The von Hippel-Lindau tumor suppressor protein (VHL), an E3 ubiquitin ligase, functions as a critical regulator of the oxygen-sensing pathway for targeting hypoxia-inducible factors. Recent evidence suggests that mammalian VHL may also be critical to the NF-κB signaling pathway, although the specific molecular mechanisms remain unclear. Herein, the roles of mandarin fish ( Siniperca chuatsi) VHL ( scVHL) in the NF-κB signaling pathway and mandarin fish ranavirus (MRV) replication were explored. The transcription of scVHL was induced by immune stimulation and MRV infection, indicating a potential role in innate immunity. Dual-luciferase reporter gene assays and reverse transcription quantitative PCR (RT-qPCR) results demonstrated that scVHL evoked and positively regulated the NF-κB signaling pathway. Treatment with NF-κB signaling pathway inhibitors indicated that the role of scVHL may be mediated through scIKKα, scIKKβ, scIκBα, or scp65. Co-immunoprecipitation (Co-IP) analysis identified scIκBα as a novel target protein of scVHL. Moreover, scVHL targeted scIκBα to catalyze the formation of K63-linked polyubiquitin chains to activate the NF-κB signaling pathway. Following MRV infection, NF-κB signaling remained activated, which, in turn, promoted MRV replication. These findings suggest that scVHL not only positively regulates NF-κB but also significantly enhances MRV replication. This study reveals a novel function of scVHL in NF-κB signaling and viral infection in fish.
肿瘤抑制蛋白VHL是一种E3泛素连接酶，在缺氧诱导因子的氧敏感通路中起关键调节作用。近期研究表明，哺乳动物VHL在NF-κB信号通路中发挥重要作用，但其具体的分子调控机制尚不清楚。在此，该文开展了鳜鱼VHL（ scVHL）在NF-κB信号通路和鳜蛙病毒（MRV）复制中的作用研究。研究结果显示，免疫刺激和MRV感染均可诱导 scVHL的转录，提示 scVHL可能在先天免疫中发挥重要作用。双荧光素酶报告基因实验和实时荧光定量PCR结果显示， scVHL可诱导并激活NF-κB信号通路。利用NF-κB信号通路抑制剂处理结果显示， scVHL在NF-κB信号通路中的调控作用可能靶向 scIKKα、 scIKKβ、 scIκBα或 scp65。通过免疫共沉淀进一步分析，发现 scIκBα是 scVHL的一个新的靶标蛋白，且 scVHL靶向 scIκBα催化K63连接的多聚泛素链的形成，从而激活NF-κB信号通路。在MRV感染后，NF-κB信号通路处于激活状态，NF-κB的激活可促进MRV的复制。上述结果表明， scVHL可正向调控NF-κB，显著促进MRV复制。该研究揭示了 scVHL在NF-κB信号通路和病毒感染中的新作用，有助于深入阐明氧敏感通路调控动物先天性免疫的作用机制提供理论基础。.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) are currently the most widely used anti-inflammatory medications, but their long-term use can cause damage to the gastrointestinal tract(GIT). One of the risk factors for GIT injury is exposure to a high-altitude hypoxic environment, which can lead to damage to the intestinal mucosal barrier. Taking NSAIDs in a high-altitude hypoxic environment can exacerbate GIT injury and impact gut microbiota. The aim of this study is to investigate the mechanisms by which resveratrol (RSV) intervention alleviates NSAID-induced intestinal injury in a high-altitude hypoxic environment, as well as its role in regulating gut microbiota.
METHODS: Aspirin was administered orally to rats to construct a rat model of intestinal injury induced by NSAIDs. Following the induction of intestinal injury, rats were administered RSV by gavage, and the expression levels of TLR4, NF-κB,IκB as well as Zonula Occludens-1 (ZO-1) and Occludin proteins in the different treatment groups were assessed via Western blot. Furthermore, the expression of the inflammatory factors IL-10, IL-1β, and TNF-α was evaluated using Elisa.16sRNA sequencing was employed to investigate alterations in the gut microbiota.
RESULTS: The HCk group showed elevated expression of TLR4/NF-κB/IκB pathway proteins, increased expression of pro-inflammatory factors IL-1β and TNF-α, decreased expression of the anti-inflammatory factor IL-10, and expression of intestinal mucosal barrier proteins ZO-1 and Occludin. The administration of NSAIDs drugs in the plateau hypoxic environment exacerbates intestinal inflammation and damage to the intestinal mucosal barrier. After treatment with RSV intervention, the expression of TLR4/NF-κB/IκB signaling pathway proteins would be reduced, thereby lowering the expression of inflammatory factors in the HAsp group. The results of HE staining directly show the damage to the intestines and the repair of intestinal mucosa after RSV intervention. 16sRNA sequencing results show significant differences (P<0.05) in Ruminococcus, Facklamia, Parasutterella, Jeotgalicoccus, Coprococcus, and Psychrobacter between the HCk group and the Ck group. Compared to the HCk group, the HAsp group shows significant differences (P<0.05) in Facklamia, Jeotgalicoccus, Roseburia, Psychrobacter, and Alloprevotella. After RSV intervention, Clostridium_sensu_stricto bacteria significantly increase compared to the HAsp group.
CONCLUSIONS: Resveratrol can attenuate intestinal damage caused by the administration of NSAIDs at high altitude in hypoxic environments by modulating the TLR4/NF-κB/IκB signaling pathway and gut microbiota composition.

