on cells. Cytokine production was measured by ELISA kits, macrophage phagocytosis was measured by neutral red uptake assay, nitric oxide production was measured by Griess reagent. RESULTS: The hydrolysates of walnut residual protein produced by papain under the optimal conditions (5 % substrate concentration and an enzyme-substrate ratio of 10 % at temperature 60 C for 3 h), showed significant growth inhibitory activity on MCF-7. The amino acid sequence of the purified peptide was identified as CTLEW with a molecular weight of 651.2795 Da. It is a novel bio-peptide with an amphiphilic structure. CTLEW induced both apoptosis and autophagy on MCF-7 cells, inhibited the cancer cells growth of Caco-2 and HeLa significantly, but did not show any cytotoxic activity against non-cancerous IEC-6 cells. Moreover, the bio-peptide enhanced proliferation and IL-2 secretion of spleen lymphocytes, promoted phagocytosis and NO production of macrophages. CONCLUSION: These results suggested that a novel bio-peptide, CTLEW inducing apoptosis and autophagy on MCF-7 cells can be released from walnut residual protein through papain hydrolyzing under the certain condition. The bio-peptide shows selective inhibition towards cancer cells growth and immunomodulatory activity.

Oviductus Ranae is the dry oviduct of Rana chensinensis, which is also called R. chensinensis oil. Oviductus Ranae is a valuable Chinese crude drug and is recorded in the Pharmacopoeia of the People's Republic of China. The aim of this study was to investigate the immunostimulatory activity of protein hydrolysate of Oviductus Ranae (ORPH) and to assess its possible mechanism. Immunomodulatory activity of ORPH was examined in murine macrophage RAW 264.7 cells. The effect of ORPH on the phagocytic activity of macrophages was determined by the neutral red uptake assay. After treatment with ORPH, NO production levels in the culture supernatant were investigated by Griess assay. The mRNA and protein expressions of inducible nitric oxide synthase (iNOS) were detected by RT-PCR and Western blotting. The production of TNF-α, IL-1β, and IL-6 after treatment with ORPH was measured using ELISA assay. In addition, NF-κB levels were also investigated by Western blot. The results showed that ORPH enhanced the phagocytosis of macrophage, increased productions of TNF-α, IL-1β, IL-6, and NO in RAW 264.7 cells, and upregulated the mRNA and protein expression of iNOS. Besides, NF-κB, levels in RAW 264.7 cells were elevated after ORPH treatment. These findings suggested that ORPH might stimulate macrophage activities by activating the NF-κB pathway.