Research On Apoptosis Mechanism On Combined Effects Of Deoxynivalenol And Zearalenone In CTLL-2 Cells

Deoxynivalenol (Deoxynivalenol, DON) and Zearalenone (Zearalenone, ZEA) are the two most common dietary mycotoxins. ZEA and DON may not only reduce the nutritional value of the feed resulting in the slow growth and decreased reproductive performance of animals but also has immunosuppressive effects that can lead to an obvious decrease in the resistance of disease in livestock. Currently, people do not fully understand the immunotoxicity and its mechanism of ZEA and DON. In addition, under natural conditions, ZEA and DON often co-exist in feed. The research in the joint immune toxicity effect and its mechanism caused by ZEA and DON are instructive to the prevention and control of poisoning of them, especiall provides the gist for the limited standards of the both mycotoxins in feed. To reveal ZEA, DON immunotoxicity and molecular mechanism as well as their joint action, this study used CTLL-2 cells (cytotoxic T lymphocyte cell line) as the material, carried out toxicity test with different concentrations of ZEA or/and DON using CCk-8 law, ELISA, flow cytometry using Annexin V/P idual stainning and Wsstern blot techniques to detect the cytotoxicity,immune*related cytokine secretion, changes in apoptosis rate and the expression of apoptosis-related signaling proteins and other possible changes. The specific test and results are as follows:1.Virulent effect of DON, ZEA on the CTLL-2 cells and the determination of joint concentration. After exposing different concentrations of ZEA (0,2.5,5,10,20,40μg/mL), DON (0,0.25,0.5,1,2,4μg/mL) for 48 h, We investigated the effects of ZEA or DON on the growth of CTLL-2 cells using the CCK-8 assay. The results showed that ZEA or DON at higher concentration significantly inhibited the growth of CTLL-2 cells. The 0.25 μg/mL of DON group had no obvious difference while the group with more than 0.5 μg/mL of DON had significant difference compared to the control group (P< 0.01); The group of ZEA had significant difference or extremely significant difference, compared to control group (P< 0.05 or P< 0.01); The cell viability of the group with 0.25 μg/mL DON+2.5 μg/mL ZEA was significantly inhibited compared to control group (P< 0.01). According to the results of CCK-8 test and the relevant literature, we set the concentration of 0.5 μg/mL DON+5 μg/mL ZEA as the best suitable poisoning concentration.2.Effects of ZEA or/and DON on the cytokine production of CTLL-2cells. After exposing the CTLL-2 to different concentrations of ZEA (0,2.5,5,10,20,40μg/mL), DON (0, 0.25,0.5,1,2,4 μg/mL), ZEA and DON (Con,5 μg/mL ZEA,0.5 μg/mL DON,5 μg/mL ZEA and 0.5 μg/mL DON) for 48 h. We investigated the concentration of cells’supernatant GZMB, PFP, IFN-y, TNF-a of CTLL-2 cells using the ELISA. The results showed that ZEA, DON can inhibit the expression of the PFP, GZMB, IFN-y in the cells and cells’supernatant of CTLL-2, which showed the dose effect. With the increasing concentration of ZEA and DON, the concentration of TNF-a also increased, which showed a does effect. The combined toxicity of ZEA and DON showed a synergy effect.3.Effects of ZEA or/and DON on apoptosis in CTLL-2 cells. After exposing the CTLL-2 to different concentrations of ZEA (0,2.5,5,10,20,40 μg/mL), DON (0,0.25,0.5,1,2,4 μg/mL), ZEA and DON (Con,5 μg/mL ZEA,0.5 μg/mL DON,5 μg/mL ZEA and 0.5 μg/mL DON) for 48 h, We investigated the effects of ZEA or/and DON on the apoptotic rat of CTLL-2 cells using Annexin V/PI dual stainin; CTLL-2 cells were treated with 20 μg/mL of ZEA and 2 μg/mL of DON for 48 h, followed by DAPI staining. The apoptosis rate significantly increased with the increasing concentration of ZEA and DON compared with the control group (P< 0.05 or P< 0.01). The combined toxicity of ZEA and DON showed a synergy effect. The DAPI staining showed the results that the cells in treated groups had changes during apoptosis, including the condensation of chromatin and the formation of membrane-embedded apoptotic bodies.4.Research on apoptosis mechanism on combined effects of deoxynivalenol and zearalenone in CTLL-2 cells. After exposing CTLL-2 to different concentrations of ZEA (0,2.5, 5,10,20,40μg/mL), DON (0,0.25,0.5,1,2,4 μg/mL), ZEA and DON (Con,5μg/mL ZEA,0.5 μg/mL DON,5 μg/mL ZEA and 0.5 μg/mL DON) for 48 h, We investigated the effects of ZEA or/and DON on the expression of channel protein related to cell apoptosis using Wsetern blot, and investigated the effects of ZEA or DON treated with anti-FasL on the apoptotic rat of CTLL-2 cells using Annexin V/PI dual staining. The results showed that the expression of p53 was significantly elevated compared to control group(P< 0.05 or P< 0.01). The ratio of the Bax/Bcl-2 was increased with a dose effect. The combined toxicity of ZEA and DON showed the synergy effect. Meanwhile, the Western blot results showed that ZEA or DON promoted the release of cytochrome c into the cytosol and triggered mitochondria-mediated apoptosis. Consequently, the expression of caspase-9 and caspase-3 were activated, which resulted in a significant difference or extremely significant difference compared to control group (P< 0.05 or P< 0.01). The expression of FasL, Fas, Cleaved-Caspase8 were significantly elevated with the increasing concentration of ZEA and DON compared to control group (P< 0.05 or P< 0.01). The apoptotic rat of CTLL-2 cells were significantly reduced in the group with ZEA or DON treated with anti-FasL compared to ZEA or DON group (P< 0.01). The activation of p38 MAPK、JNK、Erk were significantly elevated, as a dose effect relationship. In the combined ZEA and DON group,the protein expression of Cleaved-Caspase 3, Cleaved-Caspase 8, Cleaved-Caspase 9 significantly increased (P< 0.01) compared to ZEA or DON group. The combined toxicity of ZEA and DON showed the synergy effect.In summary, ZEA or DON has the toxic effect on the CTLL-2 cells. They can inhibit the proliferation of CTLL-2 cells and the expression of related cytokines as well as reduce their lethality. The ZEA or DON is able to induce the apoptosis in CTLL-2 cells from the death receptor and affects the immune function of animal through activating the pathway of mitochondria and MAPKs. The combined toxicity of ZEA and DON showed the synergy effect.