NF-κB Inhibitor Research Articles

Integrative bioinformatic approach reveals novel melatonin-related biomarkers for Alzheimer’s disease

Background: Melatonin (MLT) can improve mitophagy, thereby ameliorating cognitive deficits in Alzheimer’s disease (AD) patients. Hence, our research focused on the potential value of MLT-related genes (MRGs) in AD through bioinformatic analysis. Methods: First, the key cells in the single-cell dataset GSE138852 were screened out based on the proportion of annotated cells and Fisher’s test between the AD and control groups. The differentially expressed genes (DEGs) in the key cell and GSE5281 datasets were identified, and the MRGs in GSE5281 were selected via weighted gene coexpression network analysis. After intersecting two sets of DEGs and MRGs, we performed Mendelian randomization analysis to identify the MRGs causally related to AD. Biomarkers were further ascertained through receiver operating characteristic curve (ROC) and expression analysis in GSE5281 and GSE48350. Furthermore, gene set enrichment analysis, immune infiltration analysis and correlation analysis with metabolic pathways were conducted, as well as construction of a regulator network and molecular docking. Results: According to the Fisher test, oligodendrocytes were regarded as key cells due to their excellent abundance in the GSE138852 dataset, in which there were 281 DEGs between the AD and control groups. After overlapping with 3,490 DEGs and 550 MRGs in GSE5281, four genes were found to be causally related to AD, namely, G protein-coupled receptor, family C, group 5, member B (GPRC5B), Methyltransferase-like protein 7 A (METTL7A), NF-κB inhibitor alpha (NFKBIA) and RAS association domain family 4(RASSF4). Moreover, GPRC5B, NFKBIA and RASSF4 were deemed biomarkers, except for METTL7A, because of their indistinctive expression between the AD and control groups. Biomarkers might be involved in oxidative phosphorylation, adipogenesis and heme metabolism. Moreover, T helper type 17 cells, natural killer cells and CD56dim natural killer cells were significantly correlated with biomarkers. Transcription factors (GATA2, POU2F2, NFKB1, etc.) can regulate the expression of biomarkers. Finally, we discovered that all biomarkers could bind to MLT with a strong binding energy. Conclusion: Our study identified three novel biomarkers related to MLT for AD, namely, GPRC5B, NFKBIA and RASSF4, providing a novel approach for the investigation and treatment of AD patients.

HO-1 represses NF-κB signaling pathway to mediate microglia polarization and phagocytosis in intracerebral hemorrhage.

Microglia polarization plays a crucial role in inflammatory injury of brain following intracerebral hemorrhage (ICH). Heme oxygenase-1 (HO-1) has demonstrated protective properties against inflammation and promote hematoma clearance after ICH. The objective of this study was to explore impacts of HO-1 on microglia polarization and phagocytosis after ICH, along with the underlying mechanism. ICH model was constructed in C57BL/6 mice. Neurological deficit of ICH mice was evaluated. HE detected pathological changes of mouse brain tissue. Immunofluorescence staining tested co-localization between HO-1 or NF-κB p65 and IBA1. The expressions of gene and proteins were detected by RT-qPCR and Western blot, respectively. Flow cytometry determined microglial polarization phenotype and neuron apoptosis. Cell viability of neuron was assessed by CCK-8. Red blood cells labeled by PKH-26 and co-cultured with microglia for examining microglial erythrophagocytosis. Both HO-1 and NF-κB p65 phosphorylation were elevated in brain tissues of ICH mice. ZnPP, a HO-1 inhibitor, could exacerbate microglial M1 polarization and nerve injury, as well as repress microglial erythrophagocytosis in vitro and hematoma clearance in vivo. On the contrary, Tat-NBD, a NF-κB inhibitor, greatly suppressed microglial M1 polarization, and induced M2 polarization and microglial erythrophagocytosis, thus improving nerve injury and hematoma clearance after ICH. Notably, it was observed that NF-κB p65 could be activated by ZnPP treatment, and the regulatory roles of ZnPP on microglial polarization and erythrophagocytosis after ICH in vivo and in vitro were all diminished by Tat-NBD. Therefore, our data demonstrated that HO-1 alleviated nerve injury and induced M2 polarization and phagocytosis of microglia after ICH via inhibiting NF-κB signaling pathway, which could provide deepen the pathological understanding of ICH and provide potential intervention targets and drug candidate for ICH.

Systems biology-enabled targeting of NF-κΒ and BCL2 overcomes microenvironment-mediated BH3-mimetic resistance in DLBCL.

