2,2’-偶氮二(2-脒基丙烷)二盐酸盐诱导的氧化应激对猪肠上皮细胞活力及线粒体功能的影响

Effects of 2,2’⁃Azobis(2⁃Amidinopropane)Dihydrochloride⁃Induced Oxidative Stress on Cell Viability and Mitochondrial Function of Porcine Intestinal Epithelial Cells

本试验旨在评估2,2’-偶氮二(2-脒基丙烷)二盐酸盐(AAPH)诱导的氧化应激对猪肠上皮细胞(IPEC⁃J2细胞)活力及线粒体功能的影响,以期建立一种稳定的体外研究肠道氧化应激的模型。以不同浓度(0、30、32、34 mmol/L)AAPH处理IPEC⁃J2细胞24 h后,检测细胞活力、细胞增殖、细胞内总活性氧(ROS)含量、线粒体膜电位、线粒体DNA(mtDNA)拷贝数以及线粒体功能相关基因[线粒体转录因子A(TFAM)、线粒体转录因子B1(TFB1M)、线粒体转录因子B2(TFB2M)、解耦连蛋白1(UCP1)、解耦连蛋白2(UCP2)、解耦连蛋白3(UCP3)]表达的变化。结果表明:AAPH对IPEC⁃J2细胞活力的影响呈剂量及作用时间依赖关系。与0 mmol/L AAPH处理相比,不同浓度(30、32、34 mmol/L)AAPH处理均显著抑制了IPEC⁃J2细胞增殖(P<0.05),细胞活力显著下降(P<0.05),细胞内总ROS含量显著升高(P<0.05),细胞线粒体功能相关基因(TFAM、TFB1M、TFB2M、UCP1、UCP3)表达显著下调(P<0.05),mtDNA拷贝数显著降低(P<0.05)。综上所述,AAPH诱导的氧化应激通过增加IPEC⁃J2细胞内总ROS含量引起细胞线粒体功能损伤,进而导致细胞活力下降,抑制细胞增殖。

The aim of this study was to evaluate the effects of 2,2’⁃azobis(2⁃amidinopropane)dihydrochlo⁃ride(AAPH)⁃induced oxidative stress on cell viability and mitochondrial function of porcine intestinal epitheli⁃al cells(IPEC⁃J2 cells),and to establish a stable in vitro oxidative damage model.IPEC⁃J2 cells were treated with different concentrations(0,30,32 and 34 mmol/L)of AAPH for 24 h,the cell viability,cell prolifera⁃tion,intracellular total reactive oxygen species(ROS)content,mitochondria membrane potential,mitochon⁃drial DNA(mtDNA)copy number and the expression of mitochondrial function⁃related genes[mitochondrial transcription factor A(TFAM),mitochondrial transcription factor B1(TFB1M),mitochondrial transcription factor B2(TFB2M),uncoupling protein 1(UCP1),uncoupling protein 2(UCP2)and uncoupling protein 3(UCP3)]were measured.The results showed that AAPH had a dosage and time dependent relationship on the viability of IPEC⁃J2 cells.Compared with the 0 mmol/L AAPH treatment,different concentrations(30,32 and 34 mmol/L)of AAPH treatment significantly inhibited the proliferation of IPEC⁃J2 cells(P<0.05),the cell viability significantly decreased(P<0.05),the intracellular total ROS content significantly increased(P<0.05),the mRNA expression of mitochondrial function⁃related genes(TFAM,TFB1M,TFB2M,UCP1 and UCP3)significantly down⁃regulated(P<0.05),and the copy number of mtDNA significantly decreased(P<0.05).In summary,AAPH⁃induced oxidative damage leads to cell mitochondria dysfunction,resulting in de⁃crease of cell viability and inhibited cell proliferation of IPEC⁃J2 cells.