Nuclear factor-κB Signaling Pathway Research Articles

Abstract PS16-04: Denosumab as an enhancer of the immune infiltrate in hormone receptor-positive early breast cancer. Subgroup analysis from the D-Biomark window-of-opportunity clinical trial (NCT03691311)

Abstract Background: The receptor activator of nuclear factor κB (RANK) signaling pathway has emerged as a therapeutic target in breast cancer (BC). Recent studies indicate that inhibition of the RANK pathway induces tumor cell differentiation and may enhance the anti-tumor immune response. The D-Biomark clinical trial aims to evaluate the antitumor (antiproliferative and proapoptotic) and immunomodulatory effects of denosumab in HER2-negative early BC. Methods: Patients with early HER2-negative BC scheduled for primary surgery were randomized in a 2:1 ratio to receive two doses of 120 mg denosumab on days 1 and 8 vs no treatment before surgery. Immunohistochemistry (IHC) was used to assess Ki67 (proliferation), cleaved caspase-3 (cell survival), RANK and RANKL. Stromal tumor infiltrating lymphocytes (TILs) and serum markers including free RANKL (sRANKL), tartrate-resistant acid phosphatase 5b (TRACP5b), and osteoprotegerin (OPG) were also analyzed. Paired t-test was used to compare values between core biopsy (biopsy A) and surgical samples (biopsy B). Subgroup analysis by intrinsic subtype was performed on 47 matched cases using PAM50. Results: Between July 2019 and May 2021, we enrolled 60 patients, 58 evaluable, including 10 triple-negative breast cancer (TNBC). The clinicopathologic characteristics of the population at the time of enrollment were well balanced, but the TNBC recruited included 5 out of 10 tumors with a more indolent behavior than the typical tumors of this lineage (apocrine tumors, low cell proliferation). Due to this and the small number of cases, no conclusions can be drawn in this subgroup. RANK expression was detected in 19 tumor cases, while 17 cases expressed RANKL. The treated group showed a decrease in sRANKL (p < 0.000), indicating denosumab activity, while the control group showed no change (p1.0). OPG levels in the experimental group showed a non-significant increase (p0.07), while TRACP5b remained unchanged. Both groups showed a non-clinically relevant increase in cell proliferation (5 percentage points), control p0.04, experimental p0.01. Subgroup analysis of tumors with RANK+ or RANKL+ tumor cells also showed no reduction in Ki67. Cell survival did not decrease in the overall cohort (control p0.05, experimental p0.24), nor in subgroups or in tumors with RANK+ or RANKL+ tumor cells. Denosumab treatment increased TILs in the overall population (control p0.06, experimental p0.0006) and in the subgroups: RANK+ tumors, RANKL+ tumors, premenopausal and postmenopausal, less so in the TNBC group. The subgroup analysis is shown in Table 1. Analysis by intrinsic subtype showed that in the experimental group there was an increase in cases with a change to luminal A lineage (17%) compared to the control group (6%), suggesting a possible cellular differentiation towards less aggressive tumors. Although the number of patients is small, these changes warrant further investigation and highlight the role of denosumab as an immune activator in these luminal tumors known for their low inflammatory infiltrate. Conclusion: Two doses of denosumab prior to surgery did not reduce proliferation or increase apoptosis. However, this short course of denosumab increased TILs in early BC, particularly in luminal tumors, and may induce tumor cell differentiation into luminal A-like tumors. Table. Citation Format: Andrea Vethencourt, Eva Trinidad, Eduard Dorca, Anna Petit, Teresa Soler-Monsó, Agostina Stradella, Cristina Capo, Ander Urriticoechea, Marta Matas, Gema Pérez-Chacon, María Jimenez, Marina Ciscar, Emilia Brizzi, Gonzalo Soria Alcaide, Gonzalo Gomez, Elena Piñeiro, Elvira Purqueras, Amparo Garcia, Ariadna Iserte, M Jesus Pla, Miriam Campos, Miguel Gil-Gil, Sonia Pernas, Eva Gonzalez-Suarez, Catalina Falo. Denosumab as an enhancer of the immune infiltrate in hormone receptor-positive early breast cancer. Subgroup analysis from the D-Biomark window-of-opportunity clinical trial (NCT03691311) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS16-04.

