NF-κB inhibitor alpha controls SARS-CoV-2 infection in ACE2-overexpressing human airway organoids.

As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor (ACE2-OE) that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. Interferon-lambda was upregulated in cells with low-level infection while the NF-kB inhibitor alpha gene (encoding IkBa) was consistently upregulated in infected cells, and its expression positively correlated with infection levels. Confocal microscopy showed more IkBa expression in infected than bystander cells, but found concurrent nuclear translocation of NF-kB that IkBa usually prevents. Overexpressing a nondegradable IkBa mutant reduced NF-kB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and underscore that the strength of the NF-kB feedback loop in infected cells controls viral replication.

IκBα controls dormancy in hematopoietic stem cells via retinoic acid during embryonic development.

Recent findings suggest that Hematopoietic Stem Cells (HSC) and progenitors arise simultaneously and independently of each other already in the embryonic aorta-gonad mesonephros region, but it is still unknown how their different features are established. Here, we uncover IκBα (Nfkbia, the inhibitor of NF-κB) as a critical regulator of HSC proliferation throughout development. IκBα balances retinoic acid signaling levels together with the epigenetic silencer, PRC2, specifically in HSCs. Loss of IκBα decreases proliferation of HSC and induces a dormancy related gene expression signature instead. Also, IκBα deficient HSCs respond with superior activation to in vitro culture and in serial transplantation. At the molecular level, chromatin regions harboring binding motifs for retinoic acid signaling are hypo-methylated for the PRC2 dependent H3K27me3 mark in IκBα deficient HSCs. Overall, we show that the proliferation index in the developing HSCs is regulated by a IκBα-PRC2 axis, which controls retinoic acid signaling.

SIAH1 Promotes the Pyroptosis of Cardiomyocytes in Diabetic Cardiomyopathy via Regulating IκB-α/NF-κВ Signaling.

Inflammation-mediated dysfunction of cardiomyocytes is the main cause of diabetic cardiomyopathy (DCM). The present study aimed to investigate the roles of siah E3 ubiquitin protein ligase 1 (SIAH1) in DCM. The online dataset GSE4172 was used to analyze the differentially expressed genes in myocardial inflammation of DCM patients. RT-qPCR was conducted to detect mRNA levels. Enzyme-Linked Immunosorbent Assay (ELISA) was performed to detect cytokine release. Western blot was used to detect protein expression. Lactate dehydrogenase (LDH) assay was used to determine cytotoxicity. In vitro ubiquitination assay was applied to determine the ubiquitination of nuclear factor kappa B inhibitor alpha (1κВ-α). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect the death of cardiomyocytes. Flow cytometry was applied for determining cardiomyocyte pyroptosis. The results showed that SIAH1 was overexpressed in human inflammatory cardiomyopathy. High expression of SIAH1 was associated with inflammatory response. SIAH1 was also overexpressed lipopolysaccharide (LPS)-induced inflammatory cardiomyopathy model in vitro. However, SIAH1 knockdown suppressed the inflammatory-related pyroptosis of cardiomyocytes. SIAH1 promoted the ubiquitination of 1κВ-α and activated nuclear factor kappa В (NF-κВ) signaling, which promoted the pyroptosis of cardiomyocytes. In conclusion, SIAH1 exacerbated the progression of human inflammatory cardiomyopathy via inducing the ubiquitination of 1κВ-α and activation of NF-κВ signaling. Therefore, SIAHI/IκB-α/NF-κB signaling may be a potential target for human inflammatory cardiomyopathy.

A novel 12-membered ring non-antibiotic macrolide EM982 attenuates cytokine production by inhibiting IKKß and IκBα phosphorylation.

