Protective Effects Of Walnut Polyphenol On Immunotoxicity Induced By Fenitrothion

Walnut polyphenol,an important natural active substance,exerts strong anti-oxidation effect and has excellent repairing effect in some diseases related to oxidative stress.Fenitrothion(FNT),an organophosphate pesticide widely used in the agricultural production,exerts neurotoxicity,gnotoxicity,hepatotoxicity and immunotoxic effects.Preliminary studies have shown that Walnut Polyphenol Extract(WPE)protected FNT-induced cytotoxicity in splenocytes.However,the mechanism of WPE alleviating fenitrothion-induced immunotoxicity are unclear.In this study,spleen lymphocytes were used as the point of penetration and after determining the protective effect of WPE on FNT immunotoxicity,the protection mechanism was invetigated.Research findings were showed as follows:1.To assess the toxicity of FNT,splenocytes were treated with FNT for 48 h,and cell proliferation assay was chosen to detect the cell viability.Results showed that compared with the control group,FNT inhibited cell growth in a dose-related manner and 10-4 M FNT resulted in significant decrease in cell viability.At the same time,in order to explore the effect of FNT exposure time on FNT toxicity,FNT exposed spleen lymphocytes for different time,cell viability was detected,and the results showed that FNT reduced cell viability in a time-dependent manner.When FNT exposure time was 30 min,it has a significant inhibitory effect on the cell viability of spleen lymphocytes.These findings indicated that FNT induced cytotoxicity in splenocytes.2.To investigate the effect of WPE on FNT induced cytotoxicity,splenocytes were treated with FNT alone or in combinations with WPE(0.5,1.0,5.0 and 10.0 ?g/mL)for 48 h,and cell viability was determined.Results showed that compared with the FNT group,WPE at 0.5-1.0 ?g/mL increased splenocyte viability in a concentration dependent manner and 1.0 ?g/mL WPE resulted in significantly protective activity.The data indicated that WPE protected splenocytes against FNT induced cytotoxicity.3.To determine the effect of WPE on FNT induced cytotoxicity in subpopulations of splenocytes,cells were cultured with FNT alone or in combinations with WPE in the presence of mitogens(Concanavalin A,Con A or lipopolysaccharides)for 48 h,and cell viability was determined.Meanwhile,after splenocytes were treated with FNT alone or in combinations with WPE for 48 h,the percentages of lymphocyte subpopulations were assessed using flow cytometry.Results showed that FNT selectively inhibited the proliferation and the proportions of splenic T cells,but WPE treatment attenuated these effects.These findings indicated that WPE protected FNT-induced cytotoxicity in splenic T cells.4.To investigate the effect of WPE on immune function of splenic lymphocyte,splenocytes were treated with FNT alone or in combinations with WPE for 48 h,and production of cytokines/granzyme-B was assessed via enzyme-linked immunosorbent assay.Results showed that FNT significantly inhibited levels of T cell-related cytokines,but had no impact on the production of interleukin-6 secreted by B cells.However,treatment with WPE significantly increased the secretion of T cell-related cytokines.The data indicated that WPE alleviated the inhibitory effects of FNT on immune function of splenic T cells.5.To investigate the mechnism of WPE preventing FNT mediated immunotoxicity in splenic T cells,splenocytes were treated with FNT alone or in combinations with WPE for 48 h,and levels of reactive oxygen and the activities of antioxidant enzymes were evaluated.Results showed that treatment with WPE significantly reduced the levels of hydroxyl radical and malondialdehyde and increased the activities of superoxide dismutase and glutathione peroxidase in the splenic T lymphocyte exposed to FNT.These findings showed that FNT mediated immunotoxicity was closely related to oxidative stress and WPE protected against immunotoxicity by its antioxidant activity.6.To assess the molecular mechanism underlying the protective effect of WPE on FNT induced immunotoxicity in splenic T cells,cells were cultured with FNT alone or in combinations with WPE for 48 h,the expressions of NADPH oxidase-2,dual oxidase-1 and Toll-like receptor-4 were determined by western blotting.Results showed that WPE at 1-10 ?g/mL significantly reduced NADPH oxidase-2,dual oxidase-1 and Toll-like receptor-4 overexpression induced by FNT in Concanavalin A stimulated splenic T-cells in a concentration dependent manner.The data indicated that WPE inhibited overactivation of NADPH oxidase-2 and dual oxidase-1 by suppressing the overexpression of Toll-like receptor-4,resulting in reduction of oxidative stress induced by FNT in splenic T-cells.The results of this study showed that oxidative stress was closely related to FNT-induced immunotoxicity in spleen lymphocytes and WPE can effectively alleviated the immunotoxicity through its anti-oxidation effect.Its main mechanism was:WPE attenuated of oxidative damage by suppression of the activation of NADPH oxidase-2 and dual oxidase-1,the latter caused by a reduction in the expression of Toll-like receptor-4,thereby reducing the immunotoxicity of FNT in T cells.