As part of the efforts to understand nuclear IκB function in NF-κB-dependent gene expression, we report an X-ray crystal structure of the IκBζ ankyrin repeat domain in complex with the dimerization domain of the NF-κB p50 homodimer. IκBζ possesses an N-terminal α helix that conveys domain folding stability. Affinity and specificity of the complex depend on a small portion of p50 at the nuclear localization signal. The model suggests that only one p50 subunit supports binding with IκBζ, and biochemical experiments confirm that IκBζ associates with DNA-bound NF-κB p50:RelA heterodimers. Comparisons of IκBζ:p50 and p50:κB DNA complex crystallographic models indicate that structural rearrangement is necessary for ternary complex formation of IκBζ and p50 with DNA.

The five NF-κB family members and three nuclear IκB proteins play important biological roles, but the mechanisms by which distinct members of these protein families contribute to selective gene transcription remain poorly understood, especially at a genome-wide scale. Using nascent transcript RNA-seq, we observed considerable overlap between p50-dependent and IκBζ-dependent genes in Toll-like receptor 4 (TLR4)-activated macrophages. Key immunoregulatory genes, including Il6, Il1b, Nos2, Lcn2, and Batf, are among the p50-IκBζ-codependent genes. IκBζ-bound genomic sites are occupied at earlier time points by NF-κB dimers. However, p50-IκBζ codependence does not coincide with preferential binding of either p50 or IκBζ, as RelA co-occupies hundreds of genomic sites with the two proteins. A common feature of p50-IκBζ-codependent genes is a nearby p50/RelA/IκBζ-cobound site exhibiting p50-dependent binding of both RelA and IκBζ. This and other results suggest that IκBζ acts in concert with RelA:p50 heterodimers. Notably, p50-IκBζ-codependent genes comprise a high percentage of genes exhibiting the greatest differential expression between TLR4-stimulated and tumor necrosis factor receptor (TNFR)-stimulated macrophages. Thus, our genome-centric analysis reveals a defined p50-IκBζ pathway that selectively activates a set of key immunoregulatory genes and serves as an important contributor to differential TNFR and TLR4 responses.

Pharmacologic inhibitors of cellular hydroxylase oxygen sensors are protective in multiple preclinical in vivo models of inflammation. However, the molecular mechanisms underlying this regulation are only partly understood, preventing clinical translation. We previously proposed a new mechanism for cellular oxygen sensing: oxygen-dependent, (likely) covalent protein oligomer (oxomer) formation. Here, we report that the oxygen sensor factor inhibiting HIF (FIH) forms an oxomer with the NF-κB inhibitor β (IκBβ). The formation of this protein complex required FIH enzymatic activity and was prevented by pharmacologic inhibitors. Oxomer formation was highly hypoxia-sensitive and very stable. No other member of the IκB protein family formed an oxomer with FIH, demonstrating that FIH-IκBβ oxomer formation was highly selective. In contrast to the known FIH-dependent oxomer formation with the deubiquitinase OTUB1, FIH-IκBβ oxomer formation did not occur via an IκBβ asparagine residue, but depended on the amino acid sequence VAERR contained within a loop between IκBβ ankyrin repeat domains 2 and 3. Oxomer formation prevented IκBβ from binding to its primary interaction partners p65 and c-Rel, subunits of NF-κB, the master regulator of the cellular transcriptional response to pro-inflammatory stimuli. We therefore propose that FIH-mediated oxomer formation with IκBβ contributes to the hypoxia-dependent regulation of inflammation.

The NF-κB (nuclear factor K-light-chain-enhancer of activated B cells) transcription factor family is critical for modulating the immune proinflammatory response throughout the body. During the resting state, inactive NF-κB is sequestered by IκB in the cytoplasm. The proteasomal degradation of IκB activates NF-κB, mediating its translocation into the nucleus to act as a nuclear transcription factor in the upregulation of proinflammatory genes. Stimuli that initiate NF-κB activation are diverse but are canonically attributed to proinflammatory cytokines and chemokines. Downstream effects of NF-κB are cell type-specific and, in the majority of cases, result in the activation of pro-inflammatory cascades. Acting as the primary immune responders of the central nervous system, microglia exhibit upregulation of NF-κB upon activation in response to pathological conditions. Under such circumstances, microglial crosstalk with other cell types in the central nervous system can induce cell death, further exacerbating the disease pathology. In this review, we will emphasize the role of NF-κB in triggering neuroinflammation mediated by microglia.