In Vitro Screening of Lactic Acid Bacteria with RAW264.7 Macrophages and the Immunoregulatory Mechanism

Lactic acid bacteria (LAB) are commonly consumed as probiotics to improve gut barrier function and boost the immune system. This study aimed to screen LAB with high immunomodulatory activity using RAW264.7 macrophages. According to the results, Limosilactobacillus reuteri AUc2301 was selected from 84 screened strains that can stimulate RAW264.7 cell proliferation. Limosilactobacillus reuteri AUc2301 significantly enhanced the phagocytosis activity of RAW264.7 cells. In the ELISA test, Limosilactobacillus reuteri AUc2301 significantly promoted the release of interleukin-6, IL-1β, the tumor necrosis factor, and nitric oxide in RAW264.7 macrophages. In addition, Limosilactobacillus reuteri AUc2301 significantly inhibited the excessive release of IL-6, IL-1β, TNF-α, prostaglandin E2 as well as NO and the high expression of cyclooxygenase-2 in RAW264.7 macrophages induced by lipopolysaccharide. In further mechanism studies, Limosilactobacillus reuteri AUc2301 could regulate the nuclear factor-κB signaling pathway in RAW264.7 macrophages. Collectively, the screened Limosilactobacillus reuteri AUc2301 showed good immunomodulatory activity in vitro, and it has the potential to be developed as a novel probiotic.

Selenium Counteracts Tight Junction Disruption and Attenuates the NF-κB-Mediated Inflammatory Response in Staphylococcus aureus-Infected Mouse Mammary Glands.

Tight junctions (TJs) are the key determinant of barrier function in the mammary gland, with their disruption being associated with the pathogenesis and progression of mastitis, especially in the case of Staphylococcus aureus (S. aureus) infection. This study investigated whether selenium (Se) could attenuate S. aureus-induced mastitis by inhibiting inflammation and protecting mammary gland TJs in mice. The expression profiles of S. aureus-infected gland tissues derived from the gene expression omnibus dataset were analyzed. We found cytokine production, cell junctions, the nuclear transcription factor-κB (NF-κB) signalling pathway, and inflammatory responses associated with the differentially expressed genes, as revealed by Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses. Se reduced the mRNA expression and production of inflammatory cytokines, including tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and decreased phosphorylation levels of the NF-κB complex. Moreover, Se alleviated structural damage and microvillus injury in mammary glands. Immunohistochemical staining revealed that Se increased the expression of Claudin-3; Western blot analysis revealed increased protein levels of Occludin and Tricellulin in the group supplemented with dietary Se. In summary, Se counteracted TJ disruption and attenuated NF-κB-mediated inflammatory responses in S. aureus-infected mouse mammary glands.

Chemokines and lymphocyte homing in Sjögren's syndrome.

Sjögren's syndrome (SS) is a chronic systemic autoimmune disease that typically presents with lymphocyte, dendritic cell, and macrophage infiltration of exocrine gland ducts and the formation of ectopic germinal centers. The interactions of lymphocyte homing receptors and addressins and chemokines and their receptors, such as α4β7/MAdCAM-1, LFA-1/ICAM-1, CXCL13/CXCR5, CCL25/CCR9, CX3CL1/CX3CR1, play important roles in the migration of inflammatory cells to the focal glands and the promotion of ectopic germinal center formation in SS. A variety of molecules have been shown to be involved in lymphocyte homing, including tumor necrosis factor-α, interferon (IFN)-α, IFN-β, and B cell activating factor. This process mainly involves the Janus kinase-signal transducer and activator of transcription signaling pathway, lymphotoxin-β receptor pathway, and nuclear factor-κB signaling pathway. These findings have led to the development of antibodies to cell adhesion molecules, antagonists of chemokines and their receptors, compounds interfering with chemokine receptor signaling, and gene therapies targeting chemokines and their receptors, providing new targets for the treatment of SS in humans. The aim of this study was to explore the relationship between lymphocyte homing and the pathogenesis of SS, and to provide a review of recent studies addressing lymphocyte homing in targeted therapy for SS.

