The Role Of MTOR-regulated Autophagy In FB₁-induced Swine Umbilical Vein Endothelial Cells Injury

Fumonisins are secondary metabolites produced in grains by pathogenic fungi such as Fusarium spp.,which mainly pollute food,soil and water in the environment and cause adverse effects on ecosystems.Among them,fumonisin B₁（FB₁）is the most widely distributed and the most toxic.It has species-specific toxic effects.FB₁ can cause pulmonary edema in pigs,but its toxicity mechanism is still in the exploratory stage.m TOR is an important regulator of cellular metabolism and plays a key role in maintaining the balance between cellular anabolism and catabolism.In this study,swine umbilical vein endothelial cells（SUVECs）were used as the research object to explore the regulatory effect of m TOR on the autophagy of SUVECs induced by FB₁,as well as the effect of autophagy on FB₁-induced cell damage.Provide a research basis for further prevention and control of the harm caused by fumonisins.In this experiment,SUVECs were cultured in vitro to establish a FB₁-infected model.High-throughput sequencing technology was used to detect gene expression and transcriptional regulation in the cells,and an interfering cell line stably expressing m TOR-sh RNA was constructed.And SUVECs were treated with m TOR inhibitor RAPA and m TOR agonist MHY1485,respectively.MDC staining,mitochondrial lysosome co-localization,MTT assay,scratch assay,EDU assay,TUNEL assay,DCFH-DA fluorescent probe assay,q PCR and Western Blot technology were used to observe the expression of autophagosome,mitophagy,cell survival,migration ability,cell proliferation,apoptosis,oxidative stress and related genes and proteins,so as to explore the regulation effect of m TOR on the autophagy of SUVECs induced by FB₁.The results of high-throughput sequencing showed that a total of 1367 differentially expressed genes were screened from SUVECs treated with 20μg/m L FB₁,including 962 up-regulated genes and 405 down-regulated genes.These EDGs are mainly enriched in GO functional items such as cellular processes,biogenesis and regulation,stimulus response,organelles,and KEGG signaling pathways such as PI3K-Akt signaling pathway,P53 signaling pathway,and Jak-STAT signaling pathway.Among them,the DEGs of autophagy-related signaling pathways were most enriched.This indicates that autophagy and autophagy-related pathways may be involved in the cytotoxicity of SUVECs induced by FB₁.In order to explore the role of mTOR in the cytotoxicity of SUVECs induced by FB₁,we constructed the p LKO.1-m TOR-sh RNA recombinant plasmid,and used lentiviral packaging technology to obtain m TOR-sh RNA interference cell lines with high m TOR interference efficiency and negative control NC-sh RNA cell lines.After treatment with 20μg/m L FB₁ for24 h,compared with the NC-sh RNA group,the expression of autophagy-related genes LC3,ATG5 and Beclin 1 in the m TOR-sh RNA group was up-regulated,the ratio of LC3II/LC3I increased,and the autophagy degradation substrate P62 was significantly down-regulated.After using MDC to specifically label autophagosomes,it was found that autophagosomes were significantly increased in the interference group compared with the FB₁ treatment group.The double fluorescence colocalization results of mitochondria and lysosomes showed that mitophagy was also significantly increased in the interference group.In contrast,the mTOR activation group showed a significant decrease in mitophagy compared with the FB₁ treatment group.These above results indicated that m TOR could inhibit the FB₁-induced autophagy of SUVECs,while the interference of m TOR could promote the increase of autophagy.To further explore the role of autophagy in FB₁-induced injury of SUVECs,NC-sh RNA and m TOR-sh RNA cells were treated with 20μg/m L FB1 for 24 h.And cell damage is then detected.The results showed that after the interference of m TOR,the cell viability and migration ability of SUVECs treated with FB₁ increased significantly.It’s indicating that autophagy can improve the repair ability of SUVECs after injury.Interfering with m TOR can reduce the blocking process of the cell cycle from G1 phase to S phase caused by FB₁,reduce the apoptosis caused by FB₁,and relieve the inhibition of FB₁ on inflammation-related factors to a certain extent.At the same time,the m TOR inhibitor RAPA was used to pretreat SUVECs.The results showed that inhibition of m TOR activity could partially restore cell proliferation inhibited by FB₁ and reduce oxidative stress.The above results indicated that m TOR-regulated autophagy played a certain protective role in the injury of SUVECs induced by FB₁.