Study on the mechanism of Fusobacterium nucleatum promoting the formation of neutrophil extracellular trap to aggravate colitis
10.3760/cma.j.cn311367-20240822-00318
- VernacularTitle:具核梭形杆菌促进中性粒细胞胞外诱捕网形成加重结肠炎的机制研究
- Author:
Liqing BAO
1
;
Zhiyue WANG
;
Yang LIU
;
Yunwei WEI
Author Information
1. 温州医科大学慈溪生物医药研究院，宁波 315302
- Publication Type:Journal Article
- Keywords:
Inflammatory bowel disease;
Fusobacterium nucleatum;
Neutrophil;
Neutrophil extracellular trap
- From:
Chinese Journal of Digestion
2025;45(3):189-198
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the mechanism of Fusobacterium nucleatum ( Fn) in regulating the formation of neutrophil extracellular trap (NET) to aggravate colitis. Methods:With a completely randomized design, 15 C57BL/6 mice were randomly divided into negative control group, dextran sulfate sodium (DSS) A group (DSS-induced colitis), and DSS+ Fn A group (DSS-induced colitis with Fn infection); another 15 C57BL/6 mice were randomly divided into DSS B group, DSS+ Fn B group, and GSK484 group (DSS-induced colitis with Fn infection and followed by intraperitoneal injection of peptidylarginine deiminase 4 (PAD4)inhibitor GSK484), with 5 mice in each group. The Fn-infected neutrophils (HL-60 cell) model and PAD4-inhibited cell model were established and divided into neutrophil-like control cell (induced with 1.25% dimethylsulfoxide), Fn+ neutrophil-like cell (neutrophil-like cells co-cultured with Fn), and GSK484 cell ( Fn+ neutrophil-like cell co-cultured with GSK484) with a completely randomized design. The neutrophil-like control and Fn+ neutrophil-like cells were divided into 2 batches to conduct experiments before and after PAD4 inhibition separately.The expression of zonula occludens-1 (ZO-1), neutrophil elastase (NE), reactive oxygen species, PAD4, and citrullinated histone H3(cit-H3) were analyzed by immunohistochemistry staining, Western blotting, and flow cytometry assay. Two-tailed t test was used for statistical analysis. Results:The results of immunohistochemical staining showed that compared with those of the DSS A group, the expression levels of NE, PAD4 and cit-H3 of the DSS+ Fn A group were upregulated, and the expression level of ZO-1 was downregulated; compared with those of the DSS+ Fn B group, the expression level of ZO-1 of the GSK484 group was upregulated, and the expression levels of cit-H3 and PAD4 were downregulated. The results of Western blotting demonstrated that, before the PAD4 inhibition, the expression levels of NE, PAD4 and cit-H3 of the Fn+ neutrophil-like cell were all higher than those of the neutrophil-like control cell (1.52±0.11 vs. 1.00±0.19, 1.21±0.06 vs. 1.00±0.07, 1.59±0.16 vs. 1.00±0.16), and the differences were statistically significant ( t=-4.13, -3.86, and -4.47; P=0.014, 0.014, and 0.018); after the PAD4 inhibition, the expression levels of PAD4, cit-H3, and NE of the GSK484 cell were all lower than those of the Fn+ neutrophil-like cell (0.95±0.09 vs. 1.27±0.04, 1.15±0.34 vs. 2.29±0.50, 1.22±0.14 vs. 1.68±0.12), and the differences were statistically significant ( t=5.61, 3.24, and 4.49; P=0.005, 0.032, and 0.011). The results of flow cytometry assay indicated that the positive rate of reactive oxygen species of the DSS+ Fn A group was higher than that of the DSS A group ((21.15±2.93)% vs. (11.14±1.42)%), and the positive rate of reactive oxygen species of the Fn+ neutrophil-like cell was also higher than that of the neutrophil-like control cell before the PAD4 inhibition ((51.69±6.94)% vs.(31.95±3.31)%), and the differences were statistically significant( t=5.33 and 4.45, P=0.006 and 0.011). Conclusion:Fn can promote neutrophil to release NET by upregulating reactive oxygen/PAD4/cit-H3 signaling pathway, which disrupt the intestinal barrier and aggravate colitis.
