&beta;-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

doi:10.3969/j.issn.1006-267x.2021.07.047

2021 , Vol. 33 >Issue 7: 4057 - 4067

DOI: https://doi.org/10.3969/j.issn.1006-267x.2021.07.047

MOLECULAR AND CELLULAR NUTRITION

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

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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.

Key words： β-coglycinin; NLRP-3; lentivirus; IPEC-J2; pyroptosis

Cite this article

WANG Lei , SUN Zhifeng , LIU Yujia , LI Siting , XIE Weina , SHAN Xinggen , TIAN Peng , YANG Yue , DING Hongyan , LI Yu , WANG Xichun , WU Jinjie . β-Conglycinin Induced Porcine Intestinal Epithelial Cell Pyroptosis via Up-Regulating Expression of NOD-Like Receptor Protein 3[J]. Chinese Journal of Animal Nutrition, 2021 , 33(7) : 4057 -4067 . DOI: 10.3969/j.issn.1006-267x.2021.07.047

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