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.

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.

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.

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.

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.

Studies Related to the Involvement of EsA in Improving Intestinal Inflammation in Acute Pancreatitis via the NF-κB Pathway.

Background: Acute pancreatitis (AP) is a clinically frequent acute abdominal condition, which refers to an inflammatory response syndrome of edema, bleeding, and even necrosis caused by abnormal activation of the pancreas's own digestive enzymes. Intestinal damage can occur early in the course of AP and is manifested by impaired intestinal mucosal barrier function, and inflammatory reactions of the intestinal mucosa, among other factors. It can cause translocation of intestinal bacteria and endotoxins, further aggravating the condition of AP. Therefore, actively protecting the intestinal mucosal barrier, controlling the progression of intestinal inflammation, and improving intestinal dynamics in the early stages of AP play an important role in enhancing the prognosis of AP. Methods: The viability and apoptosis of RAW264.7 cells treated with Esculentoside A (EsA) and/or lipopolysaccharide were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry, respectively. The expression of apoptosis-related proteins and NF-κB signaling pathway-related proteins were detected by western blot (WB). An enzyme-linked immunosorbent assay was used to measure TNF-α and IL-6 secretion. Results: In vitro experiments demonstrated that EsA not only promoted the apoptosis of inflammatory cells but also reduced the secretion of TNF-α and IL-6 in a dose-dependent manner. Additionally, it inhibited the activation of the NF-κB signaling pathway by decreasing the expression of phosphorylated-p65(p-p65) and elevating the expression of IκBα. Similarly, in vivo experiments using a rat AP model showed that EsA inhibited the expression of p-p65 elevating the expression of IκBα in the intestinal tissues of the rat AP model and promoting the apoptosis of inflammatory cells in the intestinal mucosa in vivo experiments, while improving the pathological outcome of the pancreatic and intestinal tissues. Conclusion: Our results suggest that EsA can reduce intestinal inflammation in the rat AP model and that EsA may be a candidate for treating intestinal inflammation in AP and further arresting AP progression.

Huanglian Decoction treats Henoch-Schonlein purpura nephritis by inhibiting NF-κB/NLRP3 signaling pathway and reducing renal IgA deposition.

Henoch-Schonlein purpura nephritis (HSPN) is a systemic vascular inflammatory disease. Huanglian Decoction (HLD) ameliorates renal injury in nephritis; however, the mechanism of action of HLD on HSPN has not been investigated. This study aimed to investigate the protective mechanism of HLD treatment in HSPN. The effects of HLD on HSPN biochemical indices, kidney injury and NF-κB/NLRP3 signaling pathway were analyzed by biochemical analysis, ELISA, HE and PAS staining, immunohistochemistry, immunofluorescence, and Western Blot. In addition, the effects of HLD on HSPN cells were analyzed. We found that HLD treatment significantly reduced renal tissue damage, decreased the levels of IL-17, IL-18, TNF-α, and IL-1ß, and increased the levels of TP and ALB in HSPN mice. It also inhibited the deposition of IgA, IgG, and C3 in kidney tissues and significantly decreased the expression of IκBα, p-IκBα, NLRP3, caspase-1, and IL-1ß in kidney tissues and cells. In addition, PMA treatment inhibited the above-mentioned effects of HLD. These results suggested that HLD attenuates renal injury, IgA deposition, and inflammation in HSPN mice and its mechanism of action may be related to the inhibition of the NF-κB/NLRP3 pathway.

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.

[Mechanism of Fushen Granules treat peritoneal dialysis-related peritonitis by regulating TLR4/NF-κB pathway:based on network pharmacology and animal experiments].