In Diffuse Large B-cell Lymphoma (DLBCL), elevated anti-apoptotic BCL2-family proteins (e.g., MCL1, BCL2, BCLXL) and NF-κB subunits (RelA, RelB, cRel) confer poor prognosis. Heterogeneous expression, regulatory complexity, and redundancy offsetting the inhibition of individual proteins, complicate the assignment of targeted therapy. We combined flow cytometry "fingerprinting", immunofluorescence imaging, and computational modeling to identify therapeutic vulnerabilities in DLBCL. The combined workflow predicted selective responses to BCL2 inhibition (venetoclax) and non-canonical NF-κB inhibition (Amgen16). Within the U2932 cell line we identified distinct resistance mechanisms to BCL2 inhibition in cellular sub-populations recapitulating intratumoral heterogeneity. Co-cultures with CD40L-expressing stromal cells, mimicking the tumor microenvironment (TME), induced resistance to BCL2 and BCLXL targeting BH3-mimetics via cell-type specific upregulation of BCLXL or MCL1. Computational models, validated experimentally, showed that basal NF-κB activation determined whether CD40 activation drove BH3-mimetic resistance through upregulation of RelB and BCLXL, or cRel and MCL1. High basal NF-κB activity could be overcome by inhibiting BTK to resensitize cells to BH3-mimetics in CD40L co-culture. Importantly, non-canonical NF-κB inhibition overcame heterogeneous compensatory BCL2 upregulation, restoring sensitivity to both BCL2- and BCLXL-targeting BH3-mimetics. Combined molecular fingerprinting and computational modelling provides a strategy for the precision use of BH3-mimetics and NF-κB inhibitors in DLBCL.

Aerosol inhalation of rhIL-10 improves acute lung injury in mice by affecting pulmonary neutrophil phenotypes through neutrophil-platelet aggregates.

This study investigates the therapeutic effects of recombinant human IL-10 (rhIL-10) administered via aerosol inhalation in acute lung injury (ALI), with a particular focus on neutrophils. It explores how rhIL-10, in the presence of platelets, modulates neutrophil polarization to ameliorate acute lung injury. Initially, the ALI model established in mice demonstrated that aerosol inhalation of rhIL-10 significantly mitigated the cytokine storm in the lungs, reduced pulmonary edema, and alleviated histopathological damage to lung tissue. Additionally, rhIL-10 administration was found to decrease neutrophil infiltration and platelet activation in the lungs of mice, inhibiting the formation of platelet-neutrophil aggregates (PNAs) and promoting the differentiation of neutrophils toward an anti-inflammatory phenotype in the presence of platelets. Subsequently, primary neutrophils and platelets were isolated from mouse bone marrow and blood to explore the underlying mechanisms. The results indicated that rhIL-10 promotes the expression of the signal transducer and activator of transcription 3 (STAT3) and the suppressor of cytokine signaling 3 (SOCS3) in neutrophils while inhibiting the activation of the nuclear factor kappa B (NF-κB) and the NF-κB inhibitor (IκB), which in turn enhances CD40 expression. This interaction facilitates the formation of PNAs and influences neutrophil phenotype differentiation. Furthermore, the application of the STAT3 phosphorylation inhibitor Stattic and CD40 antibody in vivo provided further validation of this potential mechanism. In conclusion, these results indicate that aerosol inhalation of rhIL-10 effectively ameliorates ALI. The underlying mechanism may involve the modulation of the neutrophil STAT/SOCS-IκB/NF-κB-CD40 signaling pathway, promoting interactions between neutrophils and platelets that facilitate the differentiation of neutrophils toward an anti-inflammatory phenotype.

Inhibition of DNA Methyltransferase DNMT1 Reverses Th2 Response Polarisation and Alleviates Allergic Rhinitis.

Type 2 T helper (Th2) cells-mediated immune response plays vital roles in allergic rhinitis (AR), and DNA methylation is previously found to be closely related to AR development. Our study aims to reveal the detail mechanism of DNA methylation affecting Th2 response in AR. Mice were stimulated with ovalbumin (OVA) to induce AR symptoms, and CD4+ T cells were subjected to Th2 induction culture. Real-time quantitative PCR, western blot, flow cytometry, and enzyme-linked immunosorbent assay were performed to analyse the activation of Th2 response. DNA methyltransferase 1 (DNMT1) was significantly upregulated in OVA-induced AR model mice, and DNMT1 knockdown alleviated AR symptoms and pathological changes of nasal mucosa tissues in the model mice. DNMT1 knockdown obviously reduced the expression of GATA binding protein 3 (GATA3), the ratio of interleukin (IL)-4+CD4+ cells and the release of Th2 cytokines, but elevated the expression of T-box expressed in T cells (T-bet), the ratio of interferon (IFN)-γ+CD4+ cells and the levels of Th1 cytokines to improve Th1/Th2 imbalance in the model mice and Th2-induced CD4+T cells. Mechanistically, DNMT1 promoted promoter methylation of forkhead box O3 (FOXO3), inhibited FOXO3 expression and activated the nuclear factor kappa-B (NF-κB)/GATA3 signalling. FOXO3 overexpression remarkably inactivated the NF-κB/GATA3 pathway and mitigated Th2 polarisation in DNMT1-deficient and Th2-conditined CD4+T cells, which was reversed by a NF-κB inhibitor. Altogether, DNMT1 downregulated FOXO3 expression to activate the NF-κB/GATA3 pathway and promote Th2 response in AR.

