Effect Of Polydatin To Relieve The Injure Of Bovine Mammary Epithelial Cells Induced By Zearalenone

Zearalenone(ZEA)is one of the common mycotoxins in feed,and has the effects of estrogen,reproductive toxicity,genotoxicity and immunotoxicity.Polydatin(PD)is the main active ingredient of Polygonum cuspidatum,which has antioxidant,anti-inflammatory and anti-tumor activity.Bovine mammary epithelial cells of lactating cows play an important role in milk quality.Whether ZEA has a damaging effect on bovine mammary epithelial cells,and whether PD can alleviate the damage of ZEA on bovine mammary epithelial cells is unknown.Therefore,to study the damage of ZEA to bovine mammary epithelial cells and the mitigation mechanism of PD will lay a theoretical foundation for the feed industry to alleviate the toxicity of ZEA.In this study,bovine mammary epithelial cells(MAC-T)was used as the research object to design an in vitro test,and the appropriate concentration of ZEA was used to treat MAC-T cells.Detection of reactive oxygen species(ROS),mitochondrial membrane potential,endoplasmic reticulum(ER)stress and apoptosis,to explore the role and mechanism of PD alleviating ZEA-induced MAC-T cells.First determine the effect of ZEA on the growth of MAC-T cells.MAC-T cells were treated with different concentrations of ZEA(0,7.5,15,30,60,90,120 and 240 ?M)for 24 h to detect changes in cell viability and lactate dehydrogenase(LDH).The results showed that compared with the control group,with the increase of ZEA concentration,MAC-T cell viability showed significant or extremely significant inhibition(P<0.01 or P<0.001).The results indicate that ZEA has a toxic effect on MAC-T cells,and the concentration of 30 ?M and above reaches a very significant difference,and 30 ?M ZEA was selected for follow-up study.The results of detecting ROS levels and changes in mitochondrial membrane potential indicate that compared with the control group,the ROS of the ZEA-treated group is significantly increased and the mitochondrial membrane potential is significantly reduced.Further study the mechanism of ZEA damage MAC-T cells.The MAC-T cells were treated with 30?M ZEA for 24 h,and the mRNA expression levels of the ER stress related gene glucose-regulated protein 78(GRP78),activating transcription factor 6(ATF6),activating transcription factor 4(ATF4),and apoptosis signal-regulating kinase 1(ASK1)and the C/EBP homologous protein(CHOP)were detected.Hoechst33342 and AnnexinV-FITC/PI double staining were used to detect the apoptosis,the activity level of Caspase-3 and the expression changes of B-cell lymphoma/leukemia-2 gene(Bcl-2)and Bcl-2 associated X protein(BAX).The results showed that compared with the control group,the mRNA expressions of GRP78,ATF6,ATF4,ASK1,and CHOP genes were up-regulated in the ZEA group;the apoptosis rate and BAX gene expression were significantly increased(P<0.01);Caspase-3 activity was significantly increased(P<0.01);Bcl-2 gene expression was down-regulated(P<0.001).Finally,this study used PD to relieve ZEA's damage to MAC-T cells.According to the damage effect of ZEA on MAC-T cells,we looked for a mitigation method from the perspective of feed additives.MAC-T cells were treated with 5,10 and 20 ?M PD for 24 h to detect cell viability.The results showed that compared with the control group,5,10,and 20 ?M PD had no significant difference in cell viability.PD and 30?M ZEA were combined to determine cell viability.The results showed that 5?M PD had a significant effect on cell viability recovery.Therefore,5?M PD was selected for follow-up study.PD detection of ZEA-induced MAC-T cell oxidative damage,ER stress and apoptosis.The results showed that compared with the ZEA group,the PD+ZEA group had significantly decreased malondialdehyde(MDA)(P<0.05),and increased glutathione(GSH)and total superoxide dismutase(T-SOD)(P<0.01);mRNA expressions of GRP78,ATF6,ATF4 and CHOP genes were all down-regulated;apoptosis rate and BAX gene expression were significantly reduced(P<0.01);gene Bcl-2 expression was up-regulated(P<0.05).In summary,the results of this study indicate that ZEA induces MAC-T cell damage,causing ER stress and apoptosis.PD can reduce the damage of MAC-T cells by reducing ER stress and apoptosis.