Background and aim

Many evidences indicated that neutrophil extracellular traps (NETs) are involved in the pathogenesis of inflammatory bowel disease (IBD). Citrullination of histones by Protein Arginine Deiminase-4 (PAD4) is central for NETs formation. This paper aimed to explore the definite role of NETs in mouse model of Crohn's disease (CD) with 2,4,6-trinitrobenzene sulfonic acid (TNBS).

Methods

The expression of NETs-associated proteins and mRNAs in colon tissue were detected by immunohistochemistry and Real-time Quantitative PCR (QPCR) respectively. Neutrophils were isolated and stimulated in vitro to form NETs. In addition, we also administered Cl-amidine, PAD4 inhibitor, resulting in less NETs formation to investigate protective effect by measuring weight loss, gross bleeding, colon length, myeloperoxidase (MPO) activity, and cytokine expression in mice.

Results

The results showed enhanced expression of Ly6G, citrullinated histone H3 (CitH3), and PAD4 in TNBS-induced colitis mice and higher ability of neutrophil to produce NETs in vitro. Blocking NETs formation through Cl-amidine effectively alleviated the clinical colitis index and tissue inflammation in TNBS mice, regulated the expression of pro- or anti-inflammatory cytokines. In addition, Cl-amidine reduced the gene expression of PAD4 and the expression of NETs-associated proteins in the colon of TNBS mice and inhibited the formation of NETs in vitro.

Conclusions

Our data showed that Cl-amidine could alleviate the clinical colitis index in TNBS mice to some extend and suggested blocking NETs formation through inhibition of PAD4 as therapeutic targets for the treatment of CD.

中文翻译：

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TNBS诱导的小鼠结肠炎中蛋白质精氨酸脱亚氨酶4抑制剂的评估。

背景和目标

许多证据表明，嗜中性粒细胞胞外陷阱（NETs）参与了炎症性肠病（IBD）的发病机理。蛋白质精氨酸脱亚氨酶4（PAD4）的组蛋白对NET的形成至关重要。本文旨在探讨NETs在含2,4,6-三硝基苯磺酸（TNBS）的克罗恩病（CD）小鼠模型中的明确作用。

方法

分别通过免疫组织化学和实时定量PCR（QPCR）检测结肠组织中NETs相关蛋白和mRNA的表达。嗜中性粒细胞被分离并在体外刺激形成NET。此外，我们还施用了PAD4抑制剂Cl-am，可减少NET的形成，从而通过测量体重减轻，大出血，结肠长度，髓过氧​​化物酶（MPO）活性和小鼠细胞因子的表达来研究保护作用。

结果

结果显示，在TNBS诱导的结肠炎小鼠中，Ly6G，瓜氨酸化的组蛋白H3（CitH3）和PAD4的表达增强，并且中性粒细胞在体外产生NET的能力更高。通过Cl-am阻断NETs的形成可有效缓解TNBS小鼠的临床结肠炎指数和组织炎症，调节促炎或抗炎细胞因子的表达。此外，Cl-am降低了TNBS小鼠结肠中PAD4的基因表达和NETs相关蛋白的表达，并在体外抑制了NETs的形成。

结论

我们的数据表明，CL-脒可以减轻在临床结肠炎索引TNBS小鼠在一定程度上通过PAD4的抑制作为用于CD的治疗的治疗靶标，并建议阻断母语形成。