Network pharmacology was employed to probe into the mechanism of Fushen Granules in treating peritoneal dialysis-rela-ted peritonitis(PDRP) in rats. The main active components of Fushen Granules were searched against the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, and their targets were predicted. PDRP-related targets were retrieved from DisGeNET and other databases. The common targets shared by the drug and the disease were identified by the online tool, and protein-protein interaction(PPI) network of the common targets. The obtained 276 common targets were imported into DAVID for GO function enrichment and KEGG pathway enrichment. The main signaling pathway of Fushen Granules in the treatment of PDRP was predicted as Toll-like receptor 4(TLR4)/nuclear factor(NF)-κB. The rat model of uremia was induced by 5/6 nephrectomy. From two weeks after operation, the rat model of peritoneal dialysis(PD) was established by intraperitoneal injection of 20 mL dialysate with 1.25% glucose every day. The sham operation group and model group received 2 mL normal saline by gavage every day. The rats in Fushen Gra-nules groups were administrated with 2 mL solutions of low-(0.54 g·kg~(-1)), medium-(1.08 g·kg~(-1)) and high-dose(2.16 g·kg~(-1)) Fushen Granules every day. The bifico group received 2 mL(113.4 mg·kg~(-1)) of bifico solution every day. At the end of the 8th week, the levels of serum creatinine(Scr) and blood urea nitrogen(BUN) in each group were measured. The serum levels of hypersensitive C reactive protein(hs-CRP), tumor necrosis factor(TNF)-α, and interleukin(IL)-6 were measured, and the pathological changes in the colon tissue were observed by hematoxylin-eosin(HE) staining. The serum levels of lipopolysaccharide(LPS) and lipopolysaccharide-binding protein(LBP) of rats were measured, and the expression levels of LBP, TLR4, NF-κB p65, inhibitor of κB kinase α(IκBα), TNF-α, and IL-1ß in the colon tissue were determined. Compared with sham operation group, the model group had abnormal structure of all layers of colon tissue, sparse and shorter intestinal villi, visible edema in mucosal layer, wider gap, obvious local inflammatory cell infiltration, significantly decreased body weight(P<0.01), and significantly increased kidney function index(Scr, BUN) content(P<0.01). Serum levels of inflammatory cytokines(hs-CRP, TNF-α, IL-6), LPS and LBP were significantly increased(P<0.01), protein expressions of LBP, TLR4, NF-κB p65, TNF-α and IL-1ß were significantly increased(P<0.01), and protein expressions of IκBα were significantly decreased(P<0.01). Compared with model group, intestinal villi damage in colonic tissue of rats in low-, medium-and high-dose Fushen Granules groups and bifico group were alleviated to different degrees, edema in submucosa was alleviated, space was narrowed, and inflammatory cell infiltration in lamina propria was reduced. The contents of renal function index(Scr, BUN) and serum inflammatory factors(hs-CRP, TNF-α, IL-6) were significantly decreased(P<0.05 or P<0.01) in medium-and high-dose Fushen Granules groups and bifico group(P<0.05 or P<0.01). Serum LPS and LBP contents in Fushen Granules group and bifico group were significantly decreased(P<0.01), protein expressions of LBP, TLR4, NF-κB p65, TNF-α and IL-1ß in Fushen Granules group were significantly decreased(P<0.05 or P<0.01), and protein expressions of IκBα were significantly increased(P<0.01). The expression of LBP protein in bifico group was significantly decreased(P<0.01). The results suggest that Fushen Granules can protect the residual renal function of PD rats, reduce the inflammatory response, and protect the colon tissue. Based on network pharmacology, TLR4/NF-κB pathway may be the main signaling pathway of Fushen granule in the treatment of PDRP. The results showed that Fushen Granules could improve intestinal inflammation and protect intestinal barrier to prevent PDRP by regulating the expression of key factors in TLR4/NF-κB pathway in colon of PD rats.

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.

Nrf-2/ROS/NF-κB pathway is modulated by cynarin in human mesenchymal stem cells in vitro from ankylosing spondylitis.

Ankylosing spondylitis (AS) is an immune chronic inflammatory disease, resulting in back pain, stiffness, and thoracolumbar kyphotic deformity. Based on the reported anti-inflammatory and antioxidant capacities of cynarin (Cyn), this study explored its protective role and molecular mechanisms in mesenchymal stem cells (MSCs) from AS. The target pathways and genes were verified using Western blotting, quantitative real-time polymerase chain reaction, and immunofluorescent staining, while molecular docking analysis was conducted. In AS-MSCs, we found that the expression levels of p-NF-κB, IL-6, IL-1ß, and TNF-α were higher and IκB-α, Nrf-2, and HO-1 were lower compared with healthy control (HC)-MSCs. With molecular docking analysis, the biding affinities between Cyn and Keap1-Nrf-2 and p65-IκB-α were predicted. The mRNA and protein expression of p-NF-κB, IL-6, IL-1ß, and TNF-α and the reactive oxygen species (ROS) generation were downregulated following Cyn administration. Meanwhile, the expression level of IκB-α, Nrf-2, and HO-1 were significantly increased after Cyn pretreatment. The results suggested that the protective mechanisms of Cyn in AS-MSCs were based on enhancing the antioxidation and suppression of excessive inflammatory responses via Nrf-2/ROS/NF-κB axis. Our findings demonstrate that Cyn is a potential candidate for alleviating inflammation in AS.

