P65 Subunit Research Articles

The Prolonged Activation of the p65 Subunit of the NF-Kappa-B Nuclear Factor Sustains the Persistent Effect of Advanced Glycation End Products on Inflammatory Sensitization in Macrophages.

Advanced glycation end products (AGEs) prime macrophages for lipopolysaccharide (LPS)-induced inflammation. We investigated the persistence of cellular AGE-sensitization to LPS, considering the nuclear content of p50 and p65 nuclear factor kappa B (NFKB) subunits and the expression of inflammatory genes. Macrophages treated with control (C) or AGE-albumin were rested for varying intervals in medium alone before being incubated with LPS. Comparisons were made using one-way ANOVA or Student t-test (n = 6). AGE-albumin primed macrophages for increased responsiveness to LPS, resulting in elevated levels of TNF, IL-6, and IL-1beta (1.5%, 9.4%, and 5.6%, respectively), compared to C-albumin. TNF, IL-6, and IL-1 beta secretion persisted for up to 24 h even after the removal of AGE-albumin (area under the curve greater by 1.6, 16, and 5.2 times, respectively). The expressions of Il6 and RelA were higher 8 h after albumin removal, and Il6 and Abca1 were higher 24 h after albumin removal. The nuclear content of p50 remained similar, but p65 showed a sustained increase (2.9 times) for up to 24 h in AGE-albumin-treated cells. The prolonged activation of the p65 subunit of NFKB contributes to the persistent effect of AGEs on macrophage inflammatory priming, which could be targeted for therapies to prevent complications based on the AGE-RAGE-NFKB axis.

Metabotropic Glutamate Receptor 8 Suppresses M1 Polarization in Microglia by Alleviating Endoplasmic Reticulum Stress and Mitochondrial Dysfunction.

Microglia-mediated neuroinflammation is a hallmark of neurodegeneration. Metabotropic glutamate receptor 8 (GRM8) has been reported to promote neuronal survival in neurodegenerative diseases, yet the effect of GRM8 on neuroinflammation is still unclear. Calcium overload-induced endoplasmic reticulum (ER)-mitochondrial miscommunication has been reported to trigger neuroinflammation in the brain. The aim of this study was to investigate putative anti-inflammatory effects of GRM8 in microglia, specifically focusing on its role in calcium overload-induced ER stress and mitochondrial dysfunction. BV2 microglial cells were pretreated with GRM8 agonist prior to lipopolysaccharide administration. Pro-inflammatory cytokine levels and the microglial polarization state in BV2 cells were then quantified. Cellular apoptosis and the viability of neuron-like PC12 cells co-cultured with BV2 cells were examined using flow cytometry and a Cell Counting Kit-8, respectively. The concentration of cAMP, inositol-1,4,5-triphosphate receptor (IP3R)-dependent calcium release, ER Ca2+ concentration, mitochondrial function as reflected by reactive oxygen species levels, ATP production, mitochondrial membrane potential, expression of ER stress-sensing protein, and phosphorylation of the nuclear factor kappa B (NF-κB) p65 subunit were also quantified in BV2 cells. GRM8 activation inhibited pro-inflammatory cytokine release and shifted microglia polarization towards an anti-inflammatory-like phenotype in BV2 cells, as well as promoting neuron-like PC12 cell survival when co-cultured with BV2 cells. Mechanistically, microglial GRM8 activation significantly inhibited cAMP production, thereby desensitizing the IP3R located within the ER. This process markedly limited IP3R-dependent calcium release, thus restoring mitochondrial function while inhibiting ER stress and subsequently deactivating NF-κB signaling. Our results indicate that GRM8 activation can protect against microglia-mediated neuroinflammation by attenuating ER stress and mitochondrial dysfunction, and that IP3R-mediated calcium signaling may play a vital role in this process. GRM8 may thus be a potential target for limiting neuroinflammation.

Analysis of Inhibition Potential of Nimbin and its Analogs against NF-κB Subunits p50 and p65: A Molecular Docking and Molecular Dynamics Study.