Antimicrobial resistance poses a serious threat to human health worldwide and its incidence continues to increase owing to the overuse of antibiotics and other factors. Macrolide antibiotics such as erythromycin (EM) have immunomodulatory effects in addition to their antibacterial activity. Long-term, low-dose administration of macrolides has shown clinical benefits in treating non-infectious inflammatory respiratory diseases. However, this practice may also increase the emergence of drug-resistant bacteria. In this study, we synthesized a series of EM derivatives, and screened them for two criteria: (i) lack of antibacterial activity and (ii) ability to suppress tumor necrosis factor-α (TNF-α) production in THP-1 cells stimulated with lipopolysaccharide. Among the 37 synthesized derivatives, we identified a novel 12-membered ring macrolide EM982 that lacked antibacterial activity against Staphylococcus aureus and suppressed the production of TNF-α and other cytokines. The effects of EM982 on Toll-like receptor 4 (TLR4) signaling were analyzed using a reporter assay and Western blotting. The reporter assay showed that EM982 suppressed the activation of transcription factors, NF-κB and/or activator protein 1 (AP-1), in HEK293 cells expressing human TLR4. Western blotting showed that EM982 inhibited the phosphorylation of both IκB kinase (IKK) ß and IκBα, which function upstream of NF-κB, whereas it did not affect the phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and c-Jun N-terminal kinase, which act upstream of AP-1. These results suggest that EM982 suppresses cytokine production by inhibiting phosphorylation of IKKß and IκBα, resulting in the inactivation of NF-κB.

Quantifying NF-κB Activation by Flow Cytometry of IκBα Degradation.

Nuclear factor-κB (NF-κB) is a crucial pro-inflammatory transcription factor whose activation is of immense interest to immunology research. Estimation of NF-κB activation through flow cytometry is not possible due to the unavailability of robust flow cytometry antibodies that can bind to its phosphorylated, active, nuclear form. In this protocol, we describe a flow cytometry assay that measures the activation of the pro-inflammatory transcription factor NF-κB in stimulated immune cells by quantifying the degradation of its upstream regulator IκBα. We demonstrate the utility of this protocol by assessment of intracellular IκBα in human primary regulatory T cells experiencing TNFR2 agonism, a process previously reported to activate NF-κB in these cells. We also show that this assay may be applied to study NF-κB activation in other cell types, such as human primary T cells and THP-1 cell-derived macrophages, when induced by their corresponding inflammatory cues. Thus, this robust and reproducible protocol will be of interest to a wide range of scientists who aim to measure NF-κB activity in medium-to-high-throughput assays. © 2024 Wiley Periodicals LLC. Basic Protocol: Quantifying inflammatory activation by flow cytometry of IκBα degradation Support Protocol 1: Isolating and expanding human regulatory T cells Support Protocol 2: Calculating IC50 from flow cytometry data using Excel.

Trim14-IκBα Signaling Regulates Chronic Inflammatory Pain in Rats and Osteoarthritis Patients.

Chronic inflammatory pain is the highest priority for people with osteoarthritis when seeking medical attention. Despite the availability of NSAIDs and glucocorticoids, central sensitization and peripheral sensitization make pain increasingly difficult to control. Previous studies have identified the ubiquitination system as an important role in the chronic inflammatory pain. Our study displayed that the E3 ubiquitin ligase tripartite motif-containing 14 (Trim14) was abnormally elevated in the serum of patients with osteoarthritis and pain, and the degree of pain was positively correlated with the degree of Trim14 elevation. Furthermore, CFA-induced inflammatory pain rat model showed that Trim14 was significantly increased in the L3-5 spinal dorsal horn (SDH) and dorsal root ganglion (DRG), and in turn the inhibitor of nuclear factor Kappa-B isoform α (IκBα) was decreased after Trim14 elevation. After intrathecal injection of Trim14 siRNA to inhibit Trim14 expression, IκBα expression was reversed and increased, and the pain behaviors and anxiety behaviors of rats were significantly relieved. Overall, these findings suggested that Trim14 may contribute to chronic inflammatory pain by degrading IκBα, and that Trim14 may become a novel therapeutic target for chronic inflammatory pain.

Cadmium promoted LPS-induced inflammation through TLR4/IκBα/NFκ-B signaling by increasing ROS-mediated incomplete autophagy.

