| T-2 toxin(T2)is the most toxic of A-type single ended sporonene toxin and widely distributed in the world.If it enters the production chain of feed production,transportation and processing,it will cause great harm to the health of animals and threaten the development of animal husbandry.Studies have shown that T2 can induce oxidative stress which is the important mechanism of cell damage.Nrf2-ARE signaling pathway is a regulatory mechanism protecting cells against oxidative stress stimulation,and nuclear factor E2 related factor 2(Nrf2)is a key factor in combating oxidative stress.At present,no study has explored whether Nrf2 has antioxidant effects and related mechanisms on the effects of oxidative stress induced by T2 on broilers.Therefore,the study aimed to explore the antioxidant mechanism of broilers derived Nrf2 against T2.The main research content is as follows:1.To investigate the toxic effect of T2 on Douglas Foster-1(DF-1)cells and its oxidative stress response,oxidative stress model of DF-1 cells was constructed by T2 exploring the effect of different concentrations of T2 on DF-1.We found that T2 decreased markedly cell viabilities,enhanced significanly LDH leakage ratio,ROS level and MDA concentration,and declined GSH concentration,indicating oxidative stress was induced by T2 in DF-1 cells.Moreover,T2 induced up-regulation of intracellular antioxidative enzymes activities and the corresponding mRNA expression levels,which showed that the oxidative stress process was regulated by the Nrf2-ARE signaling pathway and initiated downstream antioxidative enzyme lines(Gpx-1、Nqo1、Hmox1)involved in the cellular antioxidative effect against T2.2.Nrf2 proteins overexpressing and knockdown DF-1 cell lines were constructed.DF-1cell lines stably overexpressing and knocking down Nrf2 expression by lentiviral transduction system and sh RNA interference was established,and the efficiency of overexpression and knockdown of mRNAs and proteins was assessed by Real-time PCR and Western blot.MTT assay was performed to assess the cell viability of the constructs mentioned above.The results showed that the mRNA and protein levels of Nrf2 in the constructed Nrf2 overexpression DF-1 cell line were significantly increased,while the mRNA and protein levels of Nrf2 in the constructed Nrf2 knockdown DF-1 cell line were significantly decreased.Furthemore,compared with the control DF-1 cell,the viabilities of the constructed cell lines were no significantly different.3.To verify Nrf2-AREsignaling pathway is an important pathway that mediates DF-1cells against oxidative stress,normal DF-1 cells,Nrf2 overexpressing and knockdown DF-1cells were treated with 50 n M of T2 for 24 h,and then the expression of oxidative stress-related indicators and downstream antioxidant factors of the Nrf2-ARE signaling pathway were detected.The results showed that overexpression of Nrf2 protein inhibited the level of oxidative stress induced by T2 and promoted the expression of antioxidant factors which were the downstream of the Nrf2-ARE signaling pathway;Knockdown with Nrf2 potentiated the oxidative stress induced by T2 and suppressed the expression of antioxidant factors which were the downstream of the Nrf2-ARE signaling pathway in DF-1 cells.It was demonstrated that the Nrf2-ARE signaling pathway regulated oxidative stress induced by T2 via modulating the expression of downstream antioxidant factors.In summary,this study revealed the toxic effects and oxidative damage caused by T2 in DF-1 cells,and we had constructed the oxidative stress model of DF-1 cells;Moreover,Through up-regulating and down-regulating of Nrf2,we verified thatthe Nrf2-ARE signaling pathway played an important role in the antioxidant stress excitation in DF-1 cells,providing a new idea for the subsequent treatment of oxidative stress-related diseases in livestock. |
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