Cancer remains the major cause of morbidity and mortality. The nuclear factor kappa-B (NF- κB) plays an indispensable role in cancer cell proliferation and drug resistance. The role of NF-κB is not only limited to tumor cell proliferation and suppression of apoptotic genes but it also induces EMT transition responsible for metastasis. Inhibition of the NF-κB pathway in cancer cells by herbal derivatives makes it a favorable yet promising target for cancer therapeutics. The purpose of the study is to explore the inhibition potential of Nimbin and its analogs against NF-κB subunits p50 and p65. In the present study, an herbal compound Nimbin and its derivative analogs were investigated to examine their impact on the p50 and p65 subunits of the NF-κB signaling pathway using in silico tools, namely molecular docking and simulation. The molecular docking analysis revealed that Nimbin and its analogs may bind to p50 and p65 subunits with dG bind values ranging from -33.23 to -50.49 Kcal/mol. Interestingly, molecular dynamic simulation for the NO5-p65 complex displayed a stable conformation and convergence when compared to the NO4-p50 complex. These results indicate that NO5 may have a potential inhibitory effect against NF-κB subunit p65, which needs to be further validated in in vitro and in vivo systems. Also, the results obtained emphasize and pave the way for exploring the Nimbin scaffold against NF-κB inhibition for cancer therapeutics.

Hydroxychavicol Inhibits In Vitro Osteoclastogenesis via the Suppression of NF-κB Signaling Pathway.

Hydroxychavicol, a primary active phenolic compound of betel leaves, previously inhibited bone loss in vivo by stimulating osteogenesis. However, the effect of hydroxychavicol on bone remodeling induced by osteoclasts is unknown. In this study, the anti-osteoclastogenic effects of hydroxychavicol and its mechanism were investigated in receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclasts. Hydroxychavicol reduced the number of tartrate resistance acid phosphatase (TRAP)-positive multinucleated, F-actin ring formation and bone-resorbing activity of osteoclasts differentiated from RAW264.7 cells in a concentration-dependent manner. Furthermore, hydroxychavicol decreased the expression of osteoclast-specific genes, including cathepsin K, MMP-9, and dendritic cell-specific transmembrane protein (DC-STAMP). For mechanistic studies, hydroxychavicol suppressed RANKL-induced expression of major transcription factors, including the nuclear factor of activated T-cells 1 (NFATc1), c-Fos, and c-Jun. At the early stage of osteoclast differentiation, hydroxychavicol blocked the phosphorylation of NF-κB subunits (p65 and Iκβα). This blockade led to the decrease of nuclear translocation of p65 induced by RANKL. In addition, the anti-osteoclastogenic effect of hydroxychavicol was confirmed by the inhibition of TRAP-positive multinucleated differentiation from human peripheral mononuclear cells (PBMCs). In conclusion, hydroxychavicol inhibits osteoclastogenesis by abrogating RANKL-induced NFATc1 expression by suppressing the NF-κB signaling pathway in vitro.

CD81 suppresses NF-κB signaling and is downregulated in hepatitis C virus expressing cells.

The tetraspanin CD81 is one of the main entry receptors for Hepatitis C virus, which is a major causative agent to develop liver cirrhosis and hepatocellular carcinoma (HCC). Here, we identify CD81 as one of few surface proteins that are downregulated in HCV expressing hepatoma cells, discovering a functional role of CD81 beyond mediating HCV entry. CD81 was downregulated at the mRNA level in hepatoma cells that replicate HCV. Kinetics of HCV expression were increased in CD81-knockout cells and accompanied by enhanced cellular growth. Furthermore, loss of CD81 compensated for inhibition of pro-survival TBK1-signaling in HCV expressing cells. Analysis of functional phenotypes that could be associated with pro-survival signaling revealed that CD81 is a negative regulator of NF-κB. Interaction of the NF-κB subunits p50 and p65 was increased in cells lacking CD81. Similarly, we witnessed an overall increase in the total levels of phosphorylated and cellular p65 upon CD81-knockout in hepatoma cells. Finally, translocation of p65 in CD81-negative hepatoma cells was markedly induced upon stimulation with TNFα or PMA. Altogether, CD81 emerges as a regulator of pro-survival NF-κB signaling. Considering the important and established role of NF-κB for HCV replication and tumorigenesis, the downregulation of CD81 by HCV and the associated increase in NF-κB signaling might be relevant for viral persistence and chronic infection.

