Walnut Polyphenol Extract Protects against Malathion- and Chlorpyrifos-Induced Immunotoxicity by Modulating TLRx-NOX-ROS.

Malathion (MT) and chlorpyrifos (CPF) are immunotoxic organophosphate pesticides that are used extensively in agriculture worldwide. Dietary polyphenols protect against a variety of toxins. In this study, walnut polyphenol extract (WPE) prevents MT- or CPF-induced toxicity to splenic lymphocytes in vitro. WPE promotes the proliferation of MT-exposed splenocytes, as indicated by increases in the proportions of splenic T-lymphocyte subpopulations (CD3+, CD4+, and CD8+ T cells) and levels of T-cell-related cytokines interleukin (IL)-2, interferon-γ, IL-4, and granzyme B, and decreases the apoptosis-associated proteins Bax and p53. WPE also significantly enhances the proliferation of CPF-exposed splenic B lymphocytes (CD19+ B cells) and levels of the B-cell-related cytokine IL-6, leading to decreases of the apoptosis-associated proteins Bax and p53. These effects are related to reduced production of reactive oxygen species (ROS), as evidenced by normalized hydroxyl radical (•OH), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and glutathione (GSH) levels, which are associated with decreased expression of NADPH oxidase 2 (NOX2) and dual oxidase 1 (DUOX1). WPE inhibits the production of ROS and expression of NOX by regulating toll-like receptors 4 and 7 in MT- and CPF-exposed splenic lymphocytes. In conclusion, WPE protects against MT- or CPF-mediated immunotoxicity and inhibits oxidative damage by modulating toll-like receptor (TLR)x-NOX-ROS.

Walnut Polyphenol Extract Protects against Fenitrothion-Induced Immunotoxicity in Murine Splenic Lymphocytes.

Fenitrothion (FNT), an organophosphate pesticide, exerts an immunotoxic effect on splenocytes. Dietary polyphenol compounds exert antioxidant, anticancer and antihypertensive effects. In this study, we investigated the effect of walnut polyphenol extract (WPE) on FNT-induced immunotoxicity in splenic lymphocytes in vitro. Treatment with WPE significantly increased the proliferation of FNT-exposed splenocytes, as evidenced by increases in the proportions of splenic T lymphocytes (CD3⁺ T cells) and T-cell subsets (CD8⁺ T cells), as well as the secretion of the T-cell-related cytokines interleukin (IL)-2, interferon-γ, IL-4 and granzyme B. These effects were associated with a reduction in oxidative stress, as evidenced by changes in the levels of hydroxyl radical, superoxide dismutase, glutathione peroxidase and malondialdehyde. Moreover, WPE decreased the FNT-induced overexpression of NADPH oxidase 2 and dual oxidase 1 by regulating Toll-like receptor 4 signaling in splenic T-cells. Taken together, these findings suggest that WPE protects against FNT-mediated immunotoxicity and improves immune function by inhibiting oxidative stress.

Walnut Polyphenol Extract Attenuates Immunotoxicity Induced by 4-Pentylphenol and 3-methyl-4-nitrophenol in Murine Splenic Lymphocyte.

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.

Isolation of a novel bio-peptide from walnut residual protein inducing apoptosis and autophagy on cancer cells.

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.

[Relationship between the level of sialic acid during perinatal period and early intelligence development of full term infants].

OBJECTIVE: To investigate the correlation between the status of sialic acid (SA) during perinatal period and early intelligence development of healthy full term infant, and to explore the effect of SA on the early intelligence development. METHOD: A total of 127 pairs of healthy mothers-neonates in the Affiliated Hospital of Nantong University were recruited randomly in this prospective cohort study. The levels of SA from body fluids of mothers-neonates were measured by enzyme-linked immunosorbent assay, such as the full-term maternal and cord blood and the colostrum. The questionnaire surveys were carried out in mothers and mental development evaluation according to Children's Development Center of China (CDCC) were carried out in infants 3 to 4 months of age to obtain the mental development index (MDI) and psycho-motor development index (PDI). RESULT: A total of 120 pairs of maternal-neonatal subjects with complete data were included into statistical analysis. The levels of SA of maternal and cord blood and colostrum were (2.25 ± 0.02), (1.21 ± 0.01), and (5.01 ± 0.06) mmol/L respectively. MDI and PDI of infants 3 to 4 months of age were (99.40 ± 1.87) and (98.53 ± 1.96). The analysis using multiple linear regression indicated that MDI was associated with SA levels of cord blood and colostrum (ß = 0.636, 0.175, P < 0.05), and PDI was also associated with them (ß = 0.502, 0.262, P < 0.05). The levels of SA of cord blood and colostrums were individually divided into high-level group and low-level one according to the median level. MDI and PDI in high-level group of cord blood were both significantly higher than that in low-level group (111.85 ± 2.79) vs. (108.88 ± 2.0) , (101.08 ± 4.44) vs. (98.88 ± 2.0) P < 0.01. So were MDI and PDI in high-level group of colostrum compared with those in low-level group (111.71 ± 3.07) vs. (108.81 ± 1.56), P < 0.01; (101.29 ± 4.23) vs.(98.56 ± 1.79), P < 0.05. The analysis on correlation between the levels of maternal-neonatal body fluids showed that the level of SA of colostrum was positively correlated with that of cord blood (r = 0.507, P = 0.004). However, no correlation was found either between the level of SA of maternal and cord blood or between the level of SA of maternal blood and colostrums. Further division into high-level and low-level groups was done according to the median level of maternal blood. The levels of SA of colostrum and cord blood in high-level group were markedly higher than those in low-level one (5.12 ± 0.35) vs. (4.87 ± 0.22) and (1.21 ± 0.02) vs. (1.17 ± 0.01), P < 0.05. CONCLUSION: High levels of SA of cord blood and colostrums might be beneficial to the early intelligence development of full term infant. Abundant intake of SA during perinatal period and good function of placenta may play important role in early intelligence development.

Immunostimulatory activity of protein hydrolysate from oviductus ranae on macrophage in vitro.

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-1ß, 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-1ß, 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.

Interactions between gonadotropin-releasing hormone (GnRH) and orexin in the regulation of feeding and reproduction in goldfish (Carassius auratus).

Links between energy homeostasis and reproduction have been demonstrated in vertebrates. As a general rule, abundant food resources favor reproduction whereas low food availability induces an inhibition of reproductive processes. In both mammals and fish, gonadotropin-releasing hormone (GnRH) and orexin (OX) are hypothalamic neuropeptides that play critical roles in the regulation of sexual behavior and appetite, respectively. In order to assess possible interactions between orexin and GnRH in the control of feeding and reproduction in goldfish, we examined the effects of chicken GnRH (cGnRH-II) intracerebroventricular (ICV) injection on feeding behavior and OX brain mRNA expression as well as the effects of orexin ICV injections on spawning behavior and cGnRH-II brain mRNA expression. Treatment with cGnRH-II at doses that stimulate spawning (0.5 ng/g or 1 ng/g) resulted in a decrease in both food intake and hypothalamic orexin mRNA expression. Treatment with orexin A at doses that stimulate feeding (10 ng/g) induced an inhibition of spawning behavior and a decrease in cGnRH-II expression in the hypothalamus and optic tectum-thalamus. Our results suggest that the anorexigenic actions of cGnRH-II in goldfish might be in part mediated by OX and that orexin inhibits reproductive behavior in part via the inhibition of the GnRH system. Our data suggest the existence of a coordinated control of feeding and reproduction by the orexin and GnRH systems in goldfish.