Sesquiterpene Lactones Containing an α-Methylene-γ-Lactone Moiety Selectively Down-Regulate the Expression of Tumor Necrosis Factor Receptor 1 by Promoting Its Ectodomain Shedding in Human Lung Adenocarcinoma A549 Cells.

Alantolactone is a eudesmane-type sesquiterpene lactone containing an α-methylene-γ-lactone moiety. Previous studies showed that alantolactone inhibits the nuclear factor κB (NF-κB) signaling pathway by targeting the inhibitor of NF-κB (IκB) kinase. However, in the present study, we demonstrated that alantolactone selectively down-regulated the expression of tumor necrosis factor (TNF) receptor 1 (TNF-R1) in human lung adenocarcinoma A549 cells. Alantolactone did not affect the expression of three adaptor proteins recruited to TNF-R1. The down-regulation of TNF-R1 expression by alantolactone was suppressed by an inhibitor of TNF-α-converting enzyme. Alantolactone increased the soluble forms of TNF-R1 that were released into the culture medium as an ectodomain. The structure-activity relationship of eight eudesmane derivatives revealed that an α-methylene-γ-lactone moiety was needed to promote TNF-R1 ectodomain shedding. In addition, parthenolide and costunolide, two sesquiterpene lactones with an α-methylene-γ-lactone moiety, increased the amount of soluble TNF-R1. Therefore, the present results demonstrate that sesquiterpene lactones with an α-methylene-γ-lactone moiety can down-regulate the expression of TNF-R1 by promoting its ectodomain shedding in A549 cells.

Matrilin-3 supports neuroprotection in ischemic stroke by suppressing astrocyte-mediated neuroinflammation

In the brain, the role of matrilin-3, an extracellular matrix component in cartilage, is unknown. Here, we identify that matrilin-3 decreased in reactive astrocytes but was unchanged in neurons after ischemic stroke in animals. Importantly, it declined in serum of patients with acute ischemic stroke. Genetic or pharmacological inhibition or supplementation of matrilin-3 aggravates or reduces brain injury, astrocytic cell death, and glial scar, respectively, but has no direct effect on neuronal cell death. RNA sequencing demonstrates that Matn3-/- mice display an increased inflammatory response profile in the ischemic brain, including the nuclear factor κB (NF-κB) signaling pathway. Both endogenous and exogenous matrilin-3 reduce inflammatory mediators. Mechanistically, extracellular matrilin-3 enters astrocytes via caveolin-1-mediated endocytosis. Cytoplasmic matrilin-3 translocates into the nucleus by binding to NF-κB p65, suppressing inflammatory cytokine transcription. Extracellular matrilin-3 binds to BMP-2, blocking the BMP-2/Smads pathway. Thus, matrilin-3 is required for astrocytes to exert neuroprotection, at least partially, by suppressing astrocyte-mediated neuroinflammation.

NF-κB affected the serum levels of TNF-α and IL-1β via activation of the MAPK/NF-κB signaling pathway in rat model of acute pulmonary microthromboembolism.

