| Zearalenone(ZEA)is a common non-steroidal estrogen mycotoxin present in crops and is one of the most common pollutants.Lipopolysaccharide(LPS),as a common endotoxin,is a component of the outer cell wall of Gram-negative bacteria.These two toxins have an impact on human and animal health.In the process of animal feeding,ZEA as an exotoxin,LPS as endotoxin,both of which may exist in the body.At present,only the harm of single toxin is studied,and there are few studies on the interaction between ZEA and LPS.Therefore,it is necessary to explore the combined effect of different concentrations of ZEA and LPS.As a natural flavonoid,quercetin(QUE)has strong biological characteristics such as anti-oxidation,anti-inflammation and anti-disease.It is not clear whether QUE can alleviate the combined effect of ZEA and LPS.Therefore,in this study,MAC-T and IPEC-J2 cells were used as research models to clarify the effect of QUE on alleviating the combined effect of ZEA and LPS on cell damage,so as to provide theoretical support for QUE as a new feed additive.Study 1:To clarify the combined effect of ZEA and LPS on MAC-T cell injury.Cell viability,lactate dehydrogenase(LDH),reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD),glutathione(GSH),mitochondrial membrane potential(MMP),Nrf2 signaling pathway and apoptosis were detected.The results showed that compared with the control group,the cell viability was decreased when the LPS concentration was 1 μg/m L.1 μg/m L LPS was combined with different concentrations of ZEA(5,15 and 30 μM).The results showed that the combination of ZEA and LPS reduced cell viability,increased LDH level,ROS production and MDA content,decreased SOD and GSH content,and decreased MMP.At the gene level,compared with the control group,the ZEA+LPS group down-regulated the expression of Nrf2,HO-1,NQO1,Keap1 and SOD2 genes,up-regulated the expression of GRP78,CHOP and ATF6 genes,decreased the expression of Nrf2,HO-1 and NQO1 proteins,and increased the expression of GRP78,CHOP and ATF6 proteins.The results of apoptosis detection showed that compared with the control group,the combination of ZEA and LPS increased the apoptosis rate,increased the expression of Bax and Caspase3,and decreased the expression of Bcl-2protein.The above studies have confirmed that the coexistence of different concentrations of ZEA and LPS can cause damage to MAC-T cells.Study 2: It is clear that QUE can alleviate the combined effect of ZEA and LPS on MAC-T cell injury.The results showed that compared with the control group,pretreatment for 1h,60-200 μM QUE had an effect on cell viability,and the addition of 60 μM QUE reduced the LDH content increased by ZEA and LPS.Subsequently,30 μM ZEA and 1 μg/m L LPS were added after pretreatment with 60 μM QUE.The results showed that compared with ZEA+LPS group,QUE pretreatment decreased MDA content and increased SOD and GSH content.Compared with the ZEA+LPS group,the combination of QUE addition up-regulated the expression of Nrf2 and HO-1 proteins,and decreased the expression of GRP78 and CHOP proteins.The results of flow cytometry showed that the addition of QUE decreased the apoptosis rate of ZEA+LPS group.Compared with ZEA+LPS group,60 μM QUE significantly increased the expression of Bcl-2 protein and decreased the expression of Bax protein.The results of metabolomics showed that the changes of metabolites were mainly manifested in protein digestion and absorption and amino acid metabolism.This study confirmed that QUE can effectively alleviate the damage caused by the coexistence of ZEA and LPS on MAC-T cells.Study 3:It is clear that QUE can alleviate the combined effect of ZEA and LPS on IPEC-J2 cell injury.IPEC-J2 cells were treated with different concentrations of ZEA(10,20,30,40,60,80 and 100 μM),LPS(1,10,50 and 100 μg/m L)and QUE(20,40,60,80,100 and 200 μM)for 24 h.The results showed that compared with the control group,the cell viability decreased gradually with the increase of ZEA concentration,and 20 μM ZEA significantly decreased the cell viability.With the increase of LPS concentration,the cell viability decreased gradually,and when the LPS concentration was 1μg/m L,it decreased significantly.For QUE,20 μM increased cell viability,while 40-200 μM QUE decreased cell viability.Therefore,in the subsequent study,20 μM QUE was combined with 20 μM ZEA and 1 μg/m L LPS.The results showed that in IPEC-J2 cells,compared with ZEA+LPS group,the combination of QUE addition increased cell viability and decreased LDH content.At the gene level,compared with the ZEA+LPS group,the combination of QUE addition up-regulated the gene expression of Nrf2,NQO1 and SOD2,and increased the protein expression of Nrf2,HO-1 and NQO1.The determination of tight junction related genes and proteins showed that compared with the ZEA+LPS group,the combination of QUE increased the expression of Claudin,ZO-1 and Occludin genes and proteins.Compared with the ZEA+LPS group,the combination of QUE addition reduced the apoptosis rate.The expression of Bcl-2 and Bax was detected at the gene level.Compared with the ZEA+LPS group,the combination of QUE significantly reduced Bax gene expression,but there was no significant difference in Bcl-2 gene expression.QUE can also alleviate the combined effects of ZEA and LPS on IPEC-J2 cells.In summary,the results of this study show that the combined effect of different concentrations of ZEA and LPS on MAC-T and IPEC-J2 cell injury.To clarify the effect of QUE on inhibiting cell injury induced by ZEA and LPS by activating Nrf2 signaling pathway. |
PDF Full Text Request