抑制Caspase-1活化能减弱玉米赤霉烯酮诱导的猪小肠上皮细胞凋亡

Inhibiting Caspase-1 activation could attenuate zearalenone-induced apoptosis of porcine small intestine epithelial cells

为探究玉米赤霉烯酮(ZEA)对猪小肠上皮细胞(IPEC-J2)毒性机制中细胞焦亡与凋亡间的关系,用不同质量浓度的ZEA(0、5、10、15μg/m L)处理细胞24 h以及ZEA(15μg/m L)与乙酰半胱氨酸(NAC,1 mmol/L)或Z-YVAD-FMK(10μmol/L)联合作用24 h。采用CCK-8法检测存活率;微板法测定细胞上清中乳酸脱氢酶(LDH)含量水平;免疫印迹检测剪切的半胱天冬氨酸蛋白酶-1(Cleaved Caspase-1)蛋白水平;实时荧光定量PCR(qRT-PCR)和ELISA测定白细胞介素(IL)-18和IL-1β水平;流式细胞术检测活性氧(ROS)水平和细胞凋亡率;免疫荧光检测Caspase-3表达水平。结果显示,ZEA引起IPEC-J2细胞存活率降低和LDH含量升高,呈剂量依赖性(P<0.05,P<0.01)。与0μg/m L组比较,ZEA处理后细胞中剪切的Caspase-1、IL-18和IL-1β水平均明显升高(P<0.05,P<0.01)。NAC孵育显著降低了ZEA处理细胞的ROS水平、剪切的Caspase-1蛋白表达以及IL-18和IL-1β含量,较ZEA组差异显著(P<0.01)。与ZEA组相比,Z-YVAD-FMK+ZEA组细胞存活率明显升高(P<0.01),而LDH含量、Caspase-3表达以及细胞凋亡水平则显著降低(P<0.01)。结果表明,活化的Caspase-1参与了ZEA诱导的IPEC-J2细胞凋亡。

The purpose of this experiment was to explore the relation between pyroptosis and apoptosis on cytotoxicity of zearalenone(ZEA) on porcine small intestine epithelial(IPEC-J2) cells.Cells were treated with different concentrations of ZEA(0,5,10,15 μg/m L) for 24 h and ZEA(15 μg/m L) in combination with acetylcysteine(NAC,1 mmol/L) or Z-YVAD-FMK(10 μmol/L) for 24 h. The CCK-8 method was used to determine cell viability.Microplate test was adopted to detect lactate dehydrogenase(LDH) content from culture supernatant.Immunoblotting was performed to detect the cleaved Caspase-1 protein level.The levels of interleukin(IL)-18 and IL-1β were measured by real-time fluorescence quantitative PCR(q RTPCR) and enzyme-linked immunosorbent assay(ELISA).Reactive oxygen specie(ROS) level and apoptosis ratio were assayed with flow cytometry. The expression of Caspase-3 was analyzed by fluorescent immunohistochemistry.The data showed that ZEA exposure reduced cell viability and increased LDH content in a dosedependent manner in IPEC-J2 cells(P<0.05,P<0.01).The increased Cleaved Caspase-1 protein expression and the IL-18 and IL-1β levels were observed in ZEA-treated cells,being markedly different from0 μg/m L group(P<0.05,P<0.01).Incubation with NAC markedly decreased the ROS level,Cleaved Caspase-1 protein expression,as well as IL-18 and IL-1β contents in ZEA-treated cells,which had significant difference compared with ZEA group(P<0.01).Compared to the ZEA group,the cell viability in Z-YVADFMK+ZEA group was significantly increased(P<0.01),while LDH content,Caspase-3 expression,and apoptosis were notably decreased(P<0.01).The results indicate that Caspase-1 induction is involved in ZEAinduced apoptosis of IPEC-J2 cells.