Critical Role of histone deacetylase 3 in the regulation of kidney inflammation and fibrosis

Chronic kidney disease (CKD) is characterized by renal inflammation and fibrosis. However, the precise mechanisms leading to renal inflammation and fibrosis are poorly understood. In this study, we examined the role of histone deacetylase 3 (HDAC3) in the regulation of inflammation and renal fibrosis. HDAC3 is induced in the kidneys of animal models of CKD. Mice with conditional HDAC3 deletion exhibited significantly reduced fibrosis in the kidneys compared with control mice. The expression of pro-inflammatory and pro-fibrotic genes was significantly increased in the fibrotic kidneys of control mice, which were impaired in mice with HDAC3 deletion. Genetic deletion or pharmacological inhibition of HDAC3 reduced the expression of proinflammatory genes in cultured monocytes/macrophages. Mechanistically, HDAC3 deacetylates Lys122 of NF-κB p65 turning on transcription. RGFP966, a selective HDAC3 inhibitor, reduced fibrosis in cells and animal models. Taken together, our study identifies HDAC3 as a critical regulator of renal inflammation and fibrosis through deacetylation of NF-κB unlocking its transcriptional activity and suggests targeting HDAC3 could serve as a novel therapeutic strategy for CKD. Chronic kidney disease (CKD) is characterized by kidney inflammation and fibrosis. However, the precise mechanisms leading to kidney inflammation and fibrosis are poorly understood. Since histone deacetylase is involved in inflammation and fibrosis in other tissues, we examined the role of histone deacetylase 3 (HDAC3) in the regulation of inflammation and kidney fibrosis. HDAC3 is induced in the kidneys of animal models of CKD but mice with conditional HDAC3 deletion exhibit significantly reduced fibrosis in the kidneys compared with control mice. The expression of proinflammatory and profibrotic genes was significantly increased in the fibrotic kidneys of control mice, which was impaired in mice with HDAC3 deletion. Genetic deletion or pharmacological inhibition of HDAC3 reduced the expression of proinflammatory genes in cultured monocytes/macrophages. Mechanistically, HDAC3 deacetylates Lys122 of NF-κB p65 subunit turning on transcription. RGFP966, a selective HDAC3 inhibitor, reduced fibrosis in cells and in animal models by blocking NF-κB p65 binding to κB-containing DNA sequences. Thus, our study identified HDAC3 as a critical regulator of inflammation and fibrosis of the kidney through deacetylation of NF-κB unlocking its transcriptional activity. Hence, targeting HDAC3 could serve as a novel therapeutic strategy for CKD.

Mutant KRAS-activated circATXN7 fosters tumor immunoescape by sensitizing tumor-specific T cells to activation-induced cell death

Mutant KRAS (KRASMUT) is often exploited by cancers to shape tumor immunity, but the underlying mechanisms are not fully understood. Here we report that tumor-specific cytotoxic T lymphocytes (CTLs) from KRASMUT cancers are sensitive to activation-induced cell death (AICD). circATXN7, an NF-κB-interacting circular RNA, governs T cell sensitivity to AICD by inactivating NF-κB. Mechanistically, histone lactylation derived from KRASMUT tumor cell-produced lactic acid directly activates transcription of circATXN7, which binds to NF-κB p65 subunit and masks the p65 nuclear localization signal motif, thereby sequestering it in the cytoplasm. Clinically, circATXN7 upregulation in tumor-specific CTLs correlates with adverse clinical outcomes and immunotherapeutic resistance. Genetic ablation of circAtxn7 in CD8+ T cells leads to mutant-selective tumor inhibition, while also increases anti-PD1 efficacy in multiple tumor models in female mice. Furthermore, targeting circATXN7 in adoptively transferred tumor-reactive CTLs improves their antitumor activities. These findings provide insight into how lymphocyte-expressed circRNAs contribute to T-cell fate decisions and anticancer immunotherapies.

GSK3β/NF-κB -dependent transcriptional regulation of homeostatic hepatocyte Tnf production.

