Journal: Frontiers in Nutrition

Article Title: Astaxanthin alleviates altered hepatic lipid metabolism and oxidative stress in animals fed a high-sucrose diet

doi: 10.3389/fnut.2026.1781406

Figure Lengend Snippet: (A) Representative photomicrographs of p-NFκB p65 immunocytochemical staining in liver tissues (400 × magnification), and (B) quantitative immunohistochemical analysis of p-NFκB p65 expression in the liver of rats fed a reference diet (RD), RD plus ASTX (RD + ASTX), high-sucrose diet (HSD), and HSD plus ASTX (HSD + ASTX). Values (mean ± SEM, n = 6 ) are expressed as the in tegrated optical density (IOD). Values that do not share the same letter were significantly different ( p < 0.05) when one variable at a time was compared by two-way ANOVA. D, effect of Diet. I, effect of intervention. Int (D x I), interaction between diet x intervention. ns, not significant.

Article Snippet: The samples were incubated in a humid chamber first with a specific primary antibody for Nrf2 (mouse monoclonal antibody; sc-365949; Santa Cruz Biotechnology) and p-NFκB p65 (mouse monoclonal antibody; sc-136548; Santa Cruz Biotechnology) (for 14–16 h at 4 °C) and then with biotin-conjugated secondary antibody (antimouse, 1:100 dilution, Sigma) for 30 min at room temperature.

Techniques: Staining, Immunohistochemical staining, Expressing

Journal: Frontiers in Nutrition

Article Title: Astaxanthin alleviates altered hepatic lipid metabolism and oxidative stress in animals fed a high-sucrose diet

doi: 10.3389/fnut.2026.1781406

Figure Lengend Snippet: Mechanisms by which ASTX modulates liver lipid metabolism and oxidative stress in HSD-fed rats. ASTX supplementation mitigates hepatic injury by reducing steatosis and triglyceride accumulation, mainly through enhanced mitochondrial fatty acid β-oxidation (↑ CPT-1 activity) without affecting lipogenesis. In parallel, ASTX improves hepatic redox homeostasis by lowering ROS levels, increasing antioxidant enzyme activities (CAT, GST) and restoring GSH content. Increased Nrf2 and decreased p-NFκB p65 protein expression were also modulated by ASTX treatment. Solid lines indicate effects supported by the experimental data, whereas dashed lines represent suggested pathways not directly evaluated in this study.

Article Snippet: The samples were incubated in a humid chamber first with a specific primary antibody for Nrf2 (mouse monoclonal antibody; sc-365949; Santa Cruz Biotechnology) and p-NFκB p65 (mouse monoclonal antibody; sc-136548; Santa Cruz Biotechnology) (for 14–16 h at 4 °C) and then with biotin-conjugated secondary antibody (antimouse, 1:100 dilution, Sigma) for 30 min at room temperature.

Techniques: Activity Assay, Expressing

Journal: Redox Biology

Article Title: Sigma-1 receptor counteracts non-cell-autonomous poly-PR-induced astrocytic oxidative stress in C9orf72 ALS

doi: 10.1016/j.redox.2025.103875

Figure Lengend Snippet: NF-κB p65 regulates NOX4 transcription via promoter binding and facilitates poly-PR 20 -induced NOX4 expression. (A–E) RT-qPCR analysis of NOX4 mRNA levels in astrocytes following overexpression of the transcription factors SOX2 (A), FOS (B), OCT1 (C), SP3 (D), and NF-κB p65 (E). Only p65 significantly enhanced NOX4 mRNA expression. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t -test (∗∗∗ p < 0.001). N = 3 biologically independent experiments with consistent results. (F) Schematic diagram illustrating the experimental design for assessing NOX4 mRNA expression, protein levels, and luciferase reporter activity following poly-PR 20 treatment and p65 overexpression. (G–I) Co-treatment with poly-PR 20 and p65 overexpression further increased NOX4 mRNA expression (G) and protein levels (H, I) compared to poly-PR 20 treatment alone. Data are presented as mean ± SEM and analyzed using two-way ANOVA followed by Tukey's multiple comparisons test (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). N = 3 biologically independent experiments with consistent results. (J) Schematic representation of predicted NF-κB p65-responsive elements within the NOX4 promoter and design of luciferase reporter constructs (NOX4/FL-pGL3: −710 to +1; NOX4/PI-pGL3: −210 to +1). (K) Luciferase reporter assay showing that p65 enhances transcriptional activity through the NOX4 promoter. Cells were co-transfected with HA-p65 and the indicated NOX4 reporter constructs. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗∗∗∗ p < 0.0001). N = 3 biologically independent experiments with consistent results.

Article Snippet: NFκB (p65) , Merck , 06-418; RRID: AB_11214166.

