| Two experiments were conducted in this research to investigate the toxic effects of Ochratoxin A (OTA) on IPEC-J2 cells and the protective effect of antioxidant selenium (sodium selenite source) on the cells exposured to OTA.Exp.1 Impacts of OTA on the Growth and Antioxidant System Mediated by Nrf2 in IPEC-J2 CellsTwo trials were conducted to determine the effects of OTA exposed concentration and time on the survival rate of IPEC-J2 cells, and investigate the impacts and mechanism of OTA on the cells'redox balance.Firstly, a 3×9 factorial design was carried out to study the survival rate with 0,0.05, 0.1,0.2,0.4,0.8,1,1.5,2μM/L of OTA incubated with IPEC-J2 cells for 12h,24h and 48h respectively.There were 6 replicates for each treatment. The results show that OTA reduced the survival rate of IPEC-J2 by time-dose dependent manner. Concentration and time for OTA incubation, and their interaction all had significant effects on cell survival rate (P<0.01).Based on the above results, IPEC-J2 cells were cultured 24h with OTA of 0,0.1,0.5 and 1μM/L, to study the effects of different concentration of OTA on structure and functional integrity and redox state-related indexes in IPEC-J2 cells, and preliminary studied the effect of OTA on Nrf2 activity. The results showed that:1, LDH activity in medium increased linearly or quadraticly with the increasing concentration of OTA (P<0.01), whereas Na+-K+-ATPase activity of the cells linearly or quadraticly decreased (P<0.01);2, T-AOC,GSH content and activities of GPx, GST and GR of cells significantly linearly or quadraticly decreased with increaseing concentration of OTA (P<0.01). MDA content in the culture medium significantly linearly or quadraticlu increased (P<0.01). CAT and SOD activities were not affected significantly.3, mRNA expression of GSTA2, GSTO1, TrxRl, GR, GCLC and GCLM were significantly linearly or quadraticly decreased with the OTA concentration increasing (P <0.01). No significant differences were observed in GPx2. 4, With the OTA concentration increasing, Nrf2 activity first increased and then decreased gradually.The results indicated that OTA brought toxic effect to IPEC-J2 cells by time-dose dependent way and the inhibition of Nrf2-mediated antioxidant defense might be the mechanism of OTA toxicity effects.Exp.2 Protective Effects of Selenium (Sodium Selenite Source) on Antioxidant System of IPEC-J2 Cells Exposured to OTAIn this experiment, three trials were carried out to study the protective effect of selenium (sodium selenite source) on IPEC-J2 cells which were subjected to oxidative stress induced by OTA, and further investigated whether selenium could activate Nrf2 system to mitigate oxidative stress.The first trial was to investigate the effect of selenium on survival and proliferation of IPEC-J2 cells incubated and non-incubated with OTA. The results showed that selenium could improve the survival rate of cells incubated with OTA.The second trial was desiged to investigate whether selenium (sodium selenite source) could improve the antioxidant capacity of IPEC-J2 cells exposed to OTA. There were four treatments, pretreated with 0,0,4,8μM/L sodium selenite for 12h, and then the first treatment was incubated without OTA for 24h, the other three treatments were cultured with OTA (1μM/L) for 24h, represented the PBS/ethanol group, PBS/OTA group, SS4/OTA group, and SS8/OTA group respectively. LDH activity in medium, cells Na+-K+-ATPase activity, cell redox state, cell antioxidant system-associated indicators and mRNA expression of antioxidant enzymes were measured. The results showed that:1, Compared with the PBS/OTA group, pretreatment with selenium significantly reduced the LDH activity in medium(P<0.05) and significantly increased Na+-K+-ATPase activity of cells exposured to OTA(P<0.05). And SS8/OTA group was better than SS4/OTA group.2, Pretreatment with selenium trended to improve T-AOC in IPEC-J2 cells and in the SS8/OTA group reached a significant level (P<0.05). Meanwhile, compared with PBS/OTA group, pretreatment with selenium(SS4/OTA and SS8/OTA group) significantly reduced the MDA content, increased cells' GPx, GST and GR activities (P<0.05) and trended to elevate SOD activity, but the GSH content was significantly lower than those of the PBS/ethanol group and PBS/OTA group (P< 0.05).3, With selenium concentration increased in medium, mRNA relative expression of GCLC, GLCM, GPx2, GSTO1, GSTA2, TrxR1 and GR gene trended to increase, GCLC and TrxR1 even approached or exceeded those of PBS/ethanol group.Following the two trials results, a 2×3 factorial design was conducted to study the effect of cells pretreated with three concentrations of selenium (0,4,8μM/L) for 12h, and then replaced with OTA (0, 1μM/L) medium for 24h on the activation of Nrf2. The results showed that selenium added to medium reliefed the inhibition of Nrf2 in IPEC-J2 cells exposed to OTA.In summary, this research results indicated that OTA destroyed redox balance of IPEC-J2 cells, and the inhibition of Nrf2 transactivation and further inhibition of Nrf2 downstream antioxidant genes'expression were possible mechanism of its cytotoxic effects. Pretreated with selenium could protect the cells from toxic effects of OTA by promoting Nrf2/ARE system's activation.
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