| Aflatoxins B1(AFB1)is one of the secondary metabolites of Aspergillus flavus and Aspergillus parasiticus,commonly contaminate a worldwide of food and feed stuffs.AFB1 is considered accountable for the increased incidence of human and animals health impairment and economic losse.Previous research found that the toxic effects induced by AFB1 on animal immune system,may become a major reason for immunization failure and increasing predisposition to disease.However,the mechanism and pathway remain largely unclear,and some results of pig experiments are stuck in animal vivo,few studies extend to porcine lymophcyte and macrophage in vitro.Selenium(Se)is viewed as an essential trace element linked to many health benefits in humans and other mammals.The biological effects of Se are exerted through covalent bonding within the amino acid selenocysteine(Sec).Despite some studies have focus on the effects of Se in immune function,however,further work is needed to provide insight into the role of selenium and specific selenoproteins in relation to toxin exposure.The objectives of this study were to investigate the effect of selenium on the the toxic effects of AFB1 exposure in primary porcine splenocytes and porcine macrophage caused by oxidative stress;and to explore the mechanisms by which selenium affects AFB1-induced immunotoxicity,so as to provide a theoretical basis for application of Se,especially organic selenium in swine industry.Experiment 1:Aflatoxin B1 suppressed T-Cell stimulation in primary porcine splenocytesThe aim of the present study is to investigate whether AFB1-induced immunotoxicity is associated with oxidative stress and the expression of extracellular regulated protein kinases(ERK)1/2-The primary splenocytes isolated from healthy pigs were activated and proliferated by anti-pig-CD3 monoclonal antibodies(mAb)in the present experiment,which is an antigen-specific stimulant.Results indicated that cell proliferation and interleukin-2(IL-2)production were significantly suppressed by AFB1 from 4 to 8 ?g/mL in a dose-dependent manner compared to the control group.Furthermore,AFB1 significantly increased malondialdehyde(MDA)levels,decreased reduced glutathione(GSH)and total superoxide dismutase levels,and up-regulated p-ERKl/2 expression in the activated splenocytes.N-Acetyl-L-cysteine(NAC)blocked anti-CD3-induced T-cell suppression by AFB1 through increasing intracellular concentrations of GSH levels,decreasing MDA levels,and down-regulated p-ERK1/2 expression,respectively.Inhibition of the ERK1/2 expression by ERK-specific RNA attenuated the decrease of T-cell proliferation and IL-2 production induced by AFB1.It was concluded that AFB1 inhibits anti-CD3-induced lymphocyte proliferation and IL-2 production by the oxidative stress mediated ERK1/2 MAPK signaling pathway.Experiment 2:Selenium alleviates AFB1-induced immunotoxicity in primary porcine splenocytes and mechanism researchLittle has been done about the mechanisms of how Se protects against AFB1-induced immunotoxicity.The aim of this present study is to investigate the protective effects of Se against AFB1 and the underlying mechanisms.The primary splenocytes isolated from healthy pigs were stimulated by anti-pig-CD3 monoclonal antibodies and treated by various concentrations of different Se forms and AFB1.The results showed that Se supplementation alleviated the immunotoxicity of AFB1 in a dose-dependent manner,as demonstrated by increasing T-cell proliferation and interleukin-2 production.Addition of buthionine sulfoximine abrogated the protective effects of SeMet against AFB1-SeMet enhanced mRNA and protein expression of glutathione peroxidase 1(GPx1),selenoprotein S(SelS),and thioredoxin reductase 1 without and with AFB1 treatments.Furthermore,knockdown of GPx1 and SelS by GPx1-specific siRNA and SelS-specific siRNA diminished the protective effects of SeMet against AFB1-induced immunotoxicity.It is concluded that SeMet diminishes AFB1-induced immunotoxicity through increasing antioxidant ability and improving GPx1 and SelS expression in splenocytes.Experiment 3:Aflatoxin B1 suppressed the immune function in porcine 3D4/21 cellsIn order to futher research the toxic effects of AFB1 on porcine immune cells,we chose porcine 3D4/21 cells as a new model,and detected MTT,IL-1? and TNF-?mRNA levels at 48 h and 72 h.Besides,we used fluorescence microscope to detect cell morphological changes and apoptosis and flow cytometry for ROS levels,apoptosis.Results showed that different concentration of AFB1 significantly decreased the cell ability,and regulated macrophage to apoptosis.Interestingly,AFB1 at low concentration also had a significant increase on pro-inflammatory cytokines,such as IL-1? and TNF-?.All four concentration of AFB,could increased the ROS levels.NAC blocked macrophage toxicity induced by 0.1 ?g/mL AFBi through decreasing pro-inflammatory cytokines.Further,to determine the signal pathway,real-time PCR and western blot were used to detect the expression of TLR4,and we found that AFB1 improved TLR4 mRNA and protein levels,and increased p-p38 and p-p65 respectively.Inhibition of the TLR4 expression by TLR4-specific RNA attenuated the decrease of apoptosis and the release of inflammatory cytokines induced by AFB1-It is concluded that AFB1 induce macrophage apoptosis and immunotoxicity through increasing oxidative stress and activating TLR4-NF?B signal pathway.Experiment 4:Selenium supplementation mitigates AFB1-induced immunotoxicity in porcine 3D4/21 cells and mechanism researchLittle research has been done about the signal pathway how Se affects AFB1-The present study was conducted to investigate the potential mechanism in porcine 3D4/21 cells.The 3D4/21 cells were treated by various concentrations of different Se forms and AFB1.MTT results showed that organic selenium did not have toxic effect on macrophage in a specified concentration.Flow cytometry showed that SeMet could mitigate ROS levels and apoptosis levels induced by AFB1-Besides,pro-inflammatory cytokines in AFB1-treated group,such as IL-1? and TNF-?,were decrease after SeMet supplementation.AFB1 concentration in 0.1 and 0.5 ?g/mL significantly down-regulated the SelS mRNA levels and protein levels in cells,and SeMet addition could block this inhibition.Furthermore,knockdown of SelS by SelS-specific siRNA had no effects on cell ability,IL-1?,TNF-? and ROS,however SelS-specific siRNA diminished the protective effects of SeMet supplementation against AFB1-induced immunotoxicity,by increasing apoptosis.ROS levels and pro-inflammatory cytokines.At the same time,SelS knockdown enhanced AFB1-induced p38 and p65 phosphorylation.It is concluded that SeMet diminishes AFB1-induced immunotoxicity through increasing SelS expression in order to improving antioxidant ability and inhibiting TLR4-NF?B signal pathway in porcine macrophage.Experiment 5:SelS overexpression decreased the apoptosis levels of 3D4/21 cellsIn the study,the pcDNA-SelS was transfected into 3D4/21cell,and screened by G418.After the stable cell lines had been screened,the cell lines would be grouped as the pcDNA-SelS group,the pcDNA3-1 group and the control group.Then both RT-PCR technique and western blot would be used to check the expression of SelS.After treatment,the protein expression levels of SelS was analyzed,and then the rate of apoptosis,the generation of ROS were detected.Combined with the results,overexpression of SelS protein has protective effect against AFB1-induced immunotoxicity,characterized by the decrease in the apoptosis,inhibition on the production of pro-inflammatory cytokines and ROS.It can be concluded that SelS can attenuate AFB1-induced immunotoxicity on 3D4/21 cells through suppressing oxidative stress and reducing the phosphorylation of TLR4-NF?B. |
PDF Full Text Request