Pulmonary thromboembolism caused by thrombi blocking major pulmonary artery and its branches, is a frequently encountered phenomenon and an important cause of high morbidity and mortality in lung diseases and may develop into persistent pulmonary hypertension (PH). Nuclear factor-κB (NF-κB) signaling pathway had been reported participated in the formation and development of PH by promoting inflammatory response. The aim of this study was to investigate the effects of NF-κB activation on the serum levels of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in acute pulmonary microthromboembolism (APMTE) rats. Rats were randomized into five groups. APMTE group received jugular vein injection of autologous thrombus, while control group rats received normal saline injection. Pulmonary hemodynamic parameters were measured through ECHO-guided transthoracic puncture. Pulmonary vascular morphological changes were analyzed by HE. The expression changes of NF-κB and serum TNF-α、IL-1β levels were detected by enzyme-linked immunosorbent assay. Protein expression of the MAPK/NF-κB signaling pathway including p-IκBα, p-p38 MAPK, p-NF-κB p65, IκBα, p38 MAPK, and NF-κB p65 was determined using western blot analysis. Compared with control group, the expression of NF-κB in lung tissue and the levels of serum TNF-α and IL-1β rats were higher, a significant reduction in IκBα and elevation in the phosphorylation of IκBα, p38 MAPK, and NF-κB p65 were found in APMTE group rats. And UK administration reversed the APMTE-induced increase in TNF-α, IL-1β, p-IκBα, p-MAPK, and p-NF-κB protein. Furthermore, the levels of NF-κB, TNF-α, and IL-1β were positively correlated with mean pulmonary artery. And the levels of TNF-α and IL-1β were positively correlated with NF-κB. These findings suggest that the activation of MAPK/NF-κB pathway as a critical driver of increasing TNF-α and IL-1β level in APMTE rats and UK exerted protective effects against APMTE-induced PH may be related to the downregulation of the MAPK/NF-κB signaling pathway.

Anti-Inflammatory Properties of the Citrus Flavonoid Diosmetin: An Updated Review of Experimental Models.

Inflammation is an essential contributor to various human diseases. Diosmetin (3',5,7-trihydroxy-4'-methoxyflavone), a citrus flavonoid, can be used as an anti-inflammatory agent. All the information in this article was collected from various research papers from online scientific databases such as PubMed and Web of Science. These studies have demonstrated that diosmetin can slow down the progression of inflammation by inhibiting the production of inflammatory mediators through modulating related pathways, predominantly the nuclear factor-κB (NF-κB) signaling pathway. In this review, we discuss the anti-inflammatory properties of diosmetin in cellular and animal models of various inflammatory diseases for the first time. We have identified some deficiencies in current research and offer suggestions for further advancement. In conclusion, accumulating evidence so far suggests a very important role for diosmetin in the treatment of various inflammatory disorders and suggests it is a candidate worthy of in-depth investigation.

Amiodarone inhibits the Toll-like receptor 3-mediated nuclear factor κB signaling pathway by blocking organelle acidification

Toll-like receptor (TLR) agonists or pro-inflammatory cytokines converge to activate the nuclear factor κB (NF-κB) signaling pathway, which provokes inflammatory responses. In the present study, we identified amiodarone hydrochloride as a selective inhibitor of the TLR3-mediated NF-κB signaling pathway by screening the RIKEN NPDepo Chemical Library. In human umbilical vein endothelial cells (HUVEC), amiodarone selectively inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) induced by polyinosinic-polycytidylic acid (Poly(I:C)), but not tumor necrosis factor-α, interleukin-1α, or lipopolysaccharide. In response to a Poly(I:C) stimulation, amiodarone at 20 μM reduced the up-regulation of mRNA expression encoding ICAM-1, vascular cell adhesion molecule-1, and E-selectin. The nuclear translocation of the NF-κB subunit RelA was inhibited by amiodarone at 15–20 μM in Poly(I:C)-stimulated HUVEC. Amiodarone diminished the fluorescent dots of LysoTracker® Red DND-99 scattered over the cytoplasm of HUVEC. Therefore, the present study revealed that amiodarone selectively inhibited the TLR3-mediated NF-κB signaling pathway by blocking the acidification of intracellular organelles.

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 (IL1β) 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 IL1β 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.

Hawthorn Fruit (Crataegus spp.) Polysaccharides Exhibit Immunomodulatory Activity on Macrophages via TLR4/NF-κB Signaling Activation.

The immunostimulatory effects and the involved molecular mechanisms of polysaccharides from hawthorn fruit (Crataegus spp.) have not been well understood. In this study, the chemical composition, monosaccharide composition, uronic acid content, and structural features of hawthorn fruit polysaccharides (HFP) and the two collected fractions were analyzed. Both AF1-2 and AF2 have pectic-like structural features rich in galacturonic acid. AF2 showed superior proinflammatory effects on macrophages which significantly increased the secretion of pro-inflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α, but not AF1-2. AF2 was found to activate the nuclear factor-κB signaling pathway with suppressed expression of IκBα but up-regulated expression of p-IκBα and nuclear factor-κB P65. The surface binding site of AF2 on macrophage cells was characterized and toll like receptor-4 was responsible for AF2 induced activation of down-stream nuclear factor-κB signaling pathways. AF2 from hawthorn fruit could be potentially used as a natural source of immunomodulator in functional foods.

