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. 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. 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. 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 ofRana chensinensis, which is also calledR. chensinensisoil. 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.

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.

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.

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 cytokine interleukin-6 (IL-6) mediates a wide range of inflammatory and immune responses. Tumor Necrosis Factor α (TNF-α) has a myriad of pro-inflammatory effects on the skin. In this study, we investigated the localizations and expression levels of IL-6, TNF-α and NF-κB in the skin of Rana dybowskii during the breeding period and pre-hibernation. Histologically, the skin of Rana dybowskii consists of epidermis and dermis. Four kinds of cells were identified in the epidermis during the breeding period and pre-hibernation, while the dermis was composed of homogenous gel, mucous glands and granular glands. IL-6, TNF-α and NF-κB were immunolocalized in the epithelial and glandular cells in both periods. Western blotting showed that IL-6, TNF-α and NF-κB were significantly higher in the pre-hibernation compared to the breeding period. Real-Time PCR revealed that the relative mRNA levels of IL-6 and NB-κB in the pre-hibernation increased significantly compared with the breeding period, while the TNF-α mRNA expression levels were no significant difference between these two periods. These results suggested that IL-6, TNF-α and NF-κB may collectively play important roles in the skin immune system of Rana dybowskii during the breeding period and pre-hibernation.