Walnut Polyphenol Extract Attenuates Immunotoxicity Induced By 4-Pentylphenol And 3-Methyl-4-Nitrophenol In Murine Splenic Lymphocyte

Walnut polyphenols, an important natural bioactive product, exhibit a range of antioxidative, antitumor, and anti-inflammatory properties. However, the effect of walnut polyphenols on immune toxicity is unknown. Gasoline exhaust particles (GEP) and diesel exhaust particles (DEP) are considered to be some of the most important air pollutants.4-pentylphenol (PP) and 3-methyl-4-nitrophenol (PNMC), the important phenolics components of GEP and DEP, respectively. Here, this study investigated the immunotoxicity caused by PP and PNMC on splenocytes in mice. Then, this study investigated the potential beneficial effects of walnut polyphenol extract (WPE) on immunotoxicity induced by PP and PNMC in murine splenic lymphocytes and evaluated the related mechanisms. The main results showed as follows:(1) To establish the immune injury model caused by PP and PNMC on splenocytes in mice, splenocytes were treated with PP or PNMC (10-6M, 10-5M, 10-4M) for 48 h and cell viability was measured using an MTT assay. Results showed that PP and PNMC both inhibited cell growth in a dose-related manner. Since both agents significantly inhibited splenocytes survival at 10-4M, this dose was chosen in the following experiments. Overall, the suppressive effects of PP were stronger than PNMC. These findings indicated that PP and PNMC can exert cytotoxicity on splenocytes.(2) To investigate the attenuating effects of WPE on immunotoxicity induced by PP and PNMC in murine splenic lymphocytes, splenocytes cells exposed to PP or PNMC in combinations with different doses of WPE for 48h and examined by MTT assay to assess the cell viability. Results showed that treatment with WPE was shown to significantly enhance cell viability of splenocytes exposed to PP or PNMC, limiting cytotoxicity in a dose-dependent manner at concentrations of 0.01-1.Oμg/mL. As 1.0μg/mL of WPE treatment enable the recovery of the declining cell viability induced by both agents to the similar levels as the control, this concentration was chosen for use in all subsequent experiments. WPE manifested no deleterious effect on splenocytes at 1.0μg/mL. These results suggested that WPE protected against PP and PNMC induced cytotoxicity in splenocytes.(3) To further determine the protective effect of WPE on PP and PNMC induced cytotoxicity in splenocytes, cell phenotypes, e.g., B-cell (CD19), T-cells (CD3), T-cell subsets (CD4 and CD8) were quantified by flow cytometry, and production of interleukin-2, interleukin-4, and granzyme-B was assessed via ELIS A after in vitro exposed to PP or PNMC treatment with WPE for 48h. Results showed that exposure to PP or PNMC might selectively impact on splenic T-lymphocyte populations and cytokine production, and neither compound appeared to affect levels of B-cells. WPE treatment increased the percentages of splenic CD3+, 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 and PNMC. These results suggested that WPE protected against PP and PNMC induced cytotoxicity in T cell and its function.(4) To examine the mechnism of WPE attenuating PP and PNMC induced immunotoxicity in splenocytes, levels of hydroxyl radical (OH·), malondiadehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) associated with the cultured cells were evaluated. Results showed that PP and PNMC elicited increase in the OH· and MDA production, and decreased in SOD and GSH-Px activities. WPE administration decreased the formation of excessive free radicals, inhibited lipid peroxidation and increased antioxidase activities. The data indicated that WPE may provide protection against PP and PNMC mediated immunotoxicity in splenocytes by inhibiting oxidative stress.(5) Total phenolic content of WPE was quantified using the Folin-Ciocalteu phenol assay, revealing an average content of 34800±200mg gallic acid equivalents (GAE)/100g. Individual compounds were then identified by LC-MS analyses, and a total of 16 individual phenolic compounds were identified, including ellagitannins, quercetin, valoneic acid dilactone, and gallic acid.Taken together, these results suggest that walnut polyphenols significantly attenuated PP and PNMC mediated immunotoxicity in splenocytes by restoring the cell viability, splenic T cell and T-cell sub-populations, as well as splenic T-cell formation/release of cytokines/granzymes in situ. These attenuating effects of walnut polyphenols may be related to attenuate oxidative damage induced by PP and PNMC in splenocytes.