Evaluating the mode of action of perfluorooctanoic acid-induced liver tumors in male Sprague-Dawley rats using a toxicogenomic approach

The mode of action (MOA) underlying perfluorooctanoic acid (PFOA)-induced liver tumors in rats is proposed to involve peroxisome proliferator-activated receptor α (PPARα) agonism. Despite clear PPARα activation evidence in rodent livers, the mechanisms driving cell growth remain elusive. Herein, we used dose-responsive apical endpoints and transcriptomic data to examine the proposed MOA. Male Sprague-Dawley rats were treated with 0, 1, 5, and 15 mg/kg PFOA for 7, 14, and 28 days via oral gavage. We showed PFOA induced hepatomegaly along with hepatocellular hypertrophy in rats. PPARα was activated in a dose-dependent manner. Toxicogenomic analysis revealed six early biomarkers (Cyp4a1, Nr1d1, Acot1, Acot2, Ehhadh, and Vnn1) in response to PPARα activation. A transient rise in hepatocellular DNA synthesis was demonstrated while Ki-67 labeling index showed no change. Transcriptomic analysis indicated no significant enrichment in pathways related to DNA synthesis, apoptosis, or the cell cycle. Key cyclins including Ccnd1, Ccnb1, Ccna2, and Ccne2 were dose-dependently suppressed by PFOA. Oxidative stress and the nuclear factor-κB signaling pathway were unaffected. Overall, evidence for PFOA-induced hepatocellular proliferation was transient within the studied timeframe. Our findings underscore the importance of considering inter-species differences and chemical-specific effects when evaluating the carcinogenic risk of PFOA in humans.

Network pharmacology and experimental verification study on the mechanism of Hedyotis diffusa Willd in treating colorectal cancer.

This study aimed to evaluate the pharmacological mechanism of Hedyotis diffusa Willd against CRC (colorectal cancer) using network pharmacological analysis combined with experimental validation. The active components and potential targets of Hedyotis diffusa Willd were screened from the tax compliance management program public database using network pharmacology. The core anti-CRC targets were screened using a protein-protein interaction (PPI) network. The mRNA and protein expression of core target genes in normal colon and CRC tissues and their relationship with overall CRC survival were evaluated using The Cancer Genome Atlas (TCGA), Human Protein Atlas (HPA), and Gene Expression Profiling Interactive Analysis (GEPIA) databases. Functional and pathway enrichment analyses of the potential targets were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The first six core targets with stable binding were molecular-docked with the active components quercetin and β-sitosterol. Finally, the results of network pharmacology were verified using in vitro experiments. In total, 149 potential targets were identified by searching for seven types of active components and the intersection of all potential and CRC targets. PPI network analysis showed that ten target genes, including tumor protein p53 (TP53) and recombinant cyclin D1 (CCND1), were pivotal genes. GO enrichment analysis involved 2043 biological processes, 52 cellular components, and 191 molecular functions. KEGG enrichment analysis indicated that the anticancer effects of H. alba were mediated by tumor necrosis factor, interleukin-17, and nuclear factor-κB (NF-κB) signaling pathways. Validation of key targets showed that the validation results for most core genes were consistent with those in this study. Molecular docking revealed that the ten core target proteins could be well combined with quercetin and β-sitosterol and the structure remained stable after binding. The results of the in vitro experiment showed that β-sitosterol inhibited proliferation and induced apoptosis in SW620 cells. This study identified a potential target plant for CRC through network pharmacology and in vitro validation.