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.

[Effects of conditioned medium of alveolar epithelial cells on vascular endothelial cell damage].

OBJECTIVE: To observe the effect of lipopolysaccharide (LPS) induced conditioned medium of alveolar epithelial cells on the inflammatory response and cell damage of vascular endothelial cells, and explore its mechanism. METHODS: The LPS induced type II alveolar epithelial cells (A549) conditioned medium was used as a stimulus to induce human umbilical vein endothelial cells (HUVEC) damage. The cell counting kit-8 (CCK-8) was used to detect the effect of 0% (blank group), 12.5%, 25%, 50%, 75% and 100% A549 cell conditioned medium cultured for 6, 12, 24 and 48 hours on the cell viability of HUVEC. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)] and vasoactive substances [vascular endothelial growth factor (VEGF), endothelin-1 (ET-1)] in the supernatant. Phalloidin staining was used to observe the effects of A549 cells conditioned medium on cell morphology. The expressions of protein kinase B/nuclear factor-κB (AKT/NF-κB) pathway in HUVEC induced by conditioned medium was detected by Western blotting. RESULTS: Compared with the blank group, A549 cells conditioned medium with concentrations of 12.5%, 25%, and 50% had no significant effects on cell viability of HUVEC after 6, 12, and 24 hours, but the activity of HUVEC decreased significantly after 48 hours. Therefore, 12.5%, 25%, 50% A549 cell conditioned medium stimulated for 24 hours was selected as the induction condition for follow-up experiments. Compared with the blank group, the level of IL-6 was significantly increased in 12.5% and 50% conditioned medium groups (ng/L: 2 438.95±64.89, 3 036.41±96.69 vs. 1 736.75±20.99, both P < 0.05), the level of TNF-α was significantly increased in 12.5% and 25% conditioned medium groups (ng/L: 174.08±11.09, 81.37±8.17 vs. 50.03±0.26, both P < 0.01), the levels of VEGF and ET-1 were significantly increased in 12.5%, 25% and 50% conditioned medium groups [VEGF (ng/L): 173.60±41.44, 192.49±12.38, 318.89±27.90 vs. 66.68±19.65; ET-1 (ng/L): 54.88±1.37, 36.69±0.29, 24.07±0.73 vs. 10.67±0.25, all P < 0.01]. Phalloidin staining showed that HUVEC induced by 25% A549 cells conditioned medium were irregular in shape, uneven in size, disordered in arrangement, widened in gap, dense and unclear in microfilament structure and serrated in cell membrane. Furthermore, the average fluorescence intensity of 25% conditioned medium group significantly increased compared to the blank group (67 205.60±3 430.40 vs. 56 272.67±7 650.95, P < 0.05). Western blotting showed that compared with the blank group, the expression of HUVEC cells phosphonated inhibitor α of NF-κB (p-IκBα) was significantly decreased in the 12.5%, 25%, and 50% conditioned medium groups (p-IκBα/IκBα: 0.38±0.08, 0.67±0.12, 0.31±0.07 vs. 1.00±0.00, all P < 0.01), the expressions of phosphonated-AKT (p-AKT) and VEGF were significantly increased (p-AKT/AKT: 1.50±0.18, 1.42±0.27, 1.61±0.14 vs. 1.00±0.00, VEGF/GAPDH: 1.37±0.10, 1.53±0.22, 1.40±0.12 vs. 1.00±0.00, all P < 0.05), the expression of phosphonated NF-κB p65 (p-P65) was significantly increased in the 25% conditioned medium group (p-P65/P65: 1.45±0.14 vs. 1.00±0.00, P < 0.05). CONCLUSIONS: LPS induced conditional culture medium of alveolar epithelial cells induced endothelial cell damage via activating AKT/NF-κB pathway.

Triptolide mitigates the inhibition of osteogenesis induced by TNF-α in human periodontal ligament stem cells via the p-IκBα/NF-κB signaling pathway: an in-vitro study.