Role of TNFRSF12A in cell proliferation, apoptosis, and proinflammatory cytokine expression by regulating the MAPK and NF-κB pathways in thyroid cancer cells.

Tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) has been reported to be upregulated in thyroid cancer (THCA). However, the role and mechanism of TNFRSF12A in THCA remain largely unknown. TNFRSF12A expression in THCA samples was analyzed using bioinformatics analysis. CCK-8, EdU incorporation assay, TUNEL, and caspase-3 activity assay was used to detect cell proliferation and apoptosis in THCA cells. Correlated genes of TNFRSF12A were identified using LinkedOmics database and subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Western blot analysis was performed to determine proliferating cell nuclear antigen (PCNA), cyclin D1 (CCND1), Bax, and Bcl-2 expression and to analyze the effect of TNFRSF12A on mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) pathways. Results showed that TNFRSF12A was increased in THCA tissue samples and cells. KEGG analysis showed that correlated genes of TNFRSF12A were significantly enriched in MAPK and NF-κB signaling pathways. Moreover, TNFRSF12A knockdown inactivated the MAPK and NF-κB signaling pathways in THCA cells. TNFRSF12A silencing alone or combined with inhibitor of ERK (PD98059), JNK (SP600125), p38 (SB203580), or NF-κB (Bay 11-7082) impeded cell proliferation and reduced PCNA and CCND1 expression in THCA cells. Meanwhile, TNFRSF12A knockdown alone or combined with PD98059, SP600125, SB203580, or Bay 11-7082 facilitated cell apoptosis, increased caspase-3 activity, downregulated Bcl-2 expression, and upregulated Bax expression in THCA cells. TNFRSF12A knockdown alone or combined with PD98059, SP600125, SB203580, or Bay 11-7082 also decreased the expression levels of proinflammatory cytokines IL-1β, IL-6, and IL-8 in THCA cells. On the contrary, TNFRSF12A overexpression showed an opposite effect. Treatment with PD98059, SP600125, SB203580, or Bay 11-7082 reversed the effects of TNFRSF12A overexpression on cell proliferation, apoptosis, and proinflammatory cytokine expression. In conclusion, the effects of TNFRSF12A on proliferation, apoptosis, and proinflammatory cytokine expression in THCA cells were regulated by the MAPK and NF-κB pathways.

Abstract TP375: Ischemic Insult Under Hyperhomocysteinemic Condition Triggers Early Onset Of Inflammatory Response Causing Exacerbation Of Brain Injury

Hyperhomocysteinemia, a metabolic disorder characterized by systemic elevation of amino acid homocysteine, is linked to deficiency in vitamins and enzymes involved in homocysteine metabolism. The incidence of hyperhomocysteinemia is high in the elderly population due to reduced micronutrient absorption and metabolism. Recent preclinical studies in animal models of stroke showed that predisposition to hyperhomocysteinemia exacerbates ischemic brain injury and worsens behavioral deficits, establishing hyperhomocysteinemia as a potential comorbidity for stroke. These studies further showed that hyperhomocysteinemia-induced stimulation of GluN2A subunit containing NMDA receptors (GluN2A-NMDARs) in conjunction with glutamate-induced stimulation of GluN2B subunit containing NMDARs (GluN2B-NMDARs) exacerbates stroke pathology. However, the precise intracellular mechanisms involved in such exacerbation of ischemic brain injury is not known. The current study sought to investigate whether under hyperhomocysteinemic condition GluN2A-NMDAR mediated early activation of the transcription factor Nuclear Factor-κB (NFκB) and subsequent expression of the pro-inflammatory chemokine Monocyte chemoattractant protein-1 (MCP-1) enhances post-ischemic inflammatory response and exacerbation of brain damage. Adult male Wistar rats were made hyperhomocysteinemic by implantation of Alzet osmotic pumps containing L-homocysteine. The animals were hyperhomocysteinemic within 3 days with a total plasma homocysteine level of ~24 μM. Acute ischemic stroke was induced by middle cerebral artery occlusion for 60 min followed by reperfusion (6h). Brain tissues were processed for MCP-1 ELISA and RT-PCR. A significant increase in MCP-1 mRNA and protein levels were observed in the ipsilateral cortex of hyperhomocysteinemic rats compared to control rats within 6h of reperfusion. An accelerated brain damage was also observed at this time in the hyperhomocysteinemic rats. Inhibition of GluN2A-NMDAR (NVP-AAM077, i.v.) and NFκB (BMS345541, IKK inhibitor; i.p) at the onset of ischemic insult attenuated increase in MCP-1 mRNA and protein levels, as well as exacerbation of brain injury under hyperhomocysteinemic condition. The study provides compelling evidence for a novel role of GluN2A-NMDAR in promoting neuro-inflammatory response that contributes to exacerbation of stroke pathology under hyperhomocysteinemic condition.