Maintenance of hepatocyte homeostasis plays an important role in mediating the pathogenesis of many diseases. A growing body of literature has established a critical role played by tumor necrosis factor-α (TNFα) in maintaining hepatocyte homeostasis; however, the transcriptional mechanisms underlying constitutive Tnf expression are unknown. Whole liver fractions and primary hepatocytes from adult control C57BL/6 mice and the murine hepatocyte cell line AML12 were assessed for constitutive Tnf expression. Impacts of glycogen synthase kinase-3 β (GSK3β) and nuclear factor κB (NF-κB) inhibition on constitutive Tnf expression were assessed in AML12 cells. Finally, AML12 cell proliferation following GSK3β and NF-κB inhibition was evaluated. Constitutive Tnf gene expression is present in whole liver, primary hepatocytes, and cultured AML12 hepatocytes. Cytokine-induced Tnf gene expression is regulated by NF-κB activation. Pharmacological inhibition of GSK3β resulted in a time- and dose-dependent inhibition of Tnf gene expression. GSK3β inhibition decreased nuclear levels of the NF-κB subunits p65 and p50. We determined that NF-κB transcription factor subunit p65 binds to consensus sequence elements present in the murine TNFα promoter and inhibition of GSK3β decreases binding and subsequent Tnf expression. Finally, AML12 cell growth was significantly reduced following GSK3β and NF-κB inhibition. These results demonstrate that GSK3β and NF-κB are essential for mediating Tnf expression and constitutive hepatocyte cell growth. These findings add to a growing body of literature on TNFα mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in mediating response to various disease states in the liver.NEW & NOTEWORTHY Maintenance of hepatocyte homeostasis plays an important role in controlling the pathogenesis of many diseases. Our findings add to a growing body of literature on tumor necrosis factor-α (TNFα)-mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in regulating this response.

Dihydrophenanthro[b]furan derivatives from the tubers of Bletilla striata

Two pairs of new dihydrophenanthro[b]furan enantiomers blephebibnols G–H (1–2), one new dihydrophenanthro[b]furan derivative blephebibnol I (3), along with four known analogues (4–7), were isolated from the tubers of Bletilla striata. Their structures including the absolute configurations were determined by the combination of spectroscopic data analysis, ECD and NMR calculations. Compounds 1a, 1b, and 2b showed inhibition of NO production in LPS-stimulated BV-2 cells, with IC50 values ranging from 4.11 to 14.65 μM. Further mechanistic study revealed that 1a suppressed the phosphorylation of p65 subunit to regulate the NF-κB signaling pathway. In addition, some compounds displayed selective cytotoxic activities against HCT-116, HepG2, A549, or HGC27 cancer cell lines with IC50 values ranging from 0.1 to 8.23 μM.

TNFAIP3 Overexpression Inhibits Diffuse Large B-Cell Lymphoma Progression by Promoting Autophagy through TLR4/MyD88/NF-κB Signaling Pathway.

Tumor necrosis factor alpha induced protein 3 (TNFAIP3) is reportedly to have significant implications for autophagy regulation in various cancers. The current study aimed to decipher the role and mechanism of TNFAIP3 in diffuse large B-cell lymphoma (DLBCL) by modulating autophagy. Information pertaining to the differential expression and prognostic role of TNFAIP3 in DLBCL was gleaned from the Gene Expression Omnibus (GEO) database. The TNFAIP3 expression levels in human DLBCL cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Cell counting kit-8 (CCK-8) and colony formation assays were employed to determine cell proliferation. Transwell assay and flow cytometry were applied to detect cell migration and apoptosis, respectively. Immunofluorescence and transmission electron microscope were used for the assessment of cell autophagy. The levels of apoptotic markers (caspase-3, cleaved-caspase-3, Bcl-2 Associated X (Bax), and B cell lymphoma-2 (Bcl-2)), autophagy indicators (the ratio of microtubule-associated proteins 1A/1B light chain 3 II and I (LC3II/LC3I), Sequestosome (p62)), and pathway proteins (toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), Transcription Factor NF-Kappa-B P65 Subunit (p65), and phosphorylated-p65 (p-p65)) were assessed via Western blotting. Immunohistochemistry was employed to detect Ki67 expression in tumor tissues. TNFAIP3 expression in DLBCL samples was downregulated, correlating with poor prognosis. TNFAIP3 expression was also downregulated in DLBCL cells. It was found that TNFAIP3 impeded cell proliferation and migration, and enhanced apoptosis of OCI-LY3 cells. Intervention with autophagy inhibitor 3-methyladenine (3-MA) markedly reversed apoptosis of OCI-LY3 cells induced by TNFAIP3. Besides, TNFAIP3 induced autophagy via modulating the TLR4/MyD88/nuclear factor kappa B (NF-κB) signaling pathway. In vivo experiments showed that TNFAIP3 expression in DLBCL was downregulated, and upregulation of TNFAIP3 could inhibit tumor growth. TNFAIP3 inhibits DLBCL progression by inducing TLR4/MyD88/NF-κB pathway-mediated autophagy.

