Walnut Polyphenol Attenuates Oxidative Toxicity Induced By Malathion In Murine Splenic Lymphocyte

Malathion(MT)is widely used in agricultural production as a low acute toxicity organophosphorus pesticide.However,MT has a toxic effect on nervous,reproductive and immune systems,and its toxic effect was mainly induced by oxidative stress.As an important source of natural antioxidants,walnut is rich in polyphenolic compounds.It inhibited oxidative stress by inhibiting free radical production,scavenging reactive oxygen species or increasing antioxidant enzyme activity.In this study,murine spleen lymphocytes were used as the key point.Based on the study of the toxic effects of MT on spleen lymphocytes,the mitigating effect of walnut polyphenol extract(WPE)on MT toxicity was discussed as so as mechanism.The main findings were as follows:(1)WPE alleviated the toxic effects of MT on spleen lymphocytes.The cells were co-cultured with spleen lymphocytes for 48 hours,and cell viability was determined by MTT assay.The results showed that MT significantly inhibited cell growth.When the spleen lymphocytes were co-treated with pesticides and WPE(1.0,5.0 and 10.0 μg/mL),the toxicity of MT was significantly alleviated when the concentration of WPE is 1 μg/mL,and the spleen lymphocyte activity exposed to MT could be restored to the level of the control when the concentration was 10 μg/mL.(2)WPE alleviated the toxic effects of MT on T/B lymphocytes.The cells were treated with mitogen,concanavalin or lipopolysaccharide,and the T cell or B cell were proliferated in large amounts.Under the stimulation of mitogen,the cells were exposed to medium containing MT for 48 h,and cell viability was measured by MTT assay.It was found that MT significantly inhibited the growth of both T and B lymphocytes.When cells were co-cultured with concentrations of 1.0,5.0,and 10.0 μg/mL of WPE and MT,respectively,it was found that the activity of T and B lymphocytes was significantly improved,and when the concentration of WPE was 1 μg/mL,significant relief was observed.When the concentration was lOpg/mL,the cell viability could be restored to the level of the control.(3)WPE alleviated the oxidative toxicity of MT on spleen lymphocytes.After exposed MT for 48 h,the levels of reactive oxygen species and antioxidants in spleen lymphocytes were measured.The results showed that MT increased the level of reactive oxygen species,superoxide dismutase,glutathione peroxidase,glutathione,glutathione reductase,γ-glutamylcysteine synthetase and Glutathione S-transferases.When spleen lymphocytes were co-treated with MT and WPE in concentrations of 1.0,5.0,and 10.0 μg/mL,respectively,it was found that WPE reduced the active oxygen content and increased the level of antioxidant factors in a concentration-dependent manner.When the WPE concentration was 10.0μg/ml,the redox state of the cells nearly recovered to the control.(4)WPE alleviated the mechanism of oxidative toxicity of MT on spleen lymphocytes.After co-culture of MT with spleen lymphocytes for 48 h,the activity of mitochondrial respiratory chain complex was detected by enzyme-linked immunosorbent assay.The results showed that MT inhibits the levels of complex Ⅰ and complex Ⅱ and enhances complex Ⅲ activity.When the concentration of WPE was 10.0 μg/mL,the levels of complexes Ⅰ and Ⅱ were significantly up-regulated,and the level of complex Ⅰ was restored to near the normal control group.The above results indicated that MT interferes with its proliferative function by inducing oxidative toxicity to spleen lymphocytes.WPE reduced the production of reactive oxygen species by restoring the levels of mitochondrial respiratory chain compolex Ⅰ and complex Ⅱ,and restored the antioxidant defense system by increasing the levels of antioxidant factors.Furthermore,it effectively alleviates the oxidative stress induced by MT of pesticides and protects spleen lymphocytes.