The non-neuronal and non-muscular effects of botulinum toxin type A (BTXA) on scar reduction has been discovered. This study was designed to investigate the effects of BTXA on macrophages polarization during the early stage of skin repair. A skin defect model was established on the dorsal skin of SD rats. BTXA was intracutaneous injected into the edge of wound immediately as the model was established. Histological examinations were performed on scar samples. Raw 264.7 was selected as the cell model of recruited circulating macrophages, and was induced for M1 polarization by LPS. Identify the signaling pathways that primarily regulated M1 polarization and respond to BTXA treatment. Application of BTXA at early stage of injury significantly reduced the scar diameter without delaying wound closure. BTXA treatment improved fiber proliferation and arrangement, and inhibited angiogenesis in scar granular tissue. The number of M1 macrophages and the levels of pro-inflammation were decreased after treated with BTXA in scar tissues. LPS activated JAK2/STAT1 and IκB/NFκB pathways were downregulated by BTXA, as well as LPS induced M1 polarization. At early stage of skin wound healing, injection of BTXA effectively reduced the number of M1 macrophages and the levels of pro-inflammatory mediators which contributes to scar alleviation. BTXA resisted the M1 polarization of macrophages induced by LPS via deactivating the JAK2/STAT1 and IκB/NFκB pathways.

The resistance of oral squamous cell carcinoma (OSCC) cells to cisplatin remains a tough nut to crack in OSCC therapy. Homeobox A1 (HOXA1) overexpression has been detected in head and neck squamous carcinoma (HNSC). Accordingly, this study aims to explore the potential role and mechanism of HOXA1 on cisplatin resistance in OSCC. The expression of HOXA1 in HNSC and its role in overall survival (OS) rate of OSCC patients were analyzed by bioinformatic analysis. Following transfection as needed, OSCC cells were induced by different concentrations of cisplatin, and the cell viability and apoptosis were evaluated by cell counting kit-8 and flow cytometry assays. The mRNA and protein expression levels of HOXA1 and the phosphorylation of IκBα and p65 were determined by real-time quantitative PCR and western blot. HOXA1 expression level was upregulated in HNSC tissues and OSCC cells. Overexpressed HOXA1 was correlated with a low OS rate of OSCC patients. Cisplatin exerted an anti-cancer effect on OSCC cells. HOXA1 silencing or cisplatin suppressed OSCC cell viability, boosted the apoptosis, and repressed the phosphorylation of IκBα and p65. Intriguingly, the combination of HOXA1 silencing and cisplatin generated a stronger anti-cancer effect on OSCC cells than their single use. HOXA1 silencing attenuates cisplatin resistance of OSCC cells via IκB/NF-κB signaling pathway, hinting that HOXA1 is a biomarker associated with OSCC and HOXA1 silencing can enhance the sensitivity of OSCC cells to cisplatin.

Polyphenolic compounds have shown promising neuroprotective properties, making them a valuable resource for identifying prospective drug candidates to treat several neurological disorders (NDs). Numerous studies have reported that polyphenols can disrupt the nuclear factor kappa B(NF-κB) pathway by inhibiting the phosphorylation or ubiquitination of signaling molecules, which further prevents the degradation of IκB. Additionally, they prevent NF-κB translocation to the nucleus and pro-inflammatory cytokine production. Polyphenols such as curcumin, resveratrol, and pterostilbene had significant inhibitory effects on NF-κB, making them promising candidates for treating NDs. Recent experimental findings suggest that polyphenols possess a wide range of pharmacological properties. Notably, much attention has been directed towards their potential therapeutic effects in NDs such as Alzheimer\'s disease (AD), Parkinson\'s disease (PD), cerebral ischemia, anxiety, depression, autism, and spinal cord injury (SCI). Much preclinical data supporting the neurotherapeutic benefits of polyphenols has been developed. Nevertheless, this study has described the significance of polyphenols as potential neurotherapeutic agents, specifically emphasizing their impact on the NF-κB pathway. This article offers a comprehensive analysis of the involvement of polyphenols in NDs, including both preclinical and clinical perspectives.

An increased concentration of palmitate in circulation is one of the most harmful factors in obesity. The von Willebrand factor (vWF), a protein involved in haemostasis, is produced and secreted by the vascular endothelium. An increased level of vWF in obese patients is associated with thrombosis and cardiovascular disease. The aim of this study was to investigate a palmitate effect on vWF in endothelial cells and understand the mechanisms of palmitate-activated signalling. Human umbilical vein endothelial cells (HUVECs) incubated in the presence of palmitate, exhibited an increased VWF gene expression, vWF protein maturation, and stimulated vWF secretion. Cardamonin, a Nuclear Factor kappa B (NF-κB) inhibitor, abolished the palmitate effect on VWF expression. The inhibition of Toll-like receptor (TLR) 2 with C29 resulted in the TLR4 overactivation in palmitate-treated cells. Palmitate, in the presence of TLR4 inhibitor TAK-242, leads to a higher expression of TLR6, CD36, and TIRAP. The silencing of TLR4 resulted in an increase in TLR2 level and vice versa. The obtained results indicate a potential mechanism of obesity-induced thrombotic complication caused by fatty acid activation of NF-κB signalling and vWF upregulation and help to identify various compensatory mechanisms related to TLR4 signal transduction.