Network Pharmacology-Based Prediction of Active Ingredients and Mechanisms of Zanthoxyli Bungeanum Against Lung Carcinoma

Background: Zanthoxyli Bungeanum (ZB) has been reported to have an effect on lung carcinoma (LC). However, the defined pharmacological mechanism of ZB on LC has not been expounded completely because of the complicated ingredients. Objective: The aim of this work was to explore the active ingredients and mechanisms of ZB against LC by network pharmacology. Methods: In this study, systemic network pharmacology was used to explore the underlying mechanism of ZB, including pivotal components collection, target prediction, networks construction, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. At last, molecular docking was carried out to elucidate the involved pharmacological mechanisms. Results: Twenty-eight potential active compounds with 317 related targets and 598 LC-related targets were collected. Finally, 79 intersection targets were obtained use GO and KEGG pathway enrichment analyses. Based on component-target-pathway network, quercetin, β-sitosterol, and β- amyrin, and 6 targets were selected, including RAC-alpha serine/thre-onine-protein kinase (AKT1), mitogen-active protein kinase1 (MAPK1), Transcription factor p65 (RELA), Caspase-9 (CASP9), G1/S-specifi cyclin-D1 (CCND1), and PI3-kinase subunit gamma (PIK3CG); these six predicted targets were highly involved in the PI3K-AKT signaling pathway. Conclusion: The active ingredients and mechanisms of ZB against LC were firstly investigated using network pharmacology. This work provides scientific evidence to support the clinical effect of ZB on LC, new insights into the anti-LC mechanism of ZB, and guidance for further study.

Multi-Omics Profiling Identifies Microglial Annexin A2 as a Key Mediator of NF-κB Pro-inflammatory Signaling in Ischemic Reperfusion Injury

Cerebral stroke is one of the leading causes of mortality and disability worldwide. Restoring the cerebral circulation following a period of occlusion and subsequent tissue oxygenation leads to reperfusion injury. Cerebral ischemic reperfusion (I/R) injury triggers immune and inflammatory responses, apoptosis, neuronal damage, and even death. However, the cellular function and molecular mechanisms underlying cerebral I/R-induced neuronal injury are incompletely understood. By integrating proteomic, phosphoproteomic, and transcriptomic profiling in mouse hippocampi after cerebral I/R, we revealed that the differentially expressed genes and proteins mainly fall into several immune inflammatory response-related pathways. We identified that Annexin 2 (Anxa2) was exclusively upregulated in microglial cells in response to cerebral I/R invivo and oxygen-glucose deprivation and reoxygenation (OGD/R) invitro. RNA-seq analysis revealed a critical role of Anxa2 in the expression of inflammation-related genes in microglia via the NF-κB signaling. Mechanistically, microglial Anxa2 is required for nuclear translocation of the p65 subunit of NF-κB and its transcriptional activity upon OGD/R in BV2 microglial cells. Anxa2 knockdown inhibited the OGD/R-induced microglia activation and markedly reduced the expression of pro-inflammatory factors, including TNF-α, IL-1β, and IL-6. Interestingly, conditional medium derived from Anxa2-depleted BV2 cell cultures with OGD/R treatment alleviated neuronal death invitro. Altogether, our findings revealed that microglia Anxa2 plays a critical role in I/R injury by regulating NF-κB inflammatory responses in a non-cell-autonomous manner, which might be a potential target for the neuroprotection against cerebral I/R injury.

Enhancement and inactivation effect of CRISPR/Cas12a via extending hairpin activators for detection of transcription factors.

