Effect Of FB₁ On Proliferation And Apoptosis Of Porcine Intestinal Epithelial Cells

The pig industry plays a pivotal role in the domestic breeding industry in China.In the process of breeding,external stimulation often causes gastrointestinal discomfort of pigs and the production efficiency was reduced.Mycotoxins are a kind of fungal metabolites which are widely polluted.They are mainly distributed in crops and food products.Improper transportation and storage of feed often causes contamination of mycotoxin.When the contaminated feed is ingested by animals,a series of toxic reactions will occur in their body.Fumonisins are a kind of mycotoxins produced by Fusarium moniliforme and Fusarium rotarum,which is commonly found in moldy wheat and corn.Among them,FB₁ is the most toxic and the most widespread pollution.After the moldy feed is eaten by animals,it can act on the respiratory system,urinary system,digestive system and immune system,and produce toxic effects.Intestinal tract is the first organ to be exposed to food derived external stimulation.Previous studies have confirmed that FB₁ can cause the destruction of tight junction of intestinal cells in pigs,but it is not clear whether FB₁ can cause apoptosis of intestinal cells in pigs.Therefore,in this study,porcine small intestinal epithelial cells cultured in vitro were used as the research object to explore the effects of FB₁ on the proliferation and apoptosis of porcine intestinal epithelial cells（IPEC-J2）,so as to provide a theoretical basis for the study of the pathogenic mechanism of FB₁ on intestinal tract.In this study,MTT assay was applied to detect the viability of IPEC-J2 cells exposed to FB₁,the morphology alteration of IPEC-J2 cells were observed under microscope after HE staining.The effect of FB₁ on the proliferation of IPEC-J2 cells was measured by Ed U method,Real-time PCR was used to detect the m RNA expression levels of PCNA,P21,P27,CDK2,CDK4,Cyclin D1 and Cyclin E1.JC-1 staining was used to detect the changes of mitochondrial membrane potential of IPEC-J2 cells induced by FB₁,and the number of apoptosis cells was measured by Flow cytometry.Real-time PCR and Western-blot were used to assay the relative expression levels of apoptosis-related factors Caspase3,Caspase9,Bax and Bcl-2.The results showed that the cell viability of IPEC-J2 cells decreased significantly in the20μg/mL FB₁ group after 48 h treatment,especially in the 40μg/mL FB₁ group.Morphologi-cal observation of cells showed that the morphology and number of cells did not change significantly after the treatment of 0-40μg/mL FB₁ for 24 h.However,after the treatment of48 h,compared with the control group,the number of cells in the exposed group shrunk,decreased and the floating cells increased significantly.The results of Ed U proliferation test showed that the fluorescence intensity was significantly decreased with the increase of FB₁,indicating that FB₁ could inhibit the proliferation of IPEC-J2 cells.Real-time PCR results showed that FB₁ could increase the relative expression of P21 and P27 m RNA in IPEC-J2cells.The m RNA relative expression of PCNA,CDK2,CDK4,Cyclin D1 and Cyclin E1 were significantly inhibited under the FB₁ expose.After FB₁ was treated with IPEC-J2 cells for 48h,the results of JC-1 staining showed that the red fluorescence intensity decreased with the increase of FB₁,while the green fluorescence intensity showed an increasing trend,indicating that the mitochondrial membrane potential of IPEC-J2 cells decreased after FB₁ expose.Meanwhile,Flow cytometry showed that FB₁ could increase the number of apoptotic cells after 48 h treatment,and the relative expressions of Caspase3,Caspase9 and Bax were increased,while the expression of Bcl-2 m RNA was decreased.In summary,FB₁inhibited the cell cycle of IPEC-J2,affected the expression of cyclin-related factors,and inhibited cell proliferation.At the same time,FB₁ significantly reduced the mitochondrial membrane potential of cells,up-regulated the expression of pro-apoptotic genes and down-regulated the expression of anti-apoptotic genes.FB₁ induced cell apoptosis and promoting the injury of IPEC-J2.