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Over Expression, Two Tailed Test, Luciferase, Activity Assay, Construct, Reporter Assay, Transfection

Journal: Redox Biology

Article Title: Sigma-1 receptor counteracts non-cell-autonomous poly-PR-induced astrocytic oxidative stress in C9orf72 ALS

doi: 10.1016/j.redox.2025.103875

Figure Lengend Snippet: Exogenous poly-PR 20 activates p65 phosphorylation and nuclear translocation in astrocytes via the IKK/IκB pathway. (A) Western blot analysis of phosphorylated p65 (p-p65) and total p65 in astrocytes treated with poly-PR 20 . p65 phosphorylation was notably increased at 3 h post-treatment. (B) Quantification of the p-p65/p65 ratio from three biologically independent experiments, all yielding consistent results. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗ p < 0.05). (C) Immunofluorescence staining showing nuclear translocation of p65 in astrocytes treated with FITC-poly-PR 20 for 3 h. (D) Confocal microscopy with Z-stack and line-scan analysis confirmed p65 nuclear localization. Red: p65; Green: FITC-poly-PR 20 ; Blue: DAPI. (E) Semi-quantitative analysis of nuclear versus cytosolic p65 using ImageJ (version 1.53t) from confocal images. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t -test (∗∗∗ p < 0.001). Total number of cells analyzed: control group, n = 24; FITC-poly-PR 20 group, n = 24. N = 3 biologically independent experiments with consistent results. (F) Western blot analysis showing activation of IKKα/β phosphorylation following poly-PR 20 treatment. (G, H) Quantification of phosphorylated IKKα/β relative to total IKKα (G) and IKKβ (H) revealed a significant increase at 30 min post-treatment. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). N = 3 biologically independent experiments with consistent results. (I) Western blot analysis showing increased phosphorylation of IκBα in astrocytes following poly-PR 20 treatment. (J) Quantification of the p -IκBα/IκBα ratio showed a significant increase at 2 h. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). N = 3 biologically independent experiments with consistent results.

Article Snippet: NFκB (p65) , Merck , 06-418; RRID: AB_11214166.

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Immunofluorescence, Staining, Confocal Microscopy, Two Tailed Test, Control, Activation Assay

Journal: Redox Biology

Article Title: Sigma-1 receptor counteracts non-cell-autonomous poly-PR-induced astrocytic oxidative stress in C9orf72 ALS

doi: 10.1016/j.redox.2025.103875

Figure Lengend Snippet: Sigma-1R interacts with p65 and reduces its binding to the NOX4 promoter in astrocytes under exogenous poly-PR 20 peptide treatment. (A) Western blot analysis of phosphorylated p65 (p-p65) levels in astrocytes overexpressing Sigma-1R-GFP or GFP control, followed by exogenous poly-PR 20 peptide treatment. Astrocytes were transfected for 24 h and treated with exogenous poly-PR 20 peptide for 3 h. (B) Quantification of the p-p65/p65 protein ratio from three biologically independent experiments, each yielding consistent results. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t -test. (C) Schematic diagram illustrating the experimental timeline for the luciferase reporter assay following co-transfection of GFP or Sigma-1R-GFP with HA-p65, followed by exogenous poly-PR 20 peptide treatment. (D) Overexpression of Sigma-1R-GFP reduced p65-mediated activation of the NOX4 promoter. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t -test (∗ p < 0.05). N = 3 biologically independent experiments with consistent results. (E) Schematic diagram illustrating the experimental timeline for the luciferase reporter assay after clemastine and poly-PR 20 treatment. (F) Clemastine treatment also reduced p65-driven NOX4 promoter activation. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t -test (∗ p < 0.05). N = 3 biologically independent experiments with consistent results. (G) Immunoprecipitation analysis of p65 and Sigma-1R interaction in astrocytes treated with poly-PR 20 confirmed a physical interaction between Sigma-1R and p65. (H) Immunofluorescence analysis demonstrated that Sigma-1R overexpression in astrocytes attenuated p65 nuclear translocation in response to exogenous poly-PR peptide treatment. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t -test (∗∗∗∗ p < 0.0001). Total number of cells analyzed: EGFP + poly-PR 20 group, n = 18; Sigma-1R-EGFP + poly-PR 20 group, n = 13. N = 3 biologically independent experiments with consistent results.

Article Snippet: NFκB (p65) , Merck , 06-418; RRID: AB_11214166.

Techniques: Binding Assay, Western Blot, Control, Transfection, Two Tailed Test, Luciferase, Reporter Assay, Cotransfection, Over Expression, Activation Assay, Immunoprecipitation, Immunofluorescence, Translocation Assay

Journal: Redox Biology

Article Title: Sigma-1 receptor counteracts non-cell-autonomous poly-PR-induced astrocytic oxidative stress in C9orf72 ALS

doi: 10.1016/j.redox.2025.103875

Figure Lengend Snippet: Proposed model of Sigma-1R-mediated protection against exogenous poly-PR-induced oxidative stress in astrocytes. Neuron-derived poly-PR is released and internalized by astrocytes, where it activates the IKK/IκB/NF-κB pathway, driving p65 nuclear translocation, NOX4 transcription, and H 2 O 2 production. Sigma-1R overexpression interacts with p65, limiting its nuclear translocation and binding to the NOX4 promoter, thereby suppressing astrocytic oxidative stress and neuroinflammation. This model underscores the non-cell-autonomous neuron-to-astrocyte mechanism contributing to C9orf72 ALS pathogenesis.

Article Snippet: NFκB (p65) , Merck , 06-418; RRID: AB_11214166.

Techniques: Derivative Assay, Translocation Assay, Over Expression, Binding Assay