Cadmium (Cd) exposure is considered as non-infectious stressor to human and animal health. Recent studies suggest that the immunotoxicity of low dose Cd is not directly apparent, but disrupts the immune responses when infected with some bacteria or virus. But how Cd alters the adaptive immunity organ and cells remains unclear. In this study, we applied lipopolysaccharide (LPS, infectious stressor) to induced inflammation in spleen tissues and T cells, and investigated the effects after Cd exposure and the underlying mechanism. Cd exposure promoted LPS-induced the expressions of the inflammatory factors, induced abnormal initiation of autophagy, but blocked autophagic flux. The effects Cd exposure under LPS activation were reversed by the autophagy promoter Rapamycin. Under LPS activation conditions, Cd also induced oxidative stress by increasing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and reducing total antioxidant capacity (T-AOC) activity. The increased superoxide dismutase (SOD) activity after Cd exposure might be a negative feedback or passive adaptive regulation of oxidative stress. Cd-increased autophagic flux inhibition and TNF-α expression were reversed by ROS scavenger α-tocopherol (TCP). Furthermore, under LPS activation condition, Cd promoted activation of toll-like receptor 4 (TLR4)/IκBα/NFκ-B signaling pathway and increased TLR4 protein stability, which were abolished by the pretreatment of Rapamycin. The present study confirmed that, by increasing ROS-mediated inhibiting autophagic degradation of TLR4, Cd promoted LPS-induced inflammation in spleen T cells. This study identified the mechanism of autophagy in Cd-aggravated immunotoxicity under infectious stress, which could arouse public attention to synergistic toxicity of Cd and bacterial or virus infection.

CLK2 mediates IκBα-independent early termination of NF-κB activation by inducing cytoplasmic redistribution and degradation.

Activation of the NF-κB pathway is strictly regulated to prevent excessive inflammatory and immune responses. In a well-known negative feedback model, IκBα-dependent NF-κB termination is a delayed response pattern in the later stage of activation, and the mechanisms mediating the rapid termination of active NF-κB remain unclear. Here, we showed IκBα-independent rapid termination of nuclear NF-κB mediated by CLK2, which negatively regulated active NF-κB by phosphorylating the RelA/p65 subunit of NF-κB at Ser180 in the nucleus to limit its transcriptional activation through degradation and nuclear export. Depletion of CLK2 increased the production of inflammatory cytokines, reduced viral replication and increased the survival of the mice. Mechanistically, CLK2 phosphorylated RelA/p65 at Ser180 in the nucleus, leading to ubiquitin‒proteasome-mediated degradation and cytoplasmic redistribution. Importantly, a CLK2 inhibitor promoted cytokine production, reduced viral replication, and accelerated murine psoriasis. This study revealed an IκBα-independent mechanism of early-stage termination of NF-κB in which phosphorylated Ser180 RelA/p65 turned off posttranslational modifications associated with transcriptional activation, ultimately resulting in the degradation and nuclear export of RelA/p65 to inhibit excessive inflammatory activation. Our findings showed that the phosphorylation of RelA/p65 at Ser180 in the nucleus inhibits early-stage NF-κB activation, thereby mediating the negative regulation of NF-κB.

Discovery of sesquiterpenoids from the roots of Chloranthus henryi Hemsl. var. hupehensis (Pamp.) K. F. Wu and their anti-inflammatory activity by IKBα/NF-κB p65 signaling pathway suppression.