An enhancement effect for the activation of CRISPR/Cas12a (CRISPR = clustered regularly interspaced short palindromic repeats; Cas = CRISPR-associated) was discovered. That was, a hairpin model with dangling 5' end complementary to crRNA (CRISPR RNA) greatly improved the activity of CRISPR/Cas12a after extention of two random sequences. But, the corresponding intact hairpin without PAM (protospacer adjacent motif) or suboptimal PAM sequences was completely inactive to CRISPR/Cas12a because of the superhigh stability of intact hairpin. According to the finding, a CRISPR/Cas12a-based strategy coupled with a signal reported system was designed for transcription factors detection. By using mono-labeled ssDNA (single-stranded DNA) as reporter and two newly synthesized N-C (nitrogen-doped carbon) nanosheets as scavenger to eliminate the fluorescent background, the strategy realized the detection of NF-ĸB p50 (p50 subunit of nuclear factor kappa-B) with a linear detection range of 0.8 - 2000.0pM and a LOD of 0.5pM. The discovery of "enhancement and inactivation effect" not only deepened insight into CRISPR/Cas12a but also broadened the practical application of CRISPR/Cas systems for the molecular detection and disease diagnostics.

Coagulation Factor Xa Has No Effects on the Expression of PAR1, PAR2, and PAR4 and No Proinflammatory Effects on HL-1 Cells.

Atrial fibrillation (AF), characterised by irregular high-frequency contractions of the atria of the heart, is of increasing clinical importance. The reasons are the increasing prevalence and thromboembolic complications caused by AF. So-called atrial remodelling is characterised, among other things, by atrial dilatation and fibrotic remodelling. As a result, AF is self-sustaining and forms a procoagulant state. But hypercoagulation not only appears to be the consequence of AF. Coagulation factors can exert influence on cells via protease-activated receptors (PAR) and thereby the procoagulation state could contribute to the development and maintenance of AF. In this work, the influence of FXa on Heart Like-1 (HL-1) cells, which are murine adult atrial cardiomyocytes (immortalized), was investigated. PAR1, PAR2, and PAR4 expression was detected. After incubations with FXa (5-50 nM; 4-24 h) or PAR1- and PAR2-agonists (20 µM; 4-24 h), no changes occurred in PAR expression or in the inflammatory signalling cascade. There were no time- or concentration-dependent changes in the phosphorylation of the MAP kinases ERK1/2 or the p65 subunit of NF-κB. In addition, there was no change in the mRNA expression of the cell adhesion molecules (ICAM-1, VCAM-1, fibronectin). Thus, FXa has no direct PAR-dependent effects on HL-1 cells. Future studies should investigate the influence of FXa on human cardiomyocytes or on other cardiac cell types like fibroblasts.

β-D-Glucan promotes NF-κB activation and ameliorates high-LET carbon-ion irradiation-induced human umbilical vein endothelial cell injury.

Heavy-ion irradiation seriously perturbs cellular homeostasis and thus damages cells. Vascular endothelial cells (ECs) play an important role in the pathological process of radiation damage. Protecting ECs from heavy-ion radiation is of great significance in the radioprotection of normal tissues. In this study, the radioprotective effect of β-D-glucan (BG) derived from Saccharomyces cerevisiae on human umbilical vein endothelial cell (EA.hy926) cytotoxicity produced by carbon-ion irradiation was examined and the probable mechanism was established. EA.hy926 cells were divided into seven groups: a control group; 1, 2, or 4 Gy radiation; and 10 μg/mL BG pretreatment for 24 h before 1, 2, or 4 Gy irradiation. Cell survival was assessed by colony formation assay. Cell cycles, apoptosis, DNA damage, and reactive oxygen species (ROS) levels were measured through flow cytometry. The level of malondialdehyde and antioxidant enzyme activities were analyzed using assay kits. The activation of NF-κB was analyzed using western blotting and a transcription factor assay kit. The expression of downstream target genes was detected by western blotting. BG pretreatment significantly increased the survival of irradiated cells, improved cell cycle progression, and decreased DNA damage and apoptosis. The levels of ROS and malondialdehyde were also decreased by BG. Further study indicated that BG increased the antioxidant enzyme activities, activated Src, and promoted NF-κB activation, especially for the p65, p50, and RelB subunits. The activated NF-κB upregulated the expression of antioxidant protein MnSOD, DNA damage-response and repair-related proteins BRCA2 and Hsp90α, and antiapoptotic protein Bcl-2. Our results demonstrated that BG protects EA.hy926 cells from high linear-energy-transfer carbon-ion irradiation damage through the upregulation of prosurvival signaling triggered by the interaction of BG with its receptor. This confirms that BG is a promising radioprotective agent for heavy-ion exposure.

