摘要
本试验旨在探讨β-伴大豆球蛋白通过上调NOD样受体蛋白-3(NLRP⁃3)表达诱导猪小肠上皮细胞(IPEC⁃J2)焦亡的作用机制。利用慢病毒转染IPEC⁃J2沉默目的基因NLRP⁃3,采用实时荧光定量PCR(RT⁃qPCR)和蛋白质印迹法(Western blot)验证目的基因沉默效果。慢病毒转染成功沉默目的基因NLRP⁃3后,将样本分为4组:A组为对照组(未转染),B组为阴性对照组(转染慢病毒空载体),C组为干扰组(转染携带NLRP⁃3干扰片段的慢病毒载体),D组为β-伴大豆球蛋白调节组(转染携带NLRP⁃3干扰片段的慢病毒载体+10 mg/mL的β-伴大豆球蛋白)。利用细胞增殖毒性检测试剂盒检测细胞活性;酶联免疫吸附试验(ELISA)检测半胱氨酸天冬氨酸蛋白酶-1(Caspase⁃1)和白细胞介素-1β(IL⁃1β)含量;脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)观察细胞阳性变化;透射电镜观察细胞形态;RT⁃qPCR和Western blot检测NLRP⁃3、凋亡相关斑点样蛋白(ASC)、Caspase⁃1、IL⁃1β和消皮素D(GSDMD)的mRNA相对表达量及蛋白表达水平。结果表明:1)与A组相比,B组的NLRP⁃3 mRNA相对表达量及蛋白表达水平无显著差异(P>0.05)。C组细胞NLRP⁃3 mRNA相对表达量及蛋白表达水平均极显著低于A组和B组(P<0.01)。2)与A组相比,C组细胞Caspase⁃1含量显著降低(P<0.05),IL⁃1β含量极显著降低(P<0.01),NLRP⁃3、ASC、Caspase⁃1、IL⁃1β、GSDMD mRNA相对表达量和蛋白表达水平显著降低(P<0.05)。3)与B组相比,C组细胞Caspase⁃1和IL⁃1β含量极显著降低(P<0.01),NLRP⁃3、ASC、Caspase⁃1、IL⁃1β、GSDMD mRNA相对表达量和蛋白表达水平显著降低(P<0.05)。4)与C组相比,D组细胞活性极显著降低(P<0.01),IL⁃1β和Caspase⁃1含量极显著升高(P<0.01),TUNEL染色显示细胞呈阳性表达,透射电镜观察可见D组细胞肿胀、细胞膜破裂、胞浆内容物流出,线粒体变性肿胀,NLRP⁃3、ASC、Caspase⁃1、IL⁃1β和GSDMD mRNA相对表达量和蛋白表达水平显著升高(P<0.05)。综上所述,β-伴大豆球蛋白上调NLRP⁃3表达,并通过NLRP⁃3/Caspase⁃1/GSDMD信号通路介导IPEC⁃J2细胞焦亡。
This study was conducted to investigate the mechanism of β-conglycinin(7 S)up-regulating NODlike receptor protein-3(NLRP-3)to cause porcine small intestinal epithelial cells(IPEC-J2)pyroptosis. Lentivirus was used to transfect IPEC-J2 to silencing the target gene NLRP-3,and the silencing effect of the target gene was verified by real-time quantitative PCR and Western blot. After successfully silencing the target NLRP-3 gene by lentivirus transfection,the samples were divided into four groups:group A was control group(nontransfected),group B was negative control group(transfected with lentivirus empty vector),group C was interference group(transfected with lentivirus vector carrying NLRP-3 interference fragment),group D was β-conglycinin regulation group(transfected with lentivirus vector carrying NLRP-3 interference fragment +10 mg/mLβ-conglycinin). CCK-8 was used to detect cell viability;enzyme linked immunosorbent assay was used to detect Caspase-1 and interleukin-1β (IL-1β)content,terminal-deoxynucleotidyl transferase mediated nick end labeling method was used to observe cell positive changes,and transmission electron microscopy was used to observe cell morphology,real-time quantitative PCR and western blot were used to detect the relative mRNA expression and protein expression levels of NLRP-3,apoptosis-associated speck-like protein containing a CARD(ASC),Caspase-1,IL-1β and gasdermin D(GSDMD). Results showed as follows:1)compared with group A,NLRP-3 mRNA relative expression level and protein expression level in the group B were no significant difference(P>0.05). NLRP-3 mRNA relative expression level and protein expression level in the group C was extremely significantly lower than that in group A and group B(P<0.01). 2)Compared with group A,the contents of Caspase-1 in group C was extremely significantly decreased(P<0.01),the contents of IL-1βin group C was significantly decreased(P<0.05),NLRP-3,ASC,Caspase-1,IL-1β and GSDMD mRNA relative expression levels and protein expression level in group C were significantly decreased(P <0.05). 3)Compared with group B,IL-1βand Caspase-1 contents in group C were extremely significantly decreased(P<0.05),NLRP-3,ASC,Caspase-1,IL-1β,and GSDMD mRNA relative expression level and protein expression level in group C were significantly decreased(P<0.05). 4)Compared with group C,cell activity in group D was significantly decreased(P<0.01),IL-1βand Caspase-1 contents in group D were significantly increased(P<0.01);TUNEL staining showed positive expression in group D;observation of the transmission electron microscope showed cells swelling,cell membrane rupture,cytoplasmic content flow out,mitochondrial degeneration and swellinging in group D;NLRP-3,ASC,Caspase-1,IL-1β and GSDMD mRNA relative expression levels and protein expression levels in group D were significantly increased(P<0.05). In conclusion, β-conglycinin up-regulates the expression of NLRP-3 and mediates the pyrolysis of IPEC-J2 through the NLRP-3/Caspase-1/GSDMD signaling pathway.
作者
王蕾
孙智峰
刘羽佳
李思婷
谢维娜
单兴根
田朋
杨悦
丁红研
李玉
王希春
吴金节
WANG Lei;SUN Zhifeng;LIU Yujia;LI Siting;XIE Weina;SHAN Xinggen;TIAN Peng;YANG Yue;DING Hongyan;LI Yu;WANG Xichun;WU Jinjie(College of Animal Science and Technology,Anhui Agricultural University,Hefei 230061,China)
出处
《动物营养学报》
CAS
CSCD
北大核心
2021年第7期4057-4067,共11页
CHINESE JOURNAL OF ANIMAL NUTRITION
基金
国家自然科学基金(31972750)。