Phytochemical analysis of Chloranthus henryi var. hupehensis roots led to the identification of a new eudesmane sesquiterpenoid dimer, 18 new sesquiterpenoids, and three known sesquiterpenoids. Among the isolates, 1 was a rare sesquiterpenoid dimer that is assembled by a unique oxygen bridge (C11-O-C8') of two highly rearranged eudesmane-type sesquiterpenes with the undescribed C16 carbon framework. (+)-2 and (-)-2 were a pair of new skeleton dinorsesquiterpenoids with a remarkable 6/6/5 tricyclic ring framework including one γ-lactone ring and the bicyclo[3.3.1]nonane core. Their structures were elucidated using spectroscopic data, single-crystal X-ray diffraction analysis, and quantum chemical computations. In the LPS-induced BV-2 microglial cell model, 17 suppressed IL-1ß and TNF-α expression with EC50 values of 6.81 and 2.76 µM, respectively, indicating its excellent efficacy in inhibiting inflammatory factors production in a dose dependent manner and without cytotoxicity. In subsequent mechanism studies, compounds 3, 16, and 17 could reduce IL-1ß and TNF-α production by inhibiting IKBα/p65 pathway activation.

Squalene epoxidase promotes the chemoresistance of colorectal cancer via (S)-2,3-epoxysqualene-activated NF-κB.

BACKGROUND: While de novo cholesterol biosynthesis plays a crucial role in chemotherapy resistance of colorectal cancer (CRC), the underlying molecular mechanism remains poorly understood. METHODS: We conducted cell proliferation assays on CRC cells with or without depletion of squalene epoxidase (SQLE), with or without 5-fluorouracil (5-FU) treatment. Additionally, a xenograft mouse model was utilized to explore the impact of SQLE on the chemosensitivity of CRC to 5-FU. RNA-sequencing analysis and immunoblotting analysis were performed to clarify the mechanism. We further explore the effect of SQLE depletion on the ubiquitin of NF-κB inhibitor alpha (IκBα) and (S)-2,3-epoxysqualene on the binding of IκBα to beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC) by using immunoprecipitation assay. In addition, a cohort of 272 CRC patients were selected for our clinical analyses. RESULTS: Mechanistically, (S)-2,3-epoxysqualene promotes IκBα degradation and subsequent NF-κB activation by enhancing the interaction between BTRC and IκBα. Activated NF-κB upregulates the expression of baculoviral IAP repeat containing 3 (BIRC3), sustains tumor cell survival after 5-FU treatment and promotes 5-FU resistance of CRC in vivo. Notably, the treatment of terbinafine, an inhibitor of SQLE commonly used as antifungal drug in clinic, enhances the sensitivity of CRC to 5-FU in vivo. Additionally, the expression of SQLE is associated with the prognosis of human CRC patients with 5-FU-based chemotherapy. CONCLUSIONS: Thus, our finding not only demonstrates a new role of SQLE in chemoresistance of CRC, but also reveals a novel mechanism of (S)-2,3-epoxysqualene-dependent NF-κB activation, implicating the combined potential of terbinafine for 5-FU-based CRC treatment.

Regulatory roles of miRNA-530 in the post-transcriptional regulation of NF-κB signaling pathway through targeted modulation of IκBα in Sebastesschlegelii.

MicroRNAs (miRNAs) are a crucial type of non-coding RNAs involved in post-transcriptional regulation. The playing essential regulatory roles in the NF-κB signaling pathway and modulate the host immune response to diverse pathogens by targeting IκBα. However, the regulatory mechanism of miRNAs in relation with IκBα in Sebastes schlegelii remains unclear. In our study, we identified two copies of IkBα gene in black rockfish (Sebastes schlegelii), namely IkBα1 and IkBα2. Moreover, we have discovered that miRNA-530 can activate the NF-κB signaling pathway by inhibiting the expression of IκBα, thereby inducing the inflammatory response. This project comprehensively investigated the interactive regulatory roles of miRNA-530 in the NF-κB signaling pathway at both cellular and in vivo levels, while also elucidating the regulatory relationships between miRNA-530 and IκBα. In conclusion, our research confirmed that miRNA-530 can target the 3'UTR region of IκBα, resulting in a decrease in the expression of IκBα at the post-transcriptional level and inhibiting its translation. The findings contribute to the understanding of the regulatory network of non-coding RNA in teleosts and its subsequent regulation of the NF-κB signaling pathway by miRNAs.

