分子与细胞营养 MOLECULAR AND CELLULAR NUTRITION

β-伴大豆球蛋白通过上调NOD样受体蛋白-3表达诱导猪小肠上皮细胞焦亡

  • 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
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  • 安徽农业大学动物科技学院, 合肥 230061
王蕾(1986-),女,河南桐柏人,硕士研究生,从事畜禽营养代谢病研究。E-mail:350939708@qq.com

收稿日期: 2020-12-31

  网络出版日期: 2021-07-06

基金资助

国家自然科学基金(31972750)

β-Conglycinin Induced Porcine Intestinal Epithelial Cell Pyroptosis via Up-Regulating Expression of NOD-Like Receptor Protein 3

  • WANG Lei ,
  • SUN Zhifeng ,
  • LIU Yujia ,
  • LI Siting ,
  • XIE Weina ,
  • SHAN Xinggen ,
  • TIAN Peng ,
  • YANG Yue ,
  • DING Hongyan ,
  • LI Yu ,
  • WANG Xichun ,
  • WU Jinjie
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  • College of Animal Science and Technology, Anhui Agricultural University, Hefei 230061, China

Received date: 2020-12-31

  Online published: 2021-07-06

Supported by

 

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、ASCCaspase-1、IL-1βGSDMD 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、ASCCaspase-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、ASCCaspase-1、IL-1βGSDMD 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。

本文引用格式

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 . β-伴大豆球蛋白通过上调NOD样受体蛋白-3表达诱导猪小肠上皮细胞焦亡[J]. 动物营养学报, 2021 , 33(7) : 4057 -4067 . DOI: 10.3969/j.issn.1006-267x.2021.07.047

Abstract

This study was conducted to investigate the mechanism of β-conglycinin (7S) up-regulating NOD-like 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 (non-transfected), 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/mL β-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-1β in 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-1β and 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-1β and 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.

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