| Deoxynivalenol (DON) and zearalenone (ZEA) are the most common mycotoxins in the feed. The mycotoxins can not only reduce the nutritional value of feed, but also inhibit the growth performance and the reproductive performance of animal. Moreover, the mycotoxins can destroy the immune system in animal and induce immunosuppression, which causes high incidence of diseases. The main target organ of mycotoxins in immune system is the spleen. The mycotoxins can cause the apoptosis of the splenic lymphocyte, and inhibit the body immune function. In this study, the toxic effects of DON and/or ZEA on the splenic lymphocytes of chicken were investigated in vitro, which will offer some theoretic evidences for further exploring the mechanism in immunotoxicity of DON and/or ZEA.By CCK-8test, the IC50of DON and ZEA to chicken splenic lymphocytes in vitro at48h were30.82±10.48μg/mL and23.91±4.96μg/mL, individually. So, it s howed that DON or ZEA affected cell proliferation activity. On this basis of IC50, the final concentrations of DON were0.2,0.8,3.2,12.5, and50μg/mL; and the fi nal concentrations of ZEA were0.1,0.4,1.6,6.25, and25μg/mL, individually. The exposure doses of DON and ZEA joint were DON0.0125μg/mL and ZEAO.00625μ g/mL, DON0.05μg/mL and ZEA0.025μg/mL, DON0.2μg/mL and ZEAO.1μg/mL, DO N0.8μg/mL and ZEA0.4μg/mL, individually. Then, the toxic effects of different dos es of DON or/and ZEA on chicken splenic lymphocytes in vitro for48h were inve stigated. The determination indicators are as follows:①Effects of DON or/and ZEA on the apoptotic rates, necrotic rates, LDH release, DNA damage, and apoptotic of morphological changes in chicken splenic lymphocytes;②Activities of SOD, CAT, GSH-Px, and contents of GSH, MDA in chicken splenic lymphocytes;③Act ivities of Ca2+-ATPase and Na+/K+-ATPase, intracellular pH, levels of mitoch ondrial membrane potential(Δφ), reactive oxygen species (ROS), intracellular [C a2+]i), and CaM relative mRNA level in chicken splenic lymphocytes;④Apoptotic genes (bcl-2and Bax bak-1, p53and caspase-3, etc.) relative mRNA1evel in chicken splenic lymphocytes(real-time PCR) and the concentrations of related apoptosis gene protein in supernatants of chicken splenic lymphocytes (ELISA).The results were as followed:①The apoptotic rates, necrotic rates, LDH release in these exposed groups were significantly higher than those in the control group (P<0.05or0.01). These indicators were significantly increased, and showed a dose-dependent effect following toxin dose increasing. Also, these indicators by combinations of DON and ZEA were always higher than those in the related DON or ZEA group in the same exposure dose. Following toxin dose increasing, DNA fragmentations in all exposed groups were more obvious than that of the control group. Typical morphological changes of apoptosis in the DON and/or ZEA groups, were nuclear shrinkage, drop-like structure of nuclear, dense nuclear chromatin, nuclear fragment. The apoptotic rates were significantly greater than the necrosis rates.②Compared with the control group, the activities of SOD, CAT, GSH-Px and GSH levels in the exposed groups decreased significantly (P<0.05or P<0.01), except for these of the low-dose groups (DON or ZEA). Following toxin dose increasing, these indicators were significantly decreased. Also, these indicators induced by the combinations of DON and ZEA, were always lower than those in the related DON or ZEA group in the same exposure dose. But following toxin dose increasing, the concentrations of MDA were increased. Compared with the control group, the concentrations of MDA increased significantly (P<0.01) except for the low-dose groups (DON and/or ZEA). Also, the concentrations of MDA induces by combinations of DON and ZEA, were always higher than those in the related DON or ZEA group in the same exposure dose.③Following toxin dose increasing, compared to the control group, intracellular ROS,[Ca2+]i, and CaM relative mRNA level increased significantly (P<0.05or0.01). Also, these indicators induced by combinations of DON and ZEA were always higher than those in the related DON or ZEA group in the same exposure dose, except that CaM relative mRNA level of DZ-3group (DON0.2μg/mL+ZEA0.1p.g/mL) was higher than that of Z1group (ZEA0.1μg/mL), but was lower than that of Dl group (DON0.2μg/mL). The mitochondrial Aφ, intracellular pH, activities of Ca2+-ATPase and Na+-/K+-ATPase decreased significantly (P<0.05or0.01) following toxin dose increasing. These indicators induced by combinations of DON and ZEA were always lower than those in the related DON or ZEA group in the same exposure dose.④Following toxin dose increasing, p53, Bax, Bak-1, and Caspase-3relative mRNA levels in the exposed groups increased significantly (P<0.05or0.01), except for the low-dose groups (DON and/or ZEA). And the ratio of Bax/Bcl-2in the exposed groups was significantly higher than that of the control group (P<0.05or0.01). These indicators induced by combinations of DON and ZEA were always higher than those in the related DON or ZEA group in the same exposure dose, except that p53relative mRNA level of DZ-4group (DON0.8μg/mL+ZEA0.4ug/mL) was higher than that of D2group(DON0.8μg/mL), but was lower than that of Z2group (ZEA0.4ug/mL), Bak-1relative mRNA level of DZ-3group (DON0.2μg/mL+ZEA0.1μg/mL) was lower than that of D1group(DON0.2μg/mL) and Z1group (ZEA0.1μg/mL). Following toxin dose increasing, the concentrations of p53of Bax, and Bak-1and Caspase-3in supernatants of chicken splenic lymphocytes gradually increased in a dose-dependent manner. There were significant differences between the exposed groups and the control group (P<0.05or0.01). Also these indicators induced by combinations of DON and ZEA were always higher than those in the related DON or ZEA group in the same exposure dose. But the concentrations of Bcl-2significantly decreased compared to the control (P<0.01), and these indicators of combinations of DON and ZEA were always higher than those in the related DON or ZEA group in the same exposure dose.Based on these results, the conclusions are as follow:①DON and/or ZEA exposure induced chiken splenic lymphocytes damage, DNA damage, and apoptosis in a dose-dependent manner, and synergistic effect lies in the administration of DON combined with ZEA. The apoptotic mechanism played a chief role in the cellular death induced by DON and/or ZEA at these doses in present study.②DON and/or ZEA exposure inhibited the activities of the anti-oxidative enzymes, which leaded to the increased oxidation products. Further, the oxidative damage in the chicken splenic lymphocytes was enhanced. The oxidative damage could be the first mechanism of apoptotic in the chicken splenic lymphocytes induced by DON and/or ZEA.③DON and/or ZEA exposure induced depletion of mitochondrial Aφ, disorder of intracellular homeostasis, i.e. intracellular acidification, and ion imbalance. The imbalance of the intracellular environment might be the second mechanism of apoptotic in the chicken splenic lymphocytes induced by DON and/or ZEA.④For Bcl-2family genes, DON and/or ZEA exposure induced increased the expression of Bax gene and the expression of Bak-1gene, but decreased expression of Bcl-2gene. Besides, DON and/or ZEA exposure also increased the expression of p53gene and the expression of Caspase-3gene, then p53and Caspase pathway was activated. The activated p53and Caspase pathway might be the third mechanism of apoptotic in the chicken splenic lymphocytes induced by DON and/or ZEA. In a word, there was an obvious synergistic effect of DON combined with ZEA on the cellular damage in the chicken splenic lymphocytes... |
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