Kaempferol attenuates inflammation in lipopolysaccharide-induced gallbladder epithelial cells by inhibiting the MAPK/NF-κB signaling pathway.

Kaempferol (KPR), a flavonoid compound found in various plants and foods, has garnered attention for its anti-inflammatory, antioxidant, and anticancer properties. In preliminary studies, KPR can modulate several signaling pathways involved in inflammation, making it a candidate for treating cholecystitis. This study aimed to explore the effects and mechanisms of KPR on lipopolysaccharide (LPS)-induced human gallbladder epithelial cells (HGBECs). To assess the impact of KPR on HGBECs, the HGBECs were divided into control, KPR, LPS, LPS + KPR, and LPS + UDCA groups. Cell viability and cytotoxicity were evaluated by MTT assay and lactate dehydrogenase (LDH) assay, respectively, and concentrations of KPR (10-200 µM) were tested. LPS-induced inflammatory responses in HGBECs were to create an in vitro model of cholecystitis. The key inflammatory markers (IL-1ß, IL-6, and TNF-α) levels were quantified using ELISA, The modulation of the MAPK/NF-κB signaling pathway was measured by western blot using specific antibodies against pathway components (p-IκBα, IκBα, p-p65, p65, p-JNK, JNK, p-ERK, ERK, p-p38, and p38). The cell viability and LDH levels in HGBECs were not significantly affected by 50 µM KPR, thus it was selected as the optimal KPR intervention concentration. KPR increased the viability of LPS-induced HGBECs. Additionally, KPR inhibited the inflammatory factors level (IL-1ß, IL-6, and TNF-α) and protein expression (iNOS and COX-2) in LPS-induced HGBECs. Furthermore, KPR reversed LPS-induced elevation of p-IκBα/IκBα, p-p65/p65, p-JNK/JNK, p-ERK/ERK, and p-p38/p38 ratios. KPR attenuates the LPS-induced inflammatory response in HGBECs, possibly by inhibiting MAPK/NF-κB signaling.

Attenuating Renal Interstitial Fibrosis by Shenqi Pill via Reducing Inflammation Response Regulated by NF-κB Pathway In Vitro and In Vivo.

INTRODUCTION: One of the most significant clinical features of chronic  kidney disease is renal interstitial fibrosis (RIF). This study aimed  to investigate the role and mechanism of Shenqi Pill (SQP) on RIF. METHODS: RIF model was established by conducting unilateral  ureteral obstruction (UUO) surgery on rat or stimulating human  kidney-2 (HK-2) cell with transforming growth factor ß1 (TGFß1).  After modeling, the rats in the SQP low dose group (SQP-L), SQP  middle dose group (SQP-M) and SQP high dose group (SQP-H)  were treated with SQP at 1.5, 3 or 6 g/kg/d, and the cells in the  TGFß1+SQP-L/M/H were treated with 2.5%, 5%, 10% SQP-containing  serum. In in vivo assays, serum creatinine (SCr) and blood urea  nitrogen (BUN) content were measured, kidney histopathology  was evaluated., and α-smooth muscle actin (α-SMA) expression  was detected by immunohistochemistry. Interleukin-1ß (IL-1ß),  interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) content,  inhibitor of kappa B alpha (IKBα) and P65 phosphorylation were  assessed. Meanwhile, cell viability, inflammatory cytokines content,  α-SMA expression, IKBα and P65 phosphorylation were detected  in vitro experiment.  Results. SQP exhibited reno-protective effect by decreasing SCr  and BUN content, improving renal interstitial damage, blunting  fibronectin (FN) and α-SMA expression in RIF rats. Similarly, after  the treatment with SQP-containing serum, viability and α-SMA  expression were remarkably decreased in TGFß1-stimulated HK-2  cell. Furthermore, SQP markedly down-regulated IL-1ß, IL-6, and  TNF-α content, IKBα and RelA (P65) phosphorylation both in vivo and in vitro.  Conclusion. SQP has a reno-protective effect against RIF in vivo and in vitro, and the effect is partly linked to nuclear factor-kappa  B (NF-κB) pathway related inflammatory response, which indicates  that SQP may be a candidate drug for RIF. DOI: 10.52547/ijkd.7546.

