Role of ROS Accumulation in Apoptosis of Jejunal Epithelial Cells Induced by Aflatoxin B1 in Piglets
[Objective]To investigate the effect of aflatoxin B1(AFB1)on the apoptosis of porcine jejunum epithelial cells(IPEC-J2)by inducing intracellular reactive oxygen species(ROS)production,providing a theoretical basis for the pathogenic mechanism of AFB1.[Methods]Cells were stimulated with AFB1 at concentrations of 0,20,40,60,80 and 100 μg/mL for 12 h.CCK-8 assay kit was used to detect cell viability and determine the toxic effect of AFB1 on IPEC-J2 cells,and then immunofluorescence assay were used to detect intracellular ROS level after the cells were stimulated with 0,10,20 and 30 μg/mL AFB1.At the same time,Real-time quantitative PCR and Western blotting were used to detect the expression of key factors related to apoptotic pathways in cells.ROS inhibitor N-acetyl-L-cysteine(NAC)was used to stimulate cells,and then the effect of AFB1 on the expression of apoptotic factors in cells was detected.[Result]The results showed that AFB1 at concentrations of 0-20 μg/mL had no significant effect on cell viability(P＞0.05),and as the concentration of AFB1 increased,the viability of IPEC-J2 cells showed a significant or extremely significant decrease trend compared with blank control group(P＜0.05 or P＜0.01).ROS immunofluorescence analysis showed that ROS level was highly dependent on AFB1 concentration,and as the concentration of AFB1 increased,the intracellular ROS level was significantly or extremely significantly increased compared with blank control group(P＜0.05 or P＜0.01).Real-time quantitative PCR and Western blotting results showed that when the concentration of AFB1 was 20 μg/mL,the expression of Caspase-3 mRNA and protein were significantly higher than that of blank control group(P＜0.05).When the concentration of AFB1 was 30 μg/mL,the expression of Bax and Bax/Bcl-2 mRNA and protein were significantly higher than that of blank control group(P＜0.05),while the protein expressions of Bax and Bcl-2 were significantly lower than those of blank control group(P＜0.05).Pretreatment of cells with ROS inhibitor NAC followed by AFB1 addition resulted in a decrease in apoptotic factors to varying degrees compared with the AFB1-only treatment group(P＜0.05).[Conclusion]AFB1 could promote the upregulation of apoptotic factors Bax,Bcl-2,and Caspase-3 in IPEC-J2 cells,and this process might be related to the accumulation of intracellular reactive oxygen species.
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