摘要
【目的】探究黄曲霉毒素B_(1)(aflatoxin B_(1),AFB_(1))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AFB_(1)致病机制提供理论依据。【方法】用0、20、40、60、80和100μg/mL AFB_(1)刺激细胞12 h,使用CCK-8试剂盒检测细胞存活率,以确定AFB_(1)对IPEC-J2细胞的毒性作用;使用0、10、20、30μg/mL AFB_(1)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AFB_(1)对细胞内凋亡因子表达的影响情况。【结果】0~20μg/mL AFB_(1)对细胞活性无显著影响(P>0.05),但随着AFB_(1)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AFB_(1)浓度呈高度依赖性,且随着AFB_(1)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AFB_(1)浓度为20μg/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AFB_(1)浓度为30μg/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AFB_(1)单独处理组相比,使用ROS抑制剂NAC预处理细胞后加入AFB_(1),凋亡因子呈现出不同程度的显著下降趋势(P<0.05)。【结论】AFB_(1)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。
【Objective】To investigate the effect of aflatoxin B_(1)(AFB_(1))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 AFB_(1)at concentrations of 0,20,40,60,80 and 100μg/mL for 12 h.CCK-8 assay kit was used to detect cell viability and determine the toxic effect of AFB_(1)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 30μg/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 AFB_(1)on the expression of apoptotic factors in cells was detected.【Result】The results showed that AFB_(1)at concentrations of 0-20μg/mL had no significant effect on cell viability(P>0.05),and as the concentration of AFB_(1)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 AFB_(1)concentration,and as the concentration of AFB_(1)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 AFB_(1)was 20μg/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 AFB_(1)was 30μg/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 ce
作者
张曼
王艳慧
李寒笑
刘恩赐
李庆豪
于瑞
罗琴
金鑫
ZHANG Man;WANG Yanhui;LI Hanxiao;LIU Enci;LI Qinghao;YU Rui;LUO Qin;JIN Xin(College of Veterinary Medicine,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China;College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China)
出处
《中国畜牧兽医》
CAS
CSCD
北大核心
2024年第9期3857-3866,共10页
China Animal Husbandry & Veterinary Medicine
基金
河南省自然科学基金(232300421271)
博士后科研经费(M4080004)
河南省科技攻关项目(242102110068)。
关键词
黄曲霉毒素B_(1)
活性氧
细胞凋亡
猪空肠上皮细胞
aflatoxins B_(1)
reactive oxygen species
cell apoptosis
porcine jejunum epithelial cells