HOXA1 silencing inhibits cisplatin resistance of oral squamous cell carcinoma cells via IκB/NF-κB signaling pathway.

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.

Neuroprotective Effects of Aucubin against Cerebral Ischemia and Ischemia Injury through the Inhibition of the TLR4/NF-κB Inflammatory Signaling Pathway in Gerbils.

Aucubin, an iridoid glycoside, possesses beneficial bioactivities in many diseases, but little is known about its neuroprotective effects and mechanisms in brain ischemia and reperfusion (IR) injury. This study evaluated whether aucubin exhibited neuroprotective effects against IR injury in the hippocampal CA1 region through anti-inflammatory activity in gerbils. Aucubin (10 mg/kg) was administered intraperitoneally once a day for one week prior to IR. Neuroprotective effects of aucubin were assessed by neuronal nuclei (NeuN) immunofluorescence and Floro-Jade C (FJC) histofluorescence. Microgliosis and astrogliosis were evaluated using immunohistochemistry with anti-ionized calcium binding adapter protein 1 (Iba1) and glial fibrillary acidic protein (GFAP). Protein levels of proinflammatory cytokines interleukin1 beta (IL1ß) and tumor necrosis factor alpha (TNFα) were assayed using enzyme-linked immunosorbent assay and Western blot. Changes in toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway were assessed by measuring levels of TLR4, inhibitor of NF-κB alpha (IκBα), and NF-κB p65 using Western blot. Aucubin treatment protected pyramidal neurons from IR injury. IR-induced microgliosis and astrogliosis were suppressed by aucubin treatment. IR-induced increases in IL1ß and TNFα levels were significantly alleviated by the treatment. IR-induced upregulation of TLR4 and downregulation of IκBα were significantly prevented by aucubin treatment, and IR-induced nuclear translocation of NF-κB was reversed by aucubin treatment. Briefly, aucubin exhibited neuroprotective effects against brain IR injury, which might be related to the attenuation of neuroinflammation through inhibiting the TLR-4/NF-κB signaling pathway. These results suggest that aucubin pretreatment may be a potential approach for the protection of brain IR injury.

Anabolic Steroids Activate the NF-κB Pathway in Porcine Ovarian Putative Stem Cells Independently of the ZIP-9 Receptor.

Boldenone (Bdn) and nandrolone (Ndn) are anabolic androgenic steroids (AASs) that, as our previous studies have shown, may increase the risk of neoplastic transformation of porcine ovarian putative stem cells (poPSCs). The NF-κB pathway may be important in the processes of carcinogenesis and tumour progression. Therefore, in this work, we decided to test the hypothesis of whether Bdn and Ndn can activate the NF-κB pathway by acting through the membrane androgen receptor ZIP-9. For this purpose, the expression profiles of both genes involved in the NF-κB pathway and the gene coding for the ZIP-9 receptor were checked. The expression and localization of proteins of this pathway in poPSCs were also examined. Additionally, the expression of the ZIP-9 receptor and the concentration of the NF-κB1 and 2 protein complex were determined. Activation of the NF-κB pathway was primarily confirmed by an increase in the relative abundances of phosphorylated forms of RelA protein and IκBα inhibitor. Reduced quantitative profiles pinpointed not only for genes representing this pathway but also for unphosphorylated proteins, and, simultaneously, decreased concentration of the NF-κB1 and 2 complex may indicate post-activation silencing by negative feedback. However, the remarkably and sustainably diminished expression levels noticed for the SLC39A9 gene and ZIP-9 protein suggest that this receptor does not play an important role in the regulation of the NF-κB pathway.

ITF2357 regulates NF-κB signaling pathway to protect barrier integrity in retinal pigment epithelial cells.