Epigallocatechin-3-gallate protects sepsis‐induced myocardial dysfunction by inhibiting the nuclear factor-κB signaling pathway

Sepsis-induced myocardial dysfunction (SIMD) has become one of the most lethal complications of sepsis, while the treatment was limited by a shortage of pertinent drugs. Epigallocatechin-3-gallate (EGCG) is the highest content of active substances in green tea, and its application in cardiovascular diseases has broad prospects. This study was conducted to test the hypothesis that EGCG was able to inhibit lipopolysaccharide (LPS) induced myocardial dysfunction and investigate the underlying molecular mechanisms. The cardiac systolic function was assessed by echocardiography. The cardiomyocyte apoptosis was determined by TUNEL staining. The expression of inflammatory factors and apoptosis-related protein, cardiac markers were examined by Western Blot and qRT-PCR. EGCG effectively improve LPS-induced cardiac function damage, enhance left ventricular systolic function, and restore myocardial cell vitality. It can effectively inhibit the upregulation of TLR4 expression induced by LPS and inhibit IκB α/NF- κB/p65 signaling pathway, thereby inhibiting cardiomyocyte apoptosis and improving myocarditis. In conclusion, EGCG protects against SIMD through anti-inflammatory and anti-apoptosis effects; it was mediated by the inhibition of the TLR4/NF-κB signal pathway. Our results demonstrated that EGCG might be a possible medicine for SIMD prevention and treatment.

CHL1 inhibits cell proliferation, migration and invasion by regulating the NF‑κB signaling pathway in colorectal cancer.

Cell adhesion molecule close homolog of L1 (CHL1) is implicated in tumorigenesis of various malignancies. However, its role and underlying molecular mechanisms in colorectal cancer (CRC) remain unclear. The present study aimed to evaluate the specific biological functions and mechanisms of CHL1, in order to provide a theoretical basis for the use of CHL1 as a biological target in CRC. CHL1 expression was originally determined in CRC cell lines. Subsequently, CHL1 overexpression was induced by plasmid transfection in HT29 and SW480 cells, and cell proliferation, migration and invasion were evaluated using the Cell Counting Kit-8, clone formation, organoids formation and Transwell assays. Immunofluorescence and western blotting were performed to assess the protein expression of E-cadherin or N-cadherin. Differentially expressed genes (DEGs) were further evaluated using RNA-sequencing (RNA-seq) in HT29 and SW480 cells following CHL1 overexpression and functional enrichment analysis. Western blotting was performed to validate the expression of proteins related to the nuclear factor κB (NF-κB) signaling pathway. The TNMplot online database revealed the significant downregulation of CHL1 in CRC tissues. The results indicated that exogenous CHL1 overexpression significantly inhibited the proliferative, organoid-forming, migratory and invasive abilities of HT29 and SW480 cells, and increased E-cadherin protein expression. Additionally, CHL1 overexpression reduced xenograft tumor growth in vivo. RNA-seq and functional analysis revealed that DEGs in CHL1 overexpressing cells were mainly enriched in the NF-κB signaling pathway. The expression of p-p65 and p-p65/p65 ratio were significantly reduced in HT29 and SW480 cells, following CHL1 overexpression. Additionally, the inhibitory effects of CHL1 overexpression on CRC cell proliferation, organoid formation, migration and invasion were partially counteracted following the overexpression of p65 expression. Overall, the present study demonstrates that CHL1 inhibits CRC cell growth, migration and invasion through the inactivation of the NF-κB signaling pathway.

Alantolactone derivatives inhibit the tumor necrosis factor α-induced nuclear factor κB pathway by a different mechanism from alantolactone

