摘要
目的探究具核梭形杆菌(Fn)调控中性粒细胞形成中性粒细胞胞外诱捕网(NET)加重结肠炎的机制。方法取第一批15只C57BL/6小鼠,采用完全随机设计法分为阴性对照组、葡聚糖硫酸钠(DSS)A组(DSS诱导结肠炎)、DSS+Fn A组(DSS诱导结肠炎并感染Fn);另取第二批15只C57BL/6小鼠,采用完全随机设计法分为DSS B组、DSS+Fn B组、GSK484组[DSS诱导结肠炎并感染Fn后腹腔注射肽酰基精氨酸脱亚氨酶4(PAD4)抑制剂GSK484];每组5只小鼠。构建Fn感染中性粒细胞(HL-60细胞)及其PAD4抑制细胞模型,采用完全随机设计法分为对照类中性粒细胞(1.25%二甲基亚砜诱导类中性粒细胞模型)、Fn类中性粒细胞(类中性粒细胞与Fn共培养模型)和GSK484细胞(类中性粒细胞感染Fn后与GSK484共培养模型),其中对照和Fn类中性粒细胞分为两批分别进行PAD4抑制前和抑制后实验。采用免疫组织化学染色、蛋白质印迹法、流式细胞术等分析小鼠或细胞中的闭锁小带蛋白1(ZO-1)、中性粒细胞弹性蛋白酶(NE)、活性氧、PAD4、瓜氨酸化组蛋白(cit-H3)的表达情况。统计学方法采用双尾t检验。结果免疫组织化学染色结果显示,相较于DSS A组,DSS+Fn A组的NE、PAD4和cit-H3表达均上调、ZO-1表达下调;相较于DSS+Fn B组,GSK484组的ZO-1表达上调、cit-H3和PAD4表达均下调。蛋白质印迹法结果显示,PAD4抑制前,Fn类中性粒细胞的NE、PAD4和cit-H3相对表达水平均高于对照类中性粒细胞(1.52±0.11比1.00±0.19、1.21±0.06比1.00±0.07、1.59±0.16比1.00±0.16),差异均有统计学意义(t=-4.13、-3.86、-4.47,P=0.014、0.014、0.018);PAD4抑制后,GSK484细胞的PAD4、cit-H3、NE相对表达量均低于Fn类中性粒细胞(0.95±0.09比1.27±0.04、1.15±0.34比2.29±0.50、1.22±0.14比1.68±0.12),差异均有统计学意义(t=5.61、3.24、4.49,P=0.005、0.032、0.011)。流式细胞术结果显示,DSS+Fn A组小鼠的活性氧阳性率高于DSS A组[(21.15±2.93)%比(11.14±1.42)%],PAD4抑制剂前的Fn类中性粒细胞的活性氧阳性率也高于对照类中性粒细胞[(51.69±6.94)%比(31.95±3.31)%],差异均有统计学意义(t=5.33、4.45,P=0.006、0.011)。结论Fn可以通过上调活性氧/PAD4/cit-H3信号促进中性粒细胞释放NET破坏肠道屏障进而加重结肠炎。
ObjectiveTo investigate the mechanism of Fusobacterium nucleatum(Fn)in regulating the formation of neutrophil extracellular trap(NET)to aggravate colitis.MethodsWith 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.ResultsThe 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.
作者
包丽清
汪之越
刘洋
魏云巍
Bao Liqing;Wang Zhiyue;Liu Yang;Wei Yunwei(Cixi Biomedical Research Institute,Wenzhou Medical University,Ningbo 315302,China;Department of Pancreatic and Gastrointestinal Surgery,Ningbo No.2 Hospital,Ningbo 315010,China)
出处
《中华消化杂志》
北大核心
2025年第3期189-198,共10页
Chinese Journal of Digestion
基金
国家自然科学基金(U23A20458、82300631)
浙江省自然科学基金(LQ23H030002)
宁波市医疗卫生高端团队重大攻坚项目(2022010101)。
