撒坝猪源E. coli高致病性毒力岛通过NLRP3/ASC/Caspase-1途径诱导IPEC-J2细胞焦亡

doi:10.11843/j.issn.0366-6964.2023.12.031

摘要/Abstract

摘要： 旨在研究撒坝猪源E. coli高致病性毒力岛(highly pathogenic virulence island, HPI)诱导IPEC-J2细胞焦亡的机制,本研究以LPS+ATP为阳性对照,将实验室前期筛选的E.coli HPI及通过CRISPR/Cas9基因敲除技术构建完成的E. coliΔHPI感染IPEC-J2细胞,观察细胞损伤情况;RT-qPCR测定各组作用0.5、3、6、9、12 h后细胞内焦亡通路中关键因子NOD样受体家族热蛋白结构域相关蛋白3 (NOD-like receptor thermal protein domain associated protein 3, NLRP3)、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a CARD, ASC)、胱天蛋白酶-1 (caspase-1)、消皮素D (gasderminD, GSDMD)及炎性因子白介素-18 (IL-18)、白介素-1β (IL-1β)的mRNA转录水平;激光共聚焦观察NLRP3/Caspase-1炎性复合体组装及表达情况;Western blot测定GSDMD及其活化形式GSDMD-N蛋白表达水平,PI染色检测胞膜完整性。结果表明:LPS+ATP处理显著促进细胞焦亡的发生,阴性对照组细胞正常生长、轮廓清晰,E. coli感染组及LPS+ATP组细胞出现变形、脱落、边界模糊等明显细胞病变效应(cytopathic effect, CPE),且HE染色显示,E.coliHPI感染相较于E. coli ΔHPI感染对细胞的损伤更严重;E. coli HPI组中的焦亡通路关键因子及炎性因子mRNA水平在3~9 h均显著(P<0.05)或极显著(P<0.01)高于E. coli ΔHPI组;NLRP3/Caspase-1炎性复合体在细胞质中发生组装,且E. coli HPI组NLRP3/Caspase-1蛋白荧光强度、GSDMD、GSDMD-N蛋白表达水平及PI染色阳性率均极显著(P<0.01)高于E. coli ΔHPI组;E. coli HPI组的CPE、HE染色、焦亡通路关键因子及炎性因子mRNA水平、GSDMD及GSDMD-N蛋白表达量、NLRP3/Caspase-1共定位情况均与LPS+ATP组相似。结果提示,撒坝猪源E.coliHPI通过上调NLRP3/ASC/Caspase-1信号通路中关键因子mRNA水平,诱导NLRP3/Caspase-1炎性复合体的组装,促进焦亡标志蛋白GSDMD及GSDMD-N的表达,并对IPEC-J2细胞膜进行打孔,从而诱导细胞发生焦亡。

关键词: 大肠杆菌, 高致病性毒力岛, 焦亡, 猪小肠上皮细胞, NLRP3/ASC/Caspase-1通路

Abstract: To study the mechanism of pyroptosis induced by highly pathogenic virulence island (HPI) of E. coli from Saba pig in IPEC-J2 cells, The E. coli HPI screened in the laboratory and the E. coli ΔHPI constructed by CRISPR/Cas9 gene knockout technology were used to infect IPEC-J2 cells to observe cell damage, LPS+ATP was used as a positive control. RT-qPCR was used to determine the mRNA transcription levels of key factors in the pyroptosis pathway, including NOD-like receptor thermal protein domain associated protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, Gasdermin D (GSDMD), and inflammatory factors interleukin-18 (IL-18) and interleukin-1β (IL-1β) at 0.5, 3, 6, 9, and 12 h after treatment in each group. Laser confocal microscopy was used to observe the assembly and expression of NLRP3/Caspase-1 inflammasome complex. Western blot was used to determine the expression levels of GSDMD and its activated form GSDMD-N protein. PI staining was used to detect membrane integrity. The results showed that LPS+ATP treatment significantly promoted the occurrence of pyroptosis. The cells in the negative control group grew normally with clear contours. The cells in the E. coli infection group and LPS+ATP group showed obvious cytopathic effects (CPE) such as deformation, detachment, and blurred boundaries. HE staining showed that E. coli HPI infection caused more severe cell damage than E. coli ΔHPI infection. The mRNA levels of key factors and inflammatory factors in the pyroptosis pathway in the E.coli HPI group were significantly (P<0.05) or extremely significantly (P<0.01) higher than those in the E. coli ΔHPI group at 3-9 h. NLRP3/Caspase-1 inflammasome complex was assembled in the cytoplasm, and the protein fluorescence intensity of NLRP3/Caspase-1, the expression levels of GSDMD and GSDMD-N protein, and the positive rate of PI staining in the E. coli HPI group were extremely significantly (P<0.01) higher than those in the E. coli ΔHPI group. The CPE, HE staining, mRNA levels of key factors and inflammatory factors in the pyroptosis pathway, expression levels of GSDMD and GSDMD-N protein, and co-localization of NLRP3/Caspase-1 in the E.coliHPI group were similar to those in the LPS+ATP group. The results suggest that E. coli HPI from Saba pig induces pyroptosis in IPEC-J2 cells by upregulating the mRNA levels of key factors in the NLRP3/ASC/Caspase-1 signaling pathway, inducing the assembly of NLRP3/Caspase-1 inflammasome complex, promoting the expression of pyroptosis marker proteins GSDMD and GSDMD-N, and punching holes in the cell membrane of IPEC-J2 cells.

Key words: Escherichia coli, highly pathogenic virulence island, pyroptosis, porcine small intestinal epithelial cells, NLRP3/ASC/Caspase-1 pathway

中图分类号:

S852.3

肖金龙, 王浩, 万全, 沈珏, 张博, 赵维薇, 邓静, 王喜, 赵汝, 肖鹏, 高洪. 撒坝猪源E. coli高致病性毒力岛通过NLRP3/ASC/Caspase-1途径诱导IPEC-J2细胞焦亡[J]. 畜牧兽医学报, 2023, 54(12): 5218-5227.

XIAO Jinlong, WANG Hao, WAN Quan, SHEN Jue, ZHANG Bo, ZHAO Weiwei, DENG Jing, WANG Xi, ZHAO Ru, XIAO Peng, GAO Hong. Induction of IPEC-J2 Pyroptosis by E.coli HPI from Saba Pig via NLRP3/ASC/Caspase-1 Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5218-5227.

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