Fangchinoline alleviates the progression of osteoarthritis through the nuclear factor kappa B signaling pathway.

Osteoarthritis is a progressive, chronic joint disease characterized by pain, stiffness, and limited mobility, which can lead to physical disability in severe cases. Owing to its complex pathological features, effective treatments for osteoarthritis are lacking. Fangchinoline is a natural alkaloid found in the tuberous roots of plants belonging to the Menispermaceae family. Fangchinoline reportedly possesses anti-inflammatory, antioxidant, and anticancer properties; however, its role in osteoarthritis progression remains unclear. In this study, we investigated the protective effects and potential mechanisms of fangchinoline against osteoarthritis. In vitro, we confirmed that fangchinoline alleviates interleukin-1β-induced cartilage inflammation, reduces the levels of metabolic factors, such as inducible nitric oxide synthase and matrix metalloproteinase-3, and modulates the expression of aggrecan, which enhances extracellular matrix synthesis. In vivo, we demonstrated that fangchinoline can ameliorate articular cartilage degeneration and reduce inflammatory destruction in a destabilization of the medial meniscus mouse model. The nuclear factor kappa B (NF-κB) signaling pathway in osteoarthritis has been a primary target for drug development, and our results suggest that fangchinoline exerts anti-inflammatory effects by inhibiting the activity of IKKα/β. Using an in vitro human cartilage culture model, we further validated that fangchinoline significantly mitigates cartilage degeneration and inflammation by modulating the NF-κB signaling pathway. This evidence highlights its dual action in preserving cartilage integrity and suppressing inflammatory responses. These findings collectively underscore fangchinoline as a potent inhibitor of NF-κB, capable of attenuating key pathological processes associated with osteoarthritis. Therefore, fangchinoline emerges as a promising therapeutic candidate for slowing the progression of osteoarthritis.

HcCnAα regulates NF-κB signaling in Hyriopsis cumingii by interacting with HcIKK and facilitating IκB phosphorylation.

Calcineurin (CN), a serine/threonine protein phosphatase regulated by Ca2+ and calmodulin, plays a crucial role in the immune response of bivalves. In this study, we examined the effects of gene silencing of the CN subunit (HcCnAα) in Hyriopsis cumingii on the expression of genes associated with the NF-κB signaling pathway, as well as the phosphorylation of the inhibitor of NF-κB (IκB), through RNA interference (RNAi), quantitative PCR (qPCR), and Western blot (WB) analyses. The IκB kinase (HcIKK) and B-cell CLL/lymphoma 10 (HcBcl10) genes of H. cumingii were cloned using rapid amplification of cDNA ends, and protein interactions with HcCnAα were investigated through yeast two-hybrid assays. The results demonstrated that RNAi-mediated knockdown of HcCnAα significantly reduced the expression of IKK, p65, and downstream immune-related genes in the NF-κB signaling pathway-Lys, The, Def, α2-M, TNF-α, and IL-17-all showing significant decreases (P<0.05). Additionally, phosphorylation of IκB was inhibited. These findings suggest that HcCnAα plays a regulatory role in the NF-κB signaling pathway, influencing the expression of downstream immune response-related genes and defense proteins. Furthermore, yeast two-hybrid assay results indicated a direct protein-protein interaction between HcIKK and HcCnAα, while no interaction was observed between HcBcl10 and HcCnAα. This study elucidates the specific molecular mechanisms by which HcCnAα regulates the immune response in H. cumingii, providing a foundational basis for further exploration of immune regulation mechanisms and the development of disease prevention strategies in bivalves.

