乙酰乙酸通过抑制Nrf2诱导奶牛乳腺上皮细胞线粒体损伤

ACAC-Induced Mitochondrial Damage in Bovine Mammary Epithelial Cells by Inhibiting the Nrf2

为探究乙酰乙酸(Acetoacetic acid,ACAC)是否通过抑制核转录因子NF-E2相关因子2(Nuclear factor E2-reate d factor2,Nrf2)导致奶牛乳腺上皮细胞线粒体损伤。在奶牛乳腺上皮细胞系(MAC-T)中添加0、0.6、1.2或1.8 mmol·L-1ACAC处理24 h;用10μmol·L-1的萝卜硫素(Sulforaphane,SFN)预处理MAC-T 24 h后,加入1.2 mmol·L-1ACAC处理24 h。流式细胞术检测活性氧(Reactive oxygen species,ROS);采用JC-1染色检测线粒体膜电位变化情况;通过q-PCR测定线粒体氧化磷酸化复合物Ⅰ-Ⅴ(Oxidative phosphorylation complexesⅠ-Ⅴ,COⅠ-Ⅴ)的基因表达情况以及线粒体生物合成调节因子Nrf2、过氧化物酶体增殖体激活受体γ-γ活化剂1-α(Peroxlsome proliferator-activated re ceptor-γ coac tlvator-1α,PGC-1α)、线粒体转录因子A (Mitoc hondrial transcription factor A,TFA M)、核呼吸因子-1 (Nuclear respiratory fac tor-1,Nrf1)、线粒体DNA(Mitochondrial DNA,mt DNA);使用试剂盒检测腺嘌呤核苷三磷酸(A denosine triphosphate,ATP)含量。结果显示,ACAC处理后,ROS的含量显著升高(P<0.05),而线粒体膜电位显著下降(P<0.05),并且COⅠ、COⅡ、COⅢ、COⅣ和COⅤ的m RNA表达水平显著降低(P<0.05),PGC-1α、TFAM、Nrf1以及Nrf2的m RNA表达量显著下降(P<0.05),mtDNA含量显著降低(P<0.05),ATP含量显著降低(P<0.05),而SFN预处理缓解了上述影响。结果表明,ACAC通过抑制Nrf2信号通路诱导了MAC-T细胞线粒体损伤。

To investigate whether acetoacetic acid(ACAC)induced mitochondrial damage of bovine mammary epithelial cells by inhibiting the nuclear transcription factor NF-E2-related factor 2(Nrf2).Bovine mammary epithelial cells(MAC-T)were treated with 0,0.6,1.2 or 1.8 mmol·L^(-1) ACAC for 24 h;MAC-T cells were pretreated with 10μmol·L^(-1) SFN for 24 h and then treated with 1.2 mmol·L^(-1) ACAC for 24 h.Flow cytometry was used to detect mitochondrial reactive oxygen species(ROS);JC-1 staining was used to detect mitochondrial membrane potential changes;the gene expression of mitochondrial oxidative phosphorylation complexⅠ-Ⅴ(COⅠ-Ⅴ)was determined by q-PCR,as well as the mitochondrial biosynthesis regulator Nrf2,peroxisome proliferator-activated receptorγ-γactivator 1-α(PGC-1α),mitochondrial transcription factor A(TFAM),Nuclear respirator-1(Nrf1),and mitochondrial DNA;the adenine nucleoside triphosphate(ATP)content was detected by the kit.The results showed that after ACAC treatment,the content of ROS was significantly increased(P<0.05),while the mitochondrial membrane potential decreased significantly(P<0.05),and the mRNA expression level of COⅠ,COⅡ,COⅢ,COⅣand COⅤwere significantly decreased(P<0.05),the mRNA expression of PGC-1α,TFAM,Nrf1 and Nrf2 were decreased(P<0.05),and the mtDNA content was decreased(P<0.05),the ATP content was decreased(P<0.05),while SFN pretreatment alleviated the above effects.The results showed that ACAC induced mitochondrial damage in MAC-T cells by inhibiting the Nrf2 signaling pathway.