Study Of Fumonisin B₁ On Autophagy And Related Pathways Of Swine Umbilical Vein Endothelial Cells

Fumonisin（FB）is one of the main mycotoxins widely present in crop products and animal feeds,and poses a great threat to the large-scale development of the breeding industry,animal food safety and human health.Fumonisin B₁is one of the most toxic fumonisins,which is related to many diseases of humans and animals,such as swine pulmonary edema,equine leukomalacia,human neural cast defects,etc.,but its mechanism of action is not clear.Based on the in vitro culture of swine umbilical vein endothelial cells（SUVEC）,the aim of this study is to explores the effect of FB₁on SUVEC cell autophagy and its related pathways,and provides a theoretical basis for FB₁toxic mechanism of pulmonary edema in pigs.In this study,SUVEC cells were cultured in vitro to study the effect of different concentrations of FB₁on autophagy.The MTT method was used to detect the effects of different concentrations of FB₁on cell viability for different times.The cell migration ability was detected by scratch experiment and used the transmission electron microscope to observe the production of autophagosomes in the cells.The q PCR and Western Blot techniques were used to detect the expression of related genes and proteins in autophagy and PI3K/AKT/mTOR and AMPK/mTOR pathways,then construct p EGFP-N1-FLAG-LC3plasmid and transfect cells to detect changes in the number and position of LC3 aggregation.In addition,the autophagy inducer rapamycin（RAPA）and the autophagy inhibitor3-methyladenine（3-MA）were added to explore the relationship between autophagy and related pathways and its role in FB₁induced SUVEC cells damage.The results showed that 40μg/m L FB₁treatment for 24 h significantly reduced the survival rate of SUVEC cells,20μg/m L FB₁treatment for 48 h significantly reduced the cell survival rate,while different concentrations of FB₁treatment significantly inhibited cell survival rate after 72 h.FB₁was used for 24 h and 48 h after the scratch treatment,and then it was observed that the cells had a strong ability to migrate to the center of the scratch at 24h,and they were scattered at 48 h.Transmission electron microscopy showed that autophagosomes with a double-layer membrane structure appeared in the FB₁treatment group;the p EGFP-N1-FLAG-LC3 plasmid was constructed and transfected into the cells,the fluorescence intensity of LC3 in the FB₁treatment group gradually increased that compared with the control group under a fluorescence microscope,and gradually moved from the cytoplasm to the autophagosome membrane.Other results of q PCR and WB showed that the expression of autophagy-related genes ATG5 and Beclin 1 increased,and the ratio of LC3II/LC3I was increased,while the expression level of P62 decreased,indicating that a certain concentration of FB₁can affect the survival and migration of SUVEC cells and induce autophagy.The study of autophagy-related pathways after the action of FB₁found that the m RNA and protein expression levels of PI3K,AKT and mTOR decreased,and the PI3K/AKT/mTOR signaling pathway was inhibited;while the expression of AMPK increased,which inhibited the expression of mTOR,AMPK The/mTOR signaling pathway is activated.After pretreatment with the autophagy inducer RAPA for 3 h,compared with the FB1 treatment group alone,the cell survival rate was significantly increased,the expression of mTOR and P62 were significantly reduced,and the level of LC3II/LC3I was increased;pretreatment with the autophagy inhibitor 3-MA6 After h,compared with the FB1treatment group alone,the cell survival rate of the inhibitor pretreatment group decreased significantly,the expression levels of PI3K and LC3II/LC3I decreased,and the expression of P62 increased.The above results indicate that FB₁may induce autophagy through PI3K/AKT/mTOR and AMPK/mTOR signaling pathways,and autophagy may play a protective role in FB₁-induced SUVEC cell damage.