The robust integrity of the retinal pigment epithelium (RPE), which contributes to the outer brain retina barrier (oBRB), is compromised in several retinal degenerative and vascular disorders, including diabetic macular edema (DME). This study evaluates the role of a new generation of histone deacetylase inhibitor (HDACi), ITF2357, in regulating outer blood-retinal barrier function and investigates the underlying mechanism of action in inhibiting TNFα-induced damage to RPE integrity. Using the immortalized RPE cell line (ARPE-19), ITF2357 was found to be non-toxic between 50 nM and 5 µM concentrations. When applied as a pre-treatment in conjunction with an inflammatory cytokine, TNFα, the HDACi was safe and effective in preventing epithelial permeability by fortifying tight junction (ZO-1, -2, -3, occludin, claudin-1, -2, -3, -5, -19) and adherens junction (E-cadherin, Nectin-1) protein expression post-TNFα stress. Mechanistically, ITF2357 depicted a late action at 24 h via attenuating IKK, IκBα, and p65 phosphorylation and ameliorated the expression of IL-1ß, IL-6, and MCP-1. Also, ITF2357 delayed IκBα synthesis and turnover. The use of Bay 11-7082 and MG132 further uncovered a possible role for ITF2357 in non-canonical NF-κB activation. Overall, this study revealed the protection effects of ITF2357 by regulating the turnover of tight and adherens junction proteins and modulating NF-κB signaling pathway in the presence of an inflammatory stressor, making it a potential therapeutic application for retinal vascular diseases such as DME with compromised outer blood-retinal barrier.

Poly-(ADP-ribose) polymerases inhibition by olaparib attenuates activities of the NLRP3 inflammasome and of NF-κB in THP-1 monocytes.

Poly-(ADP-ribose) polymerases (PARPs) are a protein family that make ADP-ribose modifications on target genes and proteins. PARP family members contribute to the pathogenesis of chronic inflammatory diseases, including atherosclerosis, in which monocytes/macrophages play important roles. PARP inhibition is protective against atherosclerosis. However, the mechanisms by which PARP inhibition exerts this beneficial effect are not well understood. Here we show that in THP-1 monocytes, inhibition of PARP by olaparib attenuated oxidized low-density lipoprotein (oxLDL)-induced protein expressions of nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing-3 (NLRP3) inflammasome components: NLRP3, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1. Consistent with this effect, olaparib decreased oxLDL-enhanced interleukin (IL)-1ß and IL-18 protein expression. Olaparib also decreased the oxLDL-mediated increase in mitochondrial reactive oxygen species. Similar to the effects of the NLRP3 inhibitor, MCC950, olaparib attenuated oxLDL-induced adhesion of monocytes to cultured human umbilical vein endothelial cells and reduced foam cell formation. Furthermore, olaparib attenuated the oxLDL-mediated activation of nuclear factor (NF)-κB through the oxLDL-mediated increase in IκBα phosphorylation and assembly of NF-κB subunits, demonstrated by co-immunoprecipitation of IκBα with RelA/p50 and RelB/p52 subunits. Moreover, PARP inhibition decreased oxLDL-mediated protein expression of a NF-κB target gene, VCAM1, encoding vascular cell adhesion molecule-1. This finding indicates an important role for NF-κB activity in PARP-mediated activation of the NLRP3 inflammasome. Thus, PARP inhibition by olaparib attenuates NF-κB and NLRP3 inflammasome activities, lessening monocyte cell adhesion and macrophage foam cell formation. These inhibitory effects of olaparib on NLRP3 activity potentially protect against atherosclerosis.

Luteolin ameliorates pentetrazole-induced seizures through the inhibition of the TLR4/NF-κB signaling pathway.