Simvastatin Differentially Modulates Glial Functions in Cultured Cortical and Hypothalamic Astrocytes Derived from Interferon α/β Receptor Knockout mice.

Astrocytes have key regulatory roles in central nervous system (CNS), integrating metabolic, inflammatory and synaptic responses. In this regard, type I interferon (IFN) receptor signaling in astrocytes can regulate synaptic plasticity. Simvastatin is a cholesterol-lowering drug that has shown anti-inflammatory properties, but its effects on astrocytes, a main source of cholesterol for neurons, remain to be elucidated. Herein, we investigated the effects of simvastatin in inflammatory and functional parameters of primary cortical and hypothalamic astrocyte cultures obtained from IFNα/β receptor knockout (IFNα/βR-/-) mice. Overall, simvastatin decreased extracellular levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), which were related to a downregulation in gene expression in hypothalamic, but not in cortical astrocytes. Moreover, there was an increase in anti-inflammatory interleukin-10 (IL-10) in both structures. Effects of simvastatin in inflammatory signaling also involved a downregulation of cyclooxygenase 2 (COX-2) gene expression as well as an upregulation of nuclear factor κB subunit p65 (NFκB p65). The expression of cytoprotective genes sirtuin 1 (SIRT1) and nuclear factor erythroid derived 2 like 2 (Nrf2) was also increased by simvastatin. In addition, simvastatin increased glutamine synthetase (GS) activity and glutathione (GSH) levels only in cortical astrocytes. Our findings provide evidence that astrocytes from different regions are important cellular targets of simvastatin in the CNS, even in the absence of IFNα/βR, which was showed by the modulation of cytokine production and release, as well as the expression of cytoprotective genes and functional parameters.

Inhibitory effects of Oncorhynchus mykiss lipids in LPS-induced RAW264.7 cells via suppression of NF-κB and MAPK pathways

Oncorhynchus mykiss, a significant aquaculture species, possesses compounds with numerous biological and pharmacological functions, including antioxidant, anticancer, anti-microbial, and anti-obesity effects. However, possible anti-inflammatory effects of lipids extracted from O. mykiss eggs on RAW264.7 cells induced by LPS have not been elucidated yet. The current study identified 13 fatty acids in lipids extracted from O. mykiss eggs that contained high amounts (51.92% of total fatty acids) of polyunsaturated fatty acids (PUFAs), especially DHA (33.66%) and EPA (7.77%). These O. mykiss lipids (100–400 μg/mL) showed significant anti-inflammatory effects by inhibiting NO and iNOS expression in LPS-stimulated RAW264.7 cells. They also inhibited expression of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, while upregulating anti-inflammatory cytokines IL-10, IL-11, and TGF-β. These lipids from O. mykiss effectively inhibited LPS-induced expression CD86 as a surface biomarker on RAW264.7 cells. Additionally, O. mykiss lipids suppressed phosphorylation of p38, JNK, and ERK1/2 and the expression of phosphorylated NF-κB subunit p65. These findings indicate that O. mykiss lipids possess anti-inflammatory properties by inhibiting NF-κB and MAPK signaling pathways.

Extracellular Vesicle Content Changes Induced by Melatonin Promote Functional Recovery of Pancreatic Beta Cells in Acute Pancreatitis.

Acute pancreatitis is an inflammatory disorder of the pancreas, which causes abnormal activation of immune cells. The macrophages were accumulated in pancreas and infiltrated into islets during the AP process to induce abnormal glucose metabolism. However, the role of macrophages in abnormal glucose metabolism remains understood. Extracellular vesicles act in the regulation of intercellular function, but whether EVs secreted by macrophages contribute to β cell failure and apoptosis in AP is unclear. Based on this, the aim of this study was to reveal the role of macrophages-EVs in AP and develop a treatment for symptoms of hyperglycemia in AP. The AP model was established and treated by various doses of melatonin to analyze the therapeutic effect. The accumulation and polarization of macrophages in the AP pancreas were observed, and the β cells were incubated with pancreatic derived EVs to analyze the role in β cell failure and apoptosis. The results showed that macrophages were recruited and polarized to M1 phenotype macrophages in the pancreas of AP mice, which obtained inflammatory EVs that contained specific miRNAs to induce β cell failure and apoptosis. Then, the EVs derived from M1 macrophages triggered β cell failure and apoptosis. Melatonin prevented polarization of macrophages to the M1 phenotype in vivo, which reduced the secretion of inflammatory EVs, changed the abundance of miRNAs in EVs, and therefore decreased inflammatory EV-mediated β cell failure and apoptosis. Our results demonstrate that similar to 20S proteasome inhibitor MG132, analyses indicated that melatonin prevented degradation of IκBα through the ubiquitylation pathway to restrict p50 subunits to the cytoplasm of macrophages, inhibited activation of the NF-κB pathway to downregulate the transcription of specific miRNAs, and reduced miRNA transport into EVs.