BACKGROUND: Triptolide is a widely utilized natural anti-inflammatory drug in clinical practice. Aim of this study was to evaluate effects of triptolide on hPDLSCs osteogenesis in an inflammatory setting and to investigate underlying mechanisms. METHODS: Using the tissue block method to obtain hPDLSCs from extracted premolar or third molar. Flow cytometry, osteogenic and adipogenic induction were carried out in order to characterise the features of the cells acquired. hPDLSC proliferative activity was assessed by CCK-8 assay to determine the effect of TNF-α and/or triptolide. The impact of triptolide on the osteogenic differentiation of hPDLSCs was investigated by ALP staining and quantification. Osteogenesis-associated genes and proteins expression level were assessed through PCR and Western blotting assay. Finally, BAY-117,082 was used to study the NF-κB pathway. RESULTS: In the group treated with TNF-α, there was an elevation in inflammation levels while osteogenic ability and the expression of both osteogenesis-associated genes and proteins decreased. In the group co-treated with TNF-α and triptolide, inflammation levels were reduced and osteogenic ability as well as the expression of both osteogenesis-associated genes and proteins were enhanced. At the end of the experiment, both triptolide and BAY-117,082 exerted similar inhibitory effects on the NF-κB pathway. CONCLUSION: The osteogenic inhibition of hPDLSCs by TNF-α can be alleviated through triptolide, with the involvement of the p-IκBα/NF-κB pathway in this mechanism.

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.

The African swine fever virus MGF300-4L protein is associated with viral pathogenicity by promoting the autophagic degradation of IKKß and increasing the stability of IκBα.

African swine fever (ASF) is a highly contagious, often fatal viral disease caused by African swine fever virus (ASFV), which imposes a substantial economic burden on the global pig industry. When screening for the virus replication-regulating genes in the left variable region of the ASFV genome, we observed a notable reduction in ASFV replication following the deletion of the MGF300-4L gene. However, the role of MGF300-4L in ASFV infection remains unexplored. In this study, we found that MGF300-4L could effectively inhibit the production of proinflammatory cytokines IL-1ß and TNF-α, which are regulated by the NF-κB signaling pathway. Mechanistically, we demonstrated that MGF300-4L interacts with IKKß and promotes its lysosomal degradation via the chaperone-mediated autophagy. Meanwhile, the interaction between MGF300-4L and IκBα competitively inhibits the binding of the E3 ligase ß-TrCP to IκBα, thereby inhibiting the ubiquitination-dependent degradation of IκBα. Remarkably, although ASFV encodes other inhibitors of NF-κB, the MGF300-4L gene-deleted ASFV (Del4L) showed reduced virulence in pigs, indicating that MGF300-4L plays a critical role in ASFV pathogenicity. Importantly, the attenuation of Del4L was associated with a significant increase in the production of IL-1ß and TNF-α early in the infection of pigs. Our findings provide insights into the functions of MGF300-4L in ASFV pathogenicity, suggesting that MGF300-4L could be a promising target for developing novel strategies and live attenuated vaccines against ASF.

Tetramethylpyrazine inhibits the inflammatory response by downregulating the TNFR1/IκB-α/NF-κB p65 pathway after spinal cord injury.

Previous studies have demonstrated that tetramethylpyrazine (TMP) can enhance the recovery of motor function in spinal cord injury (SCI) rats. However, the underlying mechanism involved in this therapeutic effect remains to be elucidated. We conducted RNA sequencing with a network pharmacology strategy to predict the targets and mechanism of TMP for SCI. The modified Allen's weight-drop method was used to construct an SCI rat model. The results indicated that the nuclear transfer factor-κB (NF-κB) pathway was identified through the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and an inflammatory response was identified through the Gene Ontology (GO) enrichment analysis. Tumor necrosis factor (TNF) was identified as a crucial target. Western blotting revealed that TMP decreased the protein expression of TNF superfamily receptor 1 (TNFR1), inhibitor κB-α (IκB-α), and NF-κB p65 in spinal cord tissues. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) demonstrated that TMP inhibited TNF-α, interleukin-1ß (IL-1ß), reactive oxygen species (ROS), and malondialdehyde (MDA) expression and enhanced superoxide dismutase (SOD) expression. Histopathological observation and behavior assessments showed that TMP improved morphology and motor function. In conclusion, TMP inhibits inflammatory response and oxidative stress, thereby exerting a neuroprotective effect that may be related to the regulation of the TNFR1/IκB-α/NF-κB p65 signaling pathway.

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.