ROS积聚在黄曲霉毒素B1诱导仔猪空肠上皮细胞凋亡中的作用

doi:10.16431/j.cnki.1671-7236.2024.09.015

摘要/Abstract

摘要： 【目的】探究黄曲霉毒素B₁(aflatoxin B₁,AFB₁)诱导细胞内活性氧(ROS)的产生对猪空肠上皮细胞(IPEC-J2)凋亡的影响，为AFB₁致病机制提供理论依据。【方法】用0、20、40、60、80和100 μg/mL AFB₁刺激细胞12 h，使用CCK-8试剂盒检测细胞存活率，以确定AFB₁对IPEC-J2细胞的毒性作用；使用0、10、20、30 μg/mL AFB₁刺激细胞后采用ROS免疫荧光分析法检测细胞内ROS产生情况；使用实时荧光定量PCR和Western blotting方法检测细胞内凋亡通路相关的关键因子mRNA和蛋白表达情况，并采用ROS抑制剂N-乙酰-半胱氨酸(N-acetyl-L-cysteine,NAC)处理细胞后检测AFB₁对细胞内凋亡因子表达的影响情况。【结果】 0～20 μg/mL AFB₁对细胞活性无显著影响(P＞0.05)，但随着AFB₁浓度升高，与空白对照组相比，IPEC-J2细胞活性显著或极显著下降(P＜0.05；P＜0.01)。ROS免疫荧光分析结果显示，ROS含量与AFB₁浓度呈高度依赖性，且随着AFB₁浓度的升高，与空白对照组相比，细胞内ROS含量显著或极显著增加(P＜0.05；P＜0.01)。实时荧光定量PCR和Western blotting结果显示，当AFB₁浓度为20 μg/mL时Caspase-3 mRNA和蛋白表达量均显著高于空白对照组(P＜0.05)；当AFB₁浓度为30 μg/mL时Caspase-3和Bax/Bcl-2 mRNA和蛋白表达量显著高于空白对照组(P＜0.05)，而Bax和Bcl-2蛋白表达量显著低于空白对照组(P＜0.05)；与AFB₁单独处理组相比，使用ROS抑制剂NAC预处理细胞后加入AFB₁，凋亡因子呈现出不同程度的显著下降趋势(P＜0.05)。【结论】 AFB₁可促进IPEC-J2细胞凋亡因子Bax、Bcl-2和Caspase-3的上调，并且这一过程与细胞内ROS的积累有关。

关键词: 黄曲霉毒素B₁; 活性氧; 细胞凋亡; 猪空肠上皮细胞

Abstract: 【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 AFB_1.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.

Key words: aflatoxins B₁; reactive oxygen species; cell apoptosis; porcine jejunum epithelial cells

中图分类号:

S852.4⁺4

张曼, 王艳慧, 李寒笑, 刘恩赐, 李庆豪, 于瑞, 罗琴, 金鑫. ROS积聚在黄曲霉毒素B₁诱导仔猪空肠上皮细胞凋亡中的作用[J]. 中国畜牧兽医, 2024, 51(9): 3857-3866.

HANG Man, WANG Yanhui, LI Hanxiao, LIU Enci, LI Qinghao, YU Rui, LUO Qin, JIN Xin. Role of ROS Accumulation in Apoptosis of Jejunal Epithelial Cells Induced by Aflatoxin B₁ in Piglets[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(9): 3857-3866.

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ROS积聚在黄曲霉毒素B₁诱导仔猪空肠上皮细胞凋亡中的作用

Role of ROS Accumulation in Apoptosis of Jejunal Epithelial Cells Induced by Aflatoxin B₁ in Piglets

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