Li Shijia, Zhu Jingchao, Song Jie, Yang Ling, Gong Yingfei, Dai Yue, Wei Zhifeng
Department of Pharmacology of Chinese Materia Medica, School of Traditional Chinese Pharmacy, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China.
Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China.
J Adv Res. 2025 Jun 27. doi: 10.1016/j.jare.2025.06.078.
The aryl hydrocarbon receptor (AhR) is a promising therapeutic target for ulcerative colitis (UC) and plays a role in regulating neutrophil function.
We aimed to investigate the effects and mechanisms of AhR on NETosis, a neutrophil-driven process that disrupts intestinal epithelial homeostasis, to support the development of anti-UC therapies.
A dextran sulphate sodium (DSS)-induced colitis mouse model was established, and bioinformatics analyses combined with multiple molecular biology techniques were used to assess changes in NETosis and signalling pathway activation.
Data from the Gene Expression Omnibus database and DSS-induced colitis mice confirmed an inverse correlation between AhR activation and NETosis in UC. In vitro experiments, including assays for double-stranded DNA release, co-localisation of myeloperoxidase with DNA and neutrophil elastase (NE)/citrullinated histone H3, histone H4 degradation, and chromatin decondensation, demonstrated that AhR activation directly inhibits NETosis. Further investigations using gene knockdown plasmids, enzyme substrate assays, and flow cytometry revealed that AhR activation reduced the NE activity-independent of pcDNA-peptidyl arginine deiminase 4-through an N-glycosylation-dependent mechanism involving the physiological NE inhibitors alpha-1 antitrypsin (AAT) and alpha-2-macroglobulin (A2M). Mechanistically, AhR functioned as an E3 ligase that bound to hexokinase 2 (HK2), promoting its K48-linked ubiquitination and degradation, thereby impairing the flux of the hexosamine biosynthesis pathway (HBP) and reducing the availability of the glycosylation precursor UDP-GlcNAc.
AhR activation suppresses NETosis by modulating HK2-mediated HBP flux and the subsequent N-glycosylation of AAT and A2M, thereby decreasing NE activity.
芳烃受体（AhR）是溃疡性结肠炎（UC）一个很有前景的治疗靶点，并且在调节中性粒细胞功能中发挥作用。
我们旨在研究AhR对中性粒细胞胞外诱捕网形成（NETosis）的影响及其机制，NETosis是一个由中性粒细胞驱动的破坏肠道上皮稳态的过程，以支持抗UC疗法的开发。
建立葡聚糖硫酸钠（DSS）诱导的结肠炎小鼠模型，并结合多种分子生物学技术进行生物信息学分析，以评估NETosis和信号通路激活的变化。
来自基因表达综合数据库和DSS诱导的结肠炎小鼠的数据证实，UC中AhR激活与NETosis呈负相关。体外实验，包括双链DNA释放测定、髓过氧化物酶与DNA和中性粒细胞弹性蛋白酶（NE）/瓜氨酸化组蛋白H3的共定位、组蛋白H4降解以及染色质解聚，表明AhR激活直接抑制NETosis。使用基因敲低质粒、酶底物测定和流式细胞术的进一步研究表明，AhR激活通过涉及生理性NE抑制剂α1抗胰蛋白酶（AAT）和α2巨球蛋白（A2M）的N-糖基化依赖性机制降低了NE活性，而与pcDNA-肽基精氨酸脱亚氨酶4无关。机制上，AhR作为一种E3连接酶，与己糖激酶2（HK2）结合，促进其K48连接的泛素化和降解，从而损害己糖胺生物合成途径（HBP）的通量并降低糖基化前体UDP-GlcNAc的可用性。
AhR激活通过调节HK2介导的HBP通量以及随后AAT和A2M的N-糖基化来抑制NETosis，从而降低NE活性。