NF-κB inhibitor PDTC involved in regulating the transplantation immunity in the pearl oyster Pinctada fucata martensii

NF-κB inhibitor PDTC involved in regulating the transplantation immunity in the pearl oyster Pinctada fucata martensii

Stem Cell Therapy Modulates Molecular Cues of Vasogenic Edema Following Ischemic Stroke: Role of Sirtuin-1 in Regulating Aquaporin-4 Expression.

Conventional post-stroke edema management strategies are limitedly successful as in multiple cases of hemorrhagic transformation is being reported. Clinically, acute-ischemic-stroke (AIS) intervention by endovascular mesenchymal stem cells (MSCs) have shown benefits by altering various signaling pathways. Our previous studies have reported that intra-arterial administration of 1*105 MSCs (IA-MSCs) were beneficial in alleviating post-stroke edema by modulating PKCδ/MMP9/AQP4 axis and helpful in preserving the integrity of blood-brain-barrier (BBB). However, the role of mitochondrial dysfunction and ROS generation post-AIS cannot be overlooked in context to the alteration of the BBB integrity and edema formation through the activation of inflammatory pathways. The anti-inflammatory activity of IA-MSCs in stroke has been reported to be regulated by sirtuin-1 (SIRT-1). Hence, the relationship between SIRT-1 and AQP4 towards regulation of post-stroke edema needs to be further explored. Therefore, the present study deciphers the molecular events towards AQP4 upregulation, mitochondrial dysfunction and BBB disruption in context to the modulation of SIRT-1/PKCδ/NFκB loop by IA-MSCs administration. Ovariectomized SD rats were subjected to focal ischemia. SIRT-1 activator, SIRT-1 inhibitor, NFkB inhibitor and IA-MSCs were administered at optimized dose. At 24h of reperfusion, behavioral tests were performed, and brains were harvested following euthanasia for molecular studies. IA-MSCs downregulated AQP4, PKCδ and NFkB expression, and upregulated SIRT-1 expression. SIRT-1 upregulation renders mitochondrial protection via reduction of oxidative stress resulting in BBB protection. IA-MSCs can modulate SIRT-1 mediated AQP4 expression via mitochondrial ROS reduction and modification of NFkB transcriptional regulation.

Ablation of the deubiquitinating enzyme cylindromatosis (CYLD) augments STAT1-mediated M1 macrophage polarization and fosters Staphylococcus aureus control

In atopic dermatitis (AD), lesional skin is frequently colonized by Staphylococcus aureus, which promotes clinical symptoms of the disease. The inflammatory milieu in the skin is characterized by a Th2 response, including M2 macrophages, which cannot eradicate S. aureus. Therefore, repolarization of macrophages toward the M1 phenotype may foster control of S. aureus. Our data show that the deubiquitinating enzyme cylindromatosis (CYLD) is strongly expressed in macrophages of AD patients and prevents the clearance of S. aureus. Mechanistically, CYLD impaired M1 macrophage polarization by K63-specific deubiquitination of STAT1 and activation of the NF-κB pathway via its interaction with TRAF6, NEMO, and RIPK2. Inhibition of STAT1 and NF-κB, independently, abolished the differences between S. aureus-infected CYLD-deficient and CYLD-competent M1 macrophages. Infection of Cyld-deficient and wild-type mice with S. aureus confirmed the protective CYLD function. Collectively, our study shows that CYLD impairs the control of S. aureus in macrophages of AD patients, identifying CYLD as a potential therapeutic target.

Bisdemethoxycurcumin and Curcumin Alleviate Inflammatory Bowel Disease by Maintaining Intestinal Epithelial Integrity and Regulating Gut Microbiota in Mice.

Curcuminoids, found in turmeric (Curcuma longa L.), include curcumin (CUR), demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). Although CUR and DMC are well-studied, the anti-inflammatory effects of BDMC remain less explored. Recent studies highlight BDMC's stronger NF-κB inhibition compared to CUR and DMC in cell models, along with its ability to target pathways associated with inflammatory bowel disease (IBD) in DSS-induced colitis mice, reflected by lower disease activity scores and reduced inflammation. This study assessed CUR and BDMC in a DSS-induced colitis mouse model. Dietary administration of CUR or BDMC strengthened tight junction (TJ) proteins, reduced inflammatory cytokine secretion, and attenuated intestinal inflammatory protein expression, thereby alleviating DSS-induced IBD in mice. Furthermore, gut microbiota and short-chain fatty acid analyses revealed that CUR and BDMC effectively regulated gut microbial imbalance and promoted the relative abundance of butyrate-producing bacteria. Furthermore, CUR showed low absorption and was primarily excreted in feces, while BDMC had higher absorption levels. In conclusion, while both BDMC and CUR have potential as adjunct therapies for IBD, BDMC at a concentration of 0.1% showed strong anti-inflammatory effects and enhanced TJ proteins, suggesting that BDMC, even at lower concentrations than CUR, holds promising therapeutic potential and prospects.