Quercetin/Anti-PD-1 Antibody Combination Therapy Regulates the Gut Microbiota, Impacts Macrophage Immunity and Reshapes the Hepatocellular Carcinoma Tumor Microenvironment.

The use of immune checkpoint inhibitors (ICIs) provides promising strategies for hepatocellular carcinoma (HCC) treatment. This study aimed to explore impact and underlying mechanism of the combination therapy of quercetin and anti-programmed cell death 1 (anti-PD-1) antibody on HCC. Orthotopically transplanted HCC tumors in mice were treated with quercetin, anti-PD-1 antibody, or a combination of both therapies. Histopathological changes and programmed cell death ligand 1 (PD-L1) expression were characterized by hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining. The diversity and differences of gut microbiota (GM) were evaluated through 16S rRNA sequencing. Levels of macrophage immunity-related cytokines were quantified by enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (RT-qPCR), and Western blot. Combination therapy reduced necrosis, fibrosis, and PD-L1 expression in liver tissues. Additionally, combination therapy reduced GM imbalance and increased abundance of Firmicutes, Actinobacteria, and Verrucomicrobiota at the phylum level as well as Dubosiella and Akkermansia at the genus level. Combination therapy improved macrophage immunity, raised the expressions of CD8a, CD4, CD11b, interleukin (IL)-10, and interferon (IFN)-γ , and declined the expressions of IL-4, IL-6, toll-like receptor 4 (TLR4), an inhibitor of nuclear factor κBα (IκBα), and the NFκB subunit p65. Upon combination therapy, expressions of M2 macrophage-related genes arginase-1 (Arg-1), IL-10, transforming growth factor-β (TGF-β), and matrix metalloproteinase-9 (MMP-9) were upregulated. Instead, M1 macrophage-related genes IL-6, IL-12a, IL-1β, and tumor necrosis factor-α (TNF-α) were downregulated. Quercetin/anti-PD-1 antibody combination therapy reshaped HCC tumor microenvironment in mice in parallel with regulating the GM and macrophage immunity.

Cancer-Preventive Activity of Argemone mexicana Linn Leaves and Its Effect on TNF-α and NF-κB Signalling

Skin cancer is the 5th most common cancer in Western countries with a surge in case occurrences making it a global burden on healthcare systems. The present study aims to evaluate the cancer-preventive activity of an ethanolic extract of Argemone mexicana Linn leaves (AML). The DMBA/TPA method was used to induce skin cancer in mice. Experimental animals were divided into three pretreatment groups of 100 mg/kg BW, 250 mg/kg BW, and 500 mg/kg BW of AML extract, and feeding was continued during the induction process. In the fourth group, 500 mg/kg BW AML extract treatment was started along with the cancer induction. The analyses were performed on the basis of the time period of in-tumour induction incidence, haematological parameters, histopathology and augmentation of TNF-α secretion and the NF-κB (p65 subunit) signalling pathway. The AML extract resisted and delayed tumour formation for up to 8 weeks in the 500 mg/kg BW pretreated group as compared to 4 weeks in the negative control group. The tumour burden varied in a dose-dependent manner in the different groups. On the 60th day, a significantly high burden (p < 0.001) was observed in the negative control group and the 100 mg/kg BW group. The study was validated by investigating the expression of TNF-α and the p65 subunit of the NF-κB signalling pathway, which were found to be reduced significantly in a dose-dependent manner and significantly reduced (p < 0.001) in the 500 mg/kg BW group as compared to negative control group. The 500 mg/kg BW pretreated group was found to have significant results in comparison to the 500 mg/kg BW post-treatment group. The study revealed the effective cancer preventive activity of Argemone mexicana Linn leaves (AML) in the mouse model and paved a pathway for molecular approaches which could be explored more in future studies.