Epilepsy represents a prevalent neurological disorder in the population, and the existing antiepileptic drugs (AEDs) often fail to adequately control seizures. Inflammation is recognized as a pivotal factor in the pathophysiology of epilepsy. Luteolin, a natural flavonoid extract, possesses anti-inflammatory properties and exhibits promising neuroprotective activity. Nevertheless, the precise molecular mechanisms underlying the antiepileptic effects of luteolin remain elusive. In this study, we established a rat model of epilepsy using pentylenetetrazole (PTZ) to induce seizures. A series of behavioral experiments were conducted to assess behavioral abilities and cognitive function. Histological techniques, including HE staining, Nissl staining, and TUNEL staining, were employed to assess hippocampal neuronal damage. Additionally, Western blotting, RT-qPCR, and ELISA were utilized to analyze the expression levels of proteins involved in the TLR4/IκBα/NF-κB signaling pathway, transcription levels of apoptotic factors, and levels of inflammatory cytokines, respectively. Luteolin exhibited a dose-dependent reduction in seizure severity, prolonged the latency period of seizures, and shortened seizure duration. Furthermore, luteolin prevented hippocampal neuronal damage in PTZ-induced epileptic rats and partially restored behavioral function and learning and memory abilities. Lastly, PTZ kindling activated the TLR4/IκBα/NF-κB pathway, leading to elevated levels of the cytokines TNF-α, IL-6 and IL-1ß, which were attenuated by luteolin. Luteolin exerted anticonvulsant and neuroprotective activities in the PTZ-induced epileptic model. Its mechanism was associated with the inhibition of the TLR4/IκBα/NF-κB pathway, alleviating the immune-inflammatory response in the post-epileptic hippocampus.

Human ß-Defensin 3 Inhibition of P. gingivalis LPS-Induced IL-1ß Production by BV-2 Microglia through Suppression of Cathepsins B and L.

Cathepsin B (CatB) is thought to be essential for the induction of Porphyromonas gingivalis lipopolysaccharide (Pg LPS)-induced Alzheimer's disease-like pathologies in mice, including interleukin-1ß (IL-1ß) production and cognitive decline. However, little is known about the role of CatB in Pg virulence factor-induced IL-1ß production by microglia. We first subjected IL-1ß-luciferase reporter BV-2 microglia to inhibitors of Toll-like receptors (TLRs), IκB kinase, and the NLRP3 inflammasome following stimulation with Pg LPS and outer membrane vesicles (OMVs). To clarify the involvement of CatB, we used several known CatB inhibitors, including CA-074Me, ZRLR, and human ß-defensin 3 (hBD3). IL-1ß production in BV-2 microglia induced by Pg LPS and OMVs was significantly inhibited by the TLR2 inhibitor C29 and the IκB kinase inhibitor wedelolactonne, but not by the NLRPs inhibitor MCC950. Both hBD3 and CA-074Me significantly inhibited Pg LPS-induced IL-1ß production in BV-2 microglia. Although CA-074Me also suppressed OMV-induced IL-1ß production, hBD3 did not inhibit it. Furthermore, both hBD3 and CA-074Me significantly blocked Pg LPS-induced nuclear NF-κB p65 translocation and IκBα degradation. In contrast, hBD3 and CA-074Me did not block OMV-induced nuclear NF-κB p65 translocation or IκBα degradation. Furthermore, neither ZRLR, a specific CatB inhibitor, nor shRNA-mediated knockdown of CatB expression had any effect on Pg virulence factor-induced IL-1ß production. Interestingly, phagocytosis of OMVs by BV-2 microglia induced IL-1ß production. Finally, the structural models generated by AlphaFold indicated that hBD3 can bind to the substrate-binding pocket of CatB, and possibly CatL as well. These results suggest that Pg LPS induces CatB/CatL-dependent synthesis and processing of pro-IL-1ß without activation of the NLRP3 inflammasome. In contrast, OMVs promote the synthesis and processing of pro-IL-1ß through CatB/CatL-independent phagocytic mechanisms. Thus, hBD3 can improve the IL-1ß-associated vicious inflammatory cycle induced by microglia through inhibition of CatB/CatL.