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.

Endoplasmic reticulum (ER) stress contribute to inflammatory bowel disease (IBD). However, the mechanistic link between toll-like receptor 4 (TLR4) and ER stress in IBD remains elusive. This study aimed to investigate the mechanism by which ER stress and TLR4 promote inflammation in IBD. IBD mouse model was established by the induction of TNBS, and Grp78 and TLR4 in intestine tissues were detected by immunohistochemistry. THP-1 cells were treated with lipopolysaccharides (LPS), ER stress inducer or inhibitor tauroursodeoxycholic acid (TUDCA), or p38 MAPK inhibitor. The activation of MAPK signaling was detected by Western blot, and the production and secretion of inflammatory factors were detected by PCR and ELISA. We found that the expression levels of TLR4 and GRP78 were significantly higher in the intestine of IBD model mice compared with control mice but were significantly lower in the intestine of IBD model mice treated with ER stress inhibitor TUDCA. ER stress inducer significantly increased while ER stress inhibitor TUDCA significantly decreased the expression and secretion of TNF-α, IL-1β and IL-8 in THP-1 cells treated by LPS. Only p38 MAPK signaling was activated in THP-1 cells treated by ER stress inducer. Furthermore, p38 inhibitor SB203580 inhibited the production and secretion of TNF-α, IL-1β and IL-8 in THP-1 cells treated with LPS. In conclusion, TLR4 promotes ER stress induced inflammation in IBD, and the effects may be mediated by p38 MAPK signaling. TLR4 and p38 MAPK signaling are novel therapeutic targets for IBD.

The NOD2 signaling pathway, which plays an important role in the mechanisms of inflammatory bowel disease (IBD) development, has been closely associated with ubiquitination. It was revealed in this study that NOD2 receptor activation could obviously affect the expression of 19 ubiquitination-related genes, with N4BP3 being the most prominently expressed and upregulated. In addition, N4BP3 knockdown was found to reduce the mRNA levels of MDP-induced inflammatory factors, while N4BP3 overexpression elevated their mRNA levels as well as the levels of phospho-ERK1/2, phospho-JNK, phospho-P38 and phospho-NF-κB P65 proteins. Immunoprecipitation tests showed that N4BP3 could pull down RIPK2 and promote its K63-linked ubiquitination. In human tissue specimen assays and mouse experiments, we found that the expression of N4BP3 was significantly elevated in Crohn’s disease (CD) patients and IBD mice, and N4BP3 knockdown reduced the dextran sulfate sodium-induced pathological score and the expression of inflammatory factors in the mouse colon tissue. In conclusion, N4BP3 is able to interact with RIPK2 and promote its K63-linked ubiquitination, to further promote the NOD2-MAPK/NF-κB pathway, thereby increasing promoting the release of inflammation factors and the degree of IBD inflammation.

Recent evidence suggests that endoplasmic reticulum (ER) stress plays a vital role in inflammatory bowel disease (IBD). Therefore, the aim of this study was to investigate the mechanism by which ER stress promotes inflammatory response in IBD. The expression of Gro-α, IL-8 and ER stress indicator Grp78 in colon tissues from patients with Crohn’s disease (CD) and colonic carcinoma was analyzed by immunohistochemistry staining. Colitis mouse model was established by the induction of trinitrobenzene sulphonic acid (TNBS), and the mice were treated with ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Then the body weight, colon length and colon inflammation were evaluated, and Grp78 and Gro-α in colon tissues were detected by immunohistochemistry. Epithelial cells of colon cancer HCT116 cells were treated with tunicamycin to induce ER stress. Grp78 was detected by Western blot, and chemokines were measured by PCR and ELISA. The expression levels of Grp78, Gro-α and IL-8 were significantly upregulated in intestinal tissues of CD patients. Mice with TNBS induced colitis had increased expression of Grp78 and Gro-α in colonic epithelia. TUDCA reduced the severity of TNBS-induced colitis. In HCT116 cells, tunicamycin increased the expression of Grp78, Gro-α and IL-8 in a concentration-dependent manner. Furthermore, p38 MAPK inhibitor significantly inhibited the upregulation of Gro-α and IL-8 induced by tunicamycin. In conclusion, ER stress promotes inflammatory response in IBD, and the effects may be mediated by the activation of p38 MAPK signaling pathway.

Protein ubiquitination is an enzymatic cascade reaction and serves as an important protein post-translational modification (PTM) that is involved in the vast majority of cellular life activities. The key enzyme in the ubiquitination process is E3 ubiquitin ligase (E3), which catalyzes the binding of ubiquitin (Ub) to the protein substrate and influences substrate specificity. In recent years, the relationship between the subfamily of neuron-expressed developmental downregulation 4 (NEDD4), which belongs to the E3 ligase system, and digestive diseases has drawn widespread attention. Numerous studies have shown that NEDD4 and NEDD4L of the NEDD4 family can regulate the digestive function, as well as a series of related physiological and pathological processes, by controlling the subsequent degradation of proteins such as PTEN, c-Myc, and P21, along with substrate ubiquitination. In this article, we reviewed the appropriate functions of NEDD4 and NEDD4L in digestive diseases including cell proliferation, invasion, metastasis, chemotherapeutic drug resistance, and multiple signaling pathways, based on the currently available research evidence for the purpose of providing new ideas for the prevention and treatment of digestive diseases.