脱氧雪腐镰刀菌烯醇诱导IPEC-J2细胞焦亡的内质网应激通路机制

Mechanism of endoplasmic reticulum stress pathway in IPEC-J2 cells induced pyroptosis by deoxynienol

[目的]本试验旨在研究脱氧雪腐镰刀菌烯(DON)诱导仔猪小肠上皮细胞(IPEC-J2)焦亡的分子机制。[方法]以500 ng·mL^(-1)DON和1μmol·mL^(-1)4-苯基丁酸(4-PBA,内质网应激抑制剂)单一及联合染毒IPEC-J2细胞24 h。采用CCK-8测细胞存活率;倒置显微镜观察细胞生长状况;透射电镜观察细胞超微结构;Western blot和RT-qPCR测内质网应激通路和细胞焦亡关键分子的基因相对表达水平和蛋白相对表达水平;流式细胞仪测定活性氧(ROS)含量。[结果]与对照组相比,DON组细胞缝隙变宽、细胞密度降低、超微结构被破坏,ROS、丙二醛(MDA)、白细胞介素1β(IL-1β)、白细胞介素18(IL-18)含量极显著升高(P<0.01),葡萄糖调节蛋白78(GRP78)、内质网核信号转导蛋白1(IRE1)、转录激活因子6(ATF6)、诱导消皮素D(GSDMD)和含半胱氨酸的天冬氨酸蛋白水解酶1(Caspase-1)mRNA相对表达水平极显著升高(P<0.01),GRP78、C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白(CHOP)、ATF6、IRE1、GSDMD和Caspase-1蛋白相对表达水平极显著升高(P<0.01),超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)的活性极显著下降(P<0.01)。与DON组相比,DON和4-PBA联合处理组的细胞数量增多,细胞缝隙变小,细胞器形态正常,ROS、MDA、IL-1β、IL-18含量以及ATF6、Caspase-1的mRNA相对表达水平极显著下降(P<0.01),GSDMD、GRP78和IRE1的mRNA相对表达水平显著下降(P<0.05),GRP78、CHOP、ATF6、IRE1、GSDMD和Caspase-1的蛋白相对表达水平极显著降低(P<0.01),SOD、GSH-Px活性极显著上升(P<0.01)。[结论]DON可通过激活内质网应激通路诱导IPEC-J2细胞焦亡。

[Objectives]The aim of this experiment was to explore the molecular mechanism of deoxynivalenol(DON)inducing pyroptosis in IPEC-J2 cells.[Methods]IPEC-J2 cells were treated with 500 ng·mL^(-1)DON and 1μmol·mL^(-1)4-phenylbutyrate acid(4-PBA,endoplasmic reticulum stress inhibitor)for 24 h.The cell viability was measured by Cell Counting Kit-8(CCK-8).The cell growth was observed by inverted microscope.The ultrastructure of cells was observed by transmission electron microscopy.Western blot(WB)and real-time fluorescent quantitative PCR(RT-qPCR)were used to measure the relative gene expression and protein expression levels of endoplasmic reticulum stress pathway and key molecules of pyroptosis.Reactive oxygen species(ROS)content was measured by flow cytometry.[Results]Compared with the control group,cell gap widened,cell density decreased,ultrastructure was destroyed,and ROS,malondialdehyde(MDA),interleukin-1β(IL-1β),interleukin-18(IL-18)contents significantly increased in DON group(P<0.01).The mRNA relative expression levels of glucose regulated protein 78(GRP78),endoplasmic reticulum(ER)to nucleus signalling 1(IRE1),activating transcription factor 6(ATF6),gasdermin D(GSDMD)and Cysteinyl aspartate specific proteinase 1(Caspase-1)significantly increased(P<0.01),and GRP78,C/EBP homologous protein(CHOP),ATF6,IRE1,GSDMD and Caspase-1 protein relative expression level significantly increased(P<0.01),while the activities of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and catalase(CAT)significantly decreased(P<0.01).Compared with DON group,DON and 4-PBA combined treatment group increased the number of cells,cell gap becomes smaller,normal organelle morphology;ROS,MDA,IL-1βand IL-18 contents and mRNA relative expression levels of ATF6 and Caspase-1 significantly decreased(P<0.01).The mRNA relative expression levels of GSDMD,GRP78 and IRE1 significantly decreased(P<0.05),GRP78,CHOP,ATF6,IRE1,GSDMD and Caspase-1 protein relative expression level significantly decreased(P<0.01),and the activities of SOD and GSH-Px significantly increased(P<0.01).[Conclusions]The experimental results indicate that DON can regulate pyroptosis in IPEC-J2 cells through endoplasmic reticulum stress pathway.