Journal: Alzheimer's & Dementia

Article Title: Endothelial NAD + depletion drives vascular senescence and neuroinflammation via mtDNA‐cGAS/STING‐CD38 signaling in Alzheimer's disease

doi: 10.1002/alz.71423

Figure Lengend Snippet: NAD + supplementation suppresses cGAS/STING pathway activation in cerebral endothelial cells of APP/PS1 mice. (A) Heatmap of differentially expressed key cGAS/STING pathway‐related genes (such as Cgas , Sting1 , Irf3 ) identified by RNA‐seq of cerebral vessel‐enriched fractions from APPtg and APPtg + NR mice ( n = 3 per group). (B, C) Representative western blot image (B) and densitometric quantification of cGAS, STING, phospho‐TBK1 Ser172 (p‐TBK1), and phospho‐IRF3 Ser396 (p‐IRF3) in cerebral vessel‐enriched fractions from APPwt, APPtg, and APPtg + NR mice (C; n = 6 per group). (D–G) Representative immunofluorescence images of hippocampus and cortex from APPtg and APPtg + NR mice showing CD31 (green) co‐stained with STING (D, red) or cGAS (F, red); quantification of STING (E) and cGAS (G) fluorescence intensity within CD31 + cerebral vessels were shown ( n = 5 or 6 mice per group); nuclei were counterstained with DAPI (blue). (H) qPCR analysis of SASP genes ( Il6 , Tnf , Il1b , Cxcl10 , Cxcl2 ) in cerebral vessel‐enriched fractions from APPtg and APPtg + NR mice ( n = 5 per group). (I) ELISA quantification of IL‐6, TNF‐α, and IL‐1β in the culture supernatants of bEnd.3 endothelial cells treated with vehicle control, NR, Aβ, or Aβ + NR ( n = 6 per group). (J) SA‐β‐galactosidase staining of bEnd.3 endothelial cells transfected with control siRNA (si‐Ctrl), Cgas siRNA (si‐ Cgas ), or Sting1 siRNA (si‐ Sting ) followed by Aβ stimulation or vehicle control; representative images show SA‐β‐gal + cells indicated by white arrows, with enlarged insets provided; the percentage of SA‐β‐gal + cells were quantified ( n = 5 per group). Data are presented as mean ± SEM. Statistical analyses were performed using one‐way ANOVA followed by Tukey's multiple comparisons test (C, E, G, I, J) or unpaired two‐tailed Student's t ‐test (H). p ‐values are indicated in the figure.

Article Snippet: For IL‐6