Alantolactone is a eudesmane-type sesquiterpene lactone that exerts various biological effects, including anti-inflammatory activity. In the present study, screening using the RIKEN Natural Products Depository chemical library identified alantolactone derivatives that inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells stimulated with proinflammatory cytokines and Toll-like receptor ligands. In human lung adenocarcinoma A549 cells stimulated with tumor necrosis factor-α (TNF-α), six alantolactone derivatives inhibited ICAM-1 expression in a dose-dependent manner and at IC50 values of 13–21 μM, whereas that of alantolactone was 5 μM. Alantolactone possesses an α-methylene-γ-lactone moiety, whereas alantolactone derivatives do not. In the nuclear factor κB (NF-κB) signaling pathway, alantolactone prevented the TNF-α-induced phosphorylation and degradation of the inhibitor of NF-κB α (IκBα) protein, and its downstream signaling pathway. In contrast, alantolactone derivatives neither reduced TNF-α-induced IκBα degradation nor the nuclear translocation of the NF-κB subunit RelA, but inhibited the binding of RelA to the ICAM-1 promoter. The inhibitory activities of alantolactone and alantolactone derivatives were attenuated by glutathione. These results indicate that alantolactone derivatives inhibit the TNF-α-induced NF-κB pathway by a different mechanism from alantolactone.

S-Nitrosylation of Septin2 Exacerbates Aortic Aneurysm and Dissection by Coupling the TIAM1-RAC1 Axis in Macrophages.

S-Nitrosylation (SNO), a prototypic redox-based posttranslational modification, is involved in cardiovascular disease. Aortic aneurysm and dissection are high-risk cardiovascular diseases without an effective cure. The aim of this study was to determine the role of SNO of Septin2 in macrophages in aortic aneurysm and dissection. Biotin-switch assay combined with liquid chromatography-tandem mass spectrometry was performed to identify the S-nitrosylated proteins in aortic tissue from both patients undergoing surgery for aortic dissection and Apoe-/- mice infused with angiotensin II. Angiotensin II-induced aortic aneurysm model and β-aminopropionitrile-induced aortic aneurysm and dissection model were used to determine the role of SNO of Septin2 (SNO-Septin2) in aortic aneurysm and dissection development. RNA-sequencing analysis was performed to recapitulate possible changes in the transcriptome profile of SNO-Septin2 in macrophages in aortic aneurysm and dissection. Liquid chromatography-tandem mass spectrometry and coimmunoprecipitation were used to uncover the TIAM1-RAC1 (Ras-related C3 botulinum toxin substrate 1) axis as the downstream target of SNO-Septin2. Both R-Ketorolac and NSC23766 treatments were used to inhibit the TIAM1-RAC1 axis. Septin2 was identified S-nitrosylated at cysteine 111 (Cys111) in both aortic tissue from patients undergoing surgery for aortic dissection and Apoe-/- mice infused with Angiotensin II. SNO-Septin2 was demonstrated driving the development of aortic aneurysm and dissection. By RNA-sequencing, SNO-Septin2 in macrophages was demonstrated to exacerbate vascular inflammation and extracellular matrix degradation in aortic aneurysm. Next, TIAM1 (T lymphoma invasion and metastasis-inducing protein 1) was identified as a SNO-Septin2 target protein. Mechanistically, compared with unmodified Septin2, SNO-Septin2 reduced its interaction with TIAM1 and activated the TIAM1-RAC1 axis and consequent nuclear factor-κB signaling pathway, resulting in stronger inflammation and extracellular matrix degradation mediated by macrophages. Consistently, both R-Ketorolac and NSC23766 treatments protected against aortic aneurysm and dissection by inhibiting the TIAM1-RAC1 axis. SNO-Septin2 drives aortic aneurysm and dissection through coupling the TIAM1-RAC1 axis in macrophages and activating the nuclear factor-κB signaling pathway-dependent inflammation and extracellular matrix degradation. Pharmacological blockade of RAC1 by R-Ketorolac or NSC23766 may therefore represent a potential treatment against aortic aneurysm and dissection.

Sasanquasaponin from Camellia oleifera Abel Exerts an Anti-Inflammatory Effect in RAW 264.7 Cells via Inhibition of the NF-κB/MAPK Signaling Pathways.

Sasanquasaponin (SQS), a secondary metabolite that is derived from Camellia seeds, reportedly possesses notable biological properties. However, the anti-inflammatory effects of SQS and its underlying mechanisms remain poorly explored. Herein, we aimed to investigate the anti-inflammatory properties of SQS against lipopolysaccharide (LPS)-induced inflammatory responses in RAW264.7 cells, focusing on the nuclear factor-κB (NF-κB) and MAPK signaling pathways. SQS was isolated using a deep eutectic solvent and D101 macroporous adsorption resin and analyzed using high-performance liquid chromatography. The viability of LPS-stimulated RAW264.7 was assessed using the CCK-8 assay. The presence of reactive oxygen species (ROS) was evaluated using 2',7'-dichlorofluorescein-diacetate. The expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were detected using reverse transcription-quantitative PCR and ELISA. Western blot was performed to analyze the protein expression of LPS-induced RAW264.7 cells. Herein, SQS exhibited anti-inflammatory activity: 30 μg/mL of SQS significantly reduced ROS generation, inhibited the LPS-induced expression of iNOS and COX-2, and attenuated the production of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. The anti-inflammatory activity was potentially mediated by inhibiting the phosphorylation of IκBα and p65 in the NF-κB signaling pathway and the phosphorylation of ERK and JNK in the MAPK signaling pathway. Accordingly, SQS could inhibit inflammation in LPS-induced RAW264.7 cells by suppressing the NF-κB and MAPK signaling pathways. This study demonstrated the potential application of SQS as an anti-inflammatory agent.

Protective effects of Prussian blue nanozyme against sepsis-induced acute lung injury by activating HO-1

Sepsis is a life-threatening condition involving dysfunctional organ responses stemming from dysregulated host immune reactions to various infections. The lungs are most prone to failure during sepsis, resulting in acute lung injury (ALI). ALI is associated with oxidative stress and inflammation, and current therapeutic strategies are limited. To develop a more specific treatment, this study aimed to synthesise Prussian blue nanozyme (PBzyme), which can reduce oxidative stress and inflammation, to alleviate ALI. PBzyme with good biosafety was synthesised using a modified hydrothermal method. PBzyme was revealed to be an activator of haem oxygenase-1 (HO-1), improving survival rate and ameliorating lung injury in mice. Zinc protoporphyrin, an inhibitor of HO-1, inhibited the prophylactic therapeutic efficacy of PBzyme on ALI, and affected the nuclear factor-κB signaling pathway and activity of HO-1. This study demonstrates that PBzyme can alleviate oxidative stress and inflammation through HO-1 and has a prophylactic therapeutic effect on ALI. This provides a new strategy and direction for the clinical treatment of sepsis-induced ALI.

Protective Effects of Four Structurally Distinct Sanshools Ameliorate Dextran Sodium Sulfate-Induced Ulcerative Colitis by Restoring Intestinal Barrier Function and Modulating the Gut Microbiota.

Hydroxy-α-sanshool (HAS), hydroxy-β-sanshool (HBS), hydroxy-γ-sanshool (HRS), and γ-sanshool (RS) are the key components from the Zanthoxylum genus, processing a range of pharmacological activities. The present study investigated the protective capacities of four sanshools on a dextran sulfate sodium (DSS)-induced model of ulcerative colitis (UC). The results showed that sanshool administration alleviated the colitis symptoms by reducing body weight loss and disease activity index (DAI) score, increasing the colon length, and improving colonic injury and the change in immune organ weight. Furthermore, sanshools enhanced the antioxidant enzyme activities, and RS exhibited the lowest effect on the improvement in total antioxidative capacity (T-AOC) and antioxidant abilities compared to the other three sanshools. The p65 nuclear factor κB (p65 NFκB) signaling pathway was inhibited to prevent hyperactivation and decreased the production of inflammatory factors. The gut barrier function in DSS-induced mice was restored by increasing goblet cell number and levels of tight junction proteins (zonula occludens-1, occludin, and claudin-1), and the levels of protein in HAS and HRS groups were higher than that in the HBS group, significantly. The analysis of gut microbiota suggested that sanshool administration significantly boosted the abundance of Lachnospiraceae, Muribaculaceae, Oscillospiraceae, and Alistipes and reduced the level of Buchnera in colitis mice. Collectively, the sanshool treatment could ameliorate colitis by resisting colon injury and regulating intestinal barrier dysfunction and gut microbiota dysbiosis; meanwhile, HRS and HAS have better improvement effects.