Increasing cellular NAD+ protects hepatocytes against palmitate-induced lipotoxicity by preventing PARP-1 inhibition and the mTORC1-p300 pathway activation.

Hepatic lipotoxicity, resulting from excessive lipid accumulation in hepatocytes, plays a central role in the pathogenesis of various metabolic liver diseases. Despite recent progress, the precise mechanisms remain incompletely understood. Employing excessive exposure to palmitate in hepatocytes as our primary experimental model and mice studies, we aimed to uncover the mechanisms behind hepatic lipotoxicity, thereby developing potential treatments. Our data reveal for the first time that exposure to palmitate leads to downregulated expression of poly(ADP-ribose) polymerase 1 (PARP-1) in hepatocytes, inhibiting its enzymatic activity. While inhibiting PARP-1 worsens palmitate-induced hepatotoxicity, preventing PARP-1 suppression, using NAD+ precursors, nicotinamide N-methyltransferase (NNMT) inhibitors, or a poly(ADP-ribose) glycohydrolase (PARG) inhibitor, prevents it. Moreover, we uncover that PARP-1 suppression contributes to palmitate-triggered mTORC1 activation, which has been previously reported by us to contribute to palmitate-induced hepatocyte cell death. Furthermore, our results identify p300 as a downstream target of mTORC1 activation upon palmitate exposure. Importantly, p300 inhibition via either pharmacological or genetic approaches protects against palmitate hepatotoxicity. Additionally, we provide evidence that the TLR4-NF-κB pathway activation in response to palmitate plays a mechanistic role in mediating palmitate-induced PARP-1 downregulation in that both TLR4 antagonist and NF-κB inhibitors prevent palmitate induced PARP-1 reduction and protect against hepatocyte cell death. In conclusion, our study presents new evidence that the PARP-1-mTORC1-p300 pathway serves as a novel molecular mechanism underlying palmitate-induced hepatic lipotoxicity. Targeting the PARP-1 pathway by increasing cellular NAD+ availability either through its precursor supplementation or by inhibiting its degradation represents a promising therapeutic approach for treating hepatic lipotoxicity.

Glycosylated Delphinidins Decrease Chemoresistance to Temozolomide by Regulating NF-κB/MGMT Signaling in Glioblastoma

Glioblastoma (GB) is a highly malignant brain tumor with a poor prognosis, with a median survival of only 14.6 months despite aggressive treatments. Resistance to chemotherapy, particularly temozolomide (TMZ), is a significant challenge. The DNA repair enzyme MGMT and glioblastoma stem cells (GSCs) often mediate this resistance. Recent studies highlight the therapeutic potential of natural compounds, particularly delphinidins, found in deep purple berries. Delphinidins are known for their ability to inhibit NF-κB signaling, a critical pathway for GB progression, chemoresistance, and MGMT expression. Our research demonstrates that glycosylated delphinidins have potential adjuvant use in the treatment of GB, offering a promising natural strategy to combat TMZ resistance. Specifically, we observed that delphinidin 3,5 di-glucoside has potent anticancer effects when used alone. Meanwhile, delphinidin 3 glucoside acted in synergy with temozolomide to decrease cell viability, highlighting its potential as an adjuvant. It also exerted a faster and more sustained inhibition of NF-κB, highlighting its potential for long-lasting therapeutic effects. These findings open new avenues for targeted therapies against glioblastoma, particularly to overcome treatment resistance.

Fluorouracil enhances the anti-pancreatic cancer effect of anti-PD-L1 antibodies via up-regulating the expression of PD-L1 in cancer cells.

Pancreatic cancer is characterized by occult onset, low early diagnosis rate, rapid progress, and poor prognosis. Due to the low response rate and low programmed cell death ligand 1 (PD-L1) expression in pancreatic cancer, the therapeutic application of PL-L1 inhibitors in pancreatic cancer is greatly limited. In vitro studies showed that the expression of PD-L1 increased in pancreatic cancer cells stimulated by fluorouracil (5-FU). We aim to explore the combined effect of 5-FU and anti-PD-L1 antibodies and to provide a reference for the clinical application of PD-L1 antibodies in pancreatic cancer. In the current study, male BALB/c mice were adopted to construct a tumor-bearing model of pancreatic cancer cells. 5-FU and anti-mouse PD-L1 antibodies were combined and administered to evaluate their synergistic effects. The enhancing immune cytotoxicity effect of 5-FU sensitizing the anti-PD-L1 antibody in vivo and in vitro was analyzed by immunohistochemistry and western blot assays. Results showed that 5-FU and anti-PD-L1 antibody combination increased the expression of PD-L1 and IFN-γ, and infiltration of CD8+ T lymphocytes in pancreatic xenograft tumor tissues, which was proven by immunohistochemistry and western analysis. Moreover, the combination with the 5-FU remarkably enhanced the immune cytotoxicity of anti-PD-L1 antibodies in mice. In vitro analysis demonstrates that 5-FU increases the expression of PD-L1 on the surface of pancreatic cancer cell lines via up-regulating nuclear factor kappa B (NF-κB) and Protein kinase B (AKT) pathways. This synergistic effect could be abolished by NF-κB and AKT inhibitors.

P0132 ACSS2 inhibition alleviates inflammatory signaling and improves epithelial integrity in Inflammatory Bowel Disease in vitro models

Abstract Background Inflammatory bowel disease (IBD), encompassing Ulcerative Colitis (UC) and Crohn’s Disease (CD), is marked by chronic gastrointestinal inflammation stemming from genetic, environmental, immunological, and microbiota factors1. Acetyl-CoA Synthetase Short Chain Family Member 2 (ACSS2) converts acetate into acetyl-CoA and is primarily cytosolic, but translocates to the nucleus under metabolic stress, where it regulates gene transcription and cellular processes2. Although the gastrointestinal microbiota influences acetate levels, the role of ACSS2 in colitis and intestinal inflammation remains poorly understood3. Methods To evaluate ACSS2's role in IBD, we employed in vitro IBD models using Caco-2 and HT-29 intestinal epithelial cell lines. ACSS2 activity was inhibited with an ACSS2 inhibitor, followed by inflammatory stimulation with 100 ng/ml Tumor Necrosis Factor-α (TNF-α) for 30 minutes or 1% Dextran Sodium Sulfate (DSS) for 6 hours. Protein expression levels were assessed via Western blot and immunohistochemistry (IHC) on paraffin-embedded samples from UC patients. The effects of ACSS2 inhibition on inflammatory signaling pathways and cell integrity were assessed using RT-qPCR, immunoblotting, and transepithelial electrical resistance (TEER) measurements. Results Treatment of Caco-2 and HT-29 cells with DSS or TNF-α induced upregulation of ACSS2 protein levels compared to controls. IHC analysis of 30 paired paraffin-embedded colon samples from UC patients revealed higher ACSS2 expression and nuclear localization in inflamed sections compared to non-inflamed regions. Inhibition of ACSS2 activity in stimulated Caco-2 and HT29 cells led to a significant decrease in the gene expression levels of pro-inflammatory cytokines such as Interleukin 8 (IL-8), IL-1β and TNF-α. Additionally, immunoblotting demonstrated reduced phosphorylation of p65 and ERK/MEK, indicating suppression of NF-κB and MAPK inflammatory signaling pathways. To assess the effect of ACSS2 inhibition on cell integrity, TEER assays revealed that ACSS2 inhibition preserved cell integrity in DSS-treated monolayers, accompanied by increased expression of tight junction proteins Occludin and ZO-1, suggesting improved epithelial integrity in the inflammatory context. Conclusion Our findings demonstrate a pro-inflammatory role for ACSS2 in IBD, mediated through its involvement in inflammatory signaling and epithelial integrity. These results highlight ACSS2 as a potential therapeutic target for IBD and warrant further investigations in more advanced models, including human-derived intestinal organoids and in vivo systems, to validate its role in disease pathogenesis and therapeutic potential.

DEF6 regulates renal ischemia reperfusion injury through suppressing the WWP2 mediated ubiquitination of PARP1.

Renal ischemia-reperfusion injury (RIRI) stands as an unavoidable complication arising from kidney surgery, profoundly intertwined with its prognosis. The role of differentially expressed in FDCP 6 homolog (DEF6) in RIRI remains elusive, despite its confirmation as a potential therapeutic target for diverse diseases. Here, we investigated the mechanism by which DEF6 regulated RIRI. RNA sequencing data and IP-MS were used to identify the expression and potential targets of DEF6 through bioinformatics analysis. To elucidate the impact of DEF6 on RIRI, both an in vivo model of RIRI in mice and an in vitro model of kidney cell hypoxia/reoxygenation were established. Biochemical and histological analyses were used to investigate the influence of DEF6 on kidney damage mediated by RIRI. We confirmed that DEF6 was upregulated during RIRI and had a close correlation with RIRI-related inflammation and apoptosis. Moreover, inhibition of DEF6 could mitigate RIRI-induced kidney damage, inflammation, and apoptosis. Through our comprehensive mechanistic investigation, we revealed that DEF6 interacts with poly ADP-ribose polymerase 1 (PARP1) and suppresses the ubiquitination of PARP1. Inhibition of DEF6 resulted in reduced cleaveage of PARP1, leading to a marked suppression of PARP1-mediated apoptosis activation. The aggravation effect on inflammation and apoptosis achieved through DEF6 was nullified by the inhibition of NF-κB and Bax/Bcl2 signaling activation through PARP1 deletion. The findings from our study indicate that DEF6 suppressed the WWP2 mediated ubiquitination of PARP1 and modulates the activation of NF-κB and Bax/Bcl2 pathway, thus involved in RIRI-induced inflammation and apoptosis. These results suggest that DEF6 holds promise as a potential therapeutic target for mitigating RIRI.

Computer-Aided Design of Self-Assembled Nanoparticles to Enhance Cancer Chemoimmunotherapy via Dual-Modulation Strategy.

The rational design of self-assembled compounds is crucial for the highly efficient development of carrier-free nanomedicines. Herein, based on computer-aided strategies, important physicochemical properties are identified to guide the rational design of self-assembled compounds. Then, the pharmacophore hybridization strategy is used to design self-assemble nanoparticles by preparing new chemical structures by combining pharmacophore groups of different bioactive compounds. Hydroxychloroquine is grafted with the lipophilic vitamin E succinate and then co-assembled with bortezomib to fabricate the nanoparticle. The nanoparticle can reduce M2-type tumor-associated macrophages (TAMs) through lysosomal alkalization and induce immunogenic cell death (ICD) and nuclear factor-κB (NF-κB) inhibition in tumor cells. In mouse models, the nanoparticles induce decreased levels of M2-type TAMs, regulatory T cells, and transforming growth factor-β (TGF-β), and increase the proportion of cytotoxicity T lymphocytes. Additionally, the nanoparticles reduce the secretion of Interleukin-6 (IL-6) by inhibiting NF-κB and enhance the programmed death ligand-1 (PD-L1) checkpoint blockade therapy. The pharmacophore hybridization-derived nanoparticle provides a dual-modulation strategy to reprogram the tumor microenvironment, which will efficiently enhance the chemoimmunotherapy against triple-negative breast cancer.

Anemonin suppresses sepsis-induced acute lung injury by inactivation of nuclear factor-kappa B and activation of nuclear factor erythroid 2-related factor-2/heme oxygenase-1 pathway.

Sepsis-induced acute lung injury (ALI) is a common acute and severe reason of death in the intensive care unit. Although the pathogenesis is complicated and multifactorial, elevated inflammation and oxidative stress are considered as fundamental mechanisms for the progression of ALI. Anemonin is a natural compound with diverse biological properties including anti-inflammatory and anti-oxidative effects. To identify whether anemonin has protective effects on sepsis-induced ALI, a mouse sepsis-induced ALI model and cellular models using the mouse alveolar macrophage MH-S cells and mouse lung epithelial MLE-12 cells were established. Our results showed that anemonin reduced lipopolysaccharide (LPS)-induced mortality, and improved sepsis-induced ALI in the mouse model, as shown by improved histopathological changes, decreased lung wet/dry weight ratio, and myeloperoxidase activity. Anemonin alleviated LPS-induced secretion of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in bronchoalveolar lavage fluid samples, as well as reversed the LPS-caused increase in malondialdehyde (MDA) content and decrease in activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) in lung tissues. In the cellular model, anemonin inhibited the LPS-induced inflammatory responses and oxidative stress in MH-S and MLE-12 cells. In addition, anemonin inhibited LPS-induced nuclear factor-kappa B (NF-κB) pathway, while enhancing the activation of nuclear factor erythroid 2-related factor-2 (Nrf2) in lung tissues, MH-S, and MLE-12 cells. NF-κB inhibition enhanced the anti-inflammatory and anti-oxidative effects of anemonin, while Nrf2 knockdown attenuated these effects of anemonin, implying the critical roles of NF-κB and Nrf2. These results indicated that anemonin suppressed sepsis-induced acute lung injury by inhibition of NF-κB and activation of Nrf2/heme oxygenase-1 pathway, suggesting that anemonin might be developed as a new therapeutic agent for the treatment of sepsis-induced ALI.