Wang Jiang,1,* Jie Yin,1,* Min Han,2,* Wenhua He,3 Yan Zhao,4 Jinyue Hu,5 Meiwei Wang,6 Sikai Wang,7 Jiafan Xu,1 Chongtian Deng,8 Jiajia Li,1 Xiaonuo Gong,1 Yueming Shen1 

1Department of Digestive Diseases, The Affiliated Changsha Central Hospital, Hengyang Medical School, University of South China, Changsha, 410000, People’s Republic of China; 2Department of Cardiovascular Diseases, The Affiliated Changsha Central Hospital, Hengyang Medical School, University of South China, Changsha, 410000, People’s Republic of China; 3Department of Gastroenterology, Jiangxi Provincial Key Laboratory of Digestive Diseases, Jiangxi Clinical Research Center for Gastroenterology, Digestive Disease Hospital, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 4Department of Pathology, The Affiliated Changsha Central Hospital, Hengyang Medical School, University of South China, Changsha, 410000, People’s Republic of China; 5Medical Research Center, The Affiliated Changsha Central Hospital, Hengyang Medical School, University of South China, Changsha, 410004, People’s Republic of China; 6The Affiliated Changsha Central Hospital, Hengyang Medical School, University of South China, Changsha, 410004, People’s Republic of China; 7Department of Oncology, The Affiliated Changsha Central Hospital, Hengyang Medical School, University of South China, Changsha, 410004, People’s Republic of China; 8The First Clinical College, Changsha Medical University, Changsha, 410219, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Yueming Shen, Department of Digestive Diseases, The Affiliated Changsha Central Hospital, Hengyang Medical School, University of South China, 161 Shaoshan Road, Changsha, 410000, People’s Republic of China, Tel +13973166482, Email 2018050504@usc.edu.cn

Purpose: N4BP3 is a ubiquitination-related gene that plays a pivotal role in neurology and neoplasia. Studies have demonstrated its essential function in axonal and dendritic branching, promoting hepatocellular carcinoma and breast cancer. Our previous research reveals that N4BP3 enhances inflammatory responses by modulating the NOD2 signaling pathway. It is crucial to investigate whether N4BP3 regulates inflammatory bowel disease (IBD) through the TLR4 signaling pathway and to elucidate the underlying mechanisms.
Methods: Lipopolysaccharides (LPS) were used to activate the TLR4 pathway in THP-1/Caco-2 cells. THP-1/Caco-2 cells were transfected with either N4BP3 overexpression or knockdown plasmids, generating N4BP3-overexpressing or N4BP3-deficient cell lines. For in vivo studies, colitis was induced in mice using dextran sodium sulfate (DSS). Additionally, negative control and N4BP3-knockdown C57BL/6 mouse models were established via intraperitoneal injection of control or N4BP3-targeting adeno-associated virus (AAV).
Results: LPS stimulation significantly upregulated N4BP3 expression in THP-1/Caco-2 cells compared to sterile water treatment (P < 0.05). In N4BP3-overexpressing cells, LPS induction led to significantly higher expression of TNF-α, IL-1β, IL-6, and IL-8 mRNA, as well as phospho-NF-κB p65 protein, compared to wild-type THP-1/Caco-2 cells (P < 0.05). Conversely, these inflammatory markers were markedly downregulated in N4BP3-knockdown THP-1 cells following LPS stimulation (P < 0.05). In DSS-induced colitis models, N4BP3-knockdown mice showed decreased phospho-NF-κB p65 but increased IκBα protein expression in colonic tissues compared to DSS-treated control mice (P < 0.05). Furthermore, we observed interaction between N4BP3 and IκBα, with N4BP3-overexpressing THP-1 cells demonstrating significantly elevated K48-linked ubiquitination levels versus controls.
Conclusion: LPS upregulates N4BP3 expression, which subsequently enhances K48-linked ubiquitination of IκBα, leading to NF-κB pathway activation, and exacerbating IBD progression. These findings suggest N4BP3 as a potential therapeutic target for developing novel IBD treatments.

Keywords: N4BP3, ubiquitination, TLR4, NF-κB, IκBα, inflammatory bowel disease