| Mycotoxin is the toxic metabolite which is produced by mold and can casue extensive harm and serious pollution.Deoxynivale(DON)is one of the most common mycotoxins,which poses a serious threat to the health of dairy cows and the development of dairy industry.Milk is mainly synthesized in mammary epithelial cells.At present,the biological toxicity of DON on mammary epithelial cells mainly focuses on cell proliferation,inflammatory response and oxidative stress,but the effect of DON on the synthesis of milk components and lactation function remains unclear.Therefore,on the one side,bovine mammary epithelial cell(MAC-T)was used as material in this study,and q RT-PCR,Western blot,immunofluorescence,ELISA,flow cytometry and other technologies were used to study the toxic effect and potential mechanism of DON on MAC-T cells,RNA-seq technology was used to study the effect of DON on MAC-T cell transcriptome;On the other side,Kunming mice were used as model animals to reveal the effects of DON on breast tissue and lactation function.The main results of this study are as follows:1.Toxic effects of DON on cell proliferation,tight junction and milk composition synthesis of bovine mammary epithelial cells(1)Effects of DON on the viability and apoptosis of MAC-T cellsAfter MAC-T was treated with 1,5,10 μM DON for 24,48 and 72 hours,CCK-8 results showed that except for the 1 μM in 24 h treatment group,the cell viability of DON treatment group decreased significantly in a dose-dependent manner.The results of cell count also showed that DON significantly reduced the number of MAC-T cells.Apoptosis results showed that different concentrations of DON could induce early apoptosis of MAC-T cells in a time and dose-dependent manner.(2)Effects of DON on cytoskeleton of MAC-T cellsThe microfilaments(F-actin)and microtubules(α-tubulin)of MAC-T were detected by immunofluorescence.The results of staining showed that the microfilaments in the control group were arranged in order and the microtubule structures were normal,but after DON treatment,the cell shrunk,the arrangement of microfilaments was in disorder,the microtubule structure became abnormal and thickened significantly around the nucleus.(3)Effects of DON on cell permeability and tight junction of MAC-T cellsThe results of lactate dehydrogenase viability showed that the activity of lactate dehydrogenase increased significantly after 5 and 10 μM DON treatment for 24 and 72 h on MAC-T cells,and showed a significant dose-dependent relationship.The results of transmembrane resistance showed that the transmembrane resistance decreased significantly after DON treatment with different concentrations for 6 h;after 24 h,the 1,5,and 10 μM DON treatment groups decreased by 14.14%,14.64% and 16.97%,respectively.The results of q RT-PCR showed that the expression levels of Claudin-3 and Claudin-4 genes were significantly decreased after 5 μM DON treatment for 24,48 and 72 h.(4)Effects of DON on milk fat synthesis and related genes of MAC-T cellsThe lipids of MAC-T cells were stained with Bodipy.The results showed that after DON treatment on MAC-T cells,the mean fluorescence intensity decreased in a time and dose-dependent manner,and the fluorescence intensity was the weakest after 10 μM DON treatment for 72 h,which was 44.64% of the group.q RT-PCR results showed that the expression of LEPTIN gene was significantly increased after 10 μM DON treatment,while the expression of FASN and ACSS2 genes was significantly decreased after 5 μM DON treatment for 48 and 72 h.(5)Effects of DON on milk protein synthesis and the related pathway proteins in MAC-T cellsThe synthesis of casein in MAC-T cells was detected by ELISA.The results showed that after MAC-T cells were treated with different concentrations of DON for 24,48 and 72 h,α-casein was significantly decreased except for 5 μM DON treatment for 24 h;β-casein was significantly decreased after 5 μM DON treatment.κ-casein decreased significantly after 10 μM DON treatment.Western blot(WB)results showed that the total protein levels of JAK2,STAT5,m TOR and 4EBP1 did not change significantly after 5 μM DON treatment in MAC-T cells for 24 h,but the phosphorylation levels were significantly reduced.(6)Effects of DON on transcriptome of MAC-T cellsThe RNA-seq results showed that after 5 μM DON treatment,1732 up-regulated genes and 2012 down-regulated genes were screened between the experimental group and the control group.The GO analysis results showed that the up-regulated differential genes were mainly enriched in functional annotations such as negative regulation of cell promotion,cellular response to growth factor stimulus and transcription factor activity,while the down-regulated genes were mainly enriched in lipid biological process,cellular modified amino acid metallic process,and structural construct construct of ribosome.The KEGG results showed that the differential genes were mainly enriched in ribosome,glutathione metabolism,steroid biosynthesis and other pathways.2.Toxic effects of DON on lactation function in Kunming mice(1)Effects of DON on lactation and the mammary gland morphology of miceKunming mice were gavaged with DON at the concentration of 2 mg/kg/d from the first day after delivery.The results showed that the lactation of female mice decreased significantly on the 5th and 7th day after delivery,while the lactation of the treatment group started to increase on the 9th day.HE staining results showed that on the 7th day after delivery,the structure of mammary gland in the control group was normal without deformation and inflammatory cell infiltration,while in the experimental group,mammary gland alveolar became smaller,interlobular stroma became thicker.On the 21 st after deliverary,the volume of mammary alveolar in the control group became smaller,while the interlobular stroma of mammary gland in the experimental group was further thickened and the number of erythrocytes increased.(2)Effects of DON on lactation related hormones of miceELISA results showed that the estrogen and progesterone levels in serum decreased significantly at the 7th day postpartum after DON treatment;Prolactin level decreased significantly at the 21 st day;The growth hormone level increased significantly at the 7th day.(3)Effects of DON on mammary gland apoptosis and the tight junction protein in miceTUNEL staining was used to stain the mammary glands of mice in the experimental group and the control group.The results showed that the apoptotic cells of mammary glands in the DON treatment group increased significantly on the 7th and 21 st day after delivery.The WB results showed that the expression of cleaved-Caspase-3 and Caspase-3 protein increased significantly and the expression of Bcl-2 protein decreased significantly after DON treatment.The results showed that the mean fluorescence intensity of Occludin protein in mammary gland decreased significantly after DON treatment.In conclusion,DON can significantly reduce the viability of MAC-T cells,induce apoptosis,destroy the cytoskeleton,destroy the cell membrane and tight junction,inhibit the expression of milk fat synthesis and related genes,and inhibit the synthesis of milk protein and the phosphorylation of related signal pathway proteins.In addition,DON can reduce lactation in mice,cause damage to structure of mammary gland,affect the level of hormones related to lactation,and induce the apoptosis of mammary gland in mice.This study can lay a theoretical foundation for clarifying the toxic effect of mycotoxin on lactation and ring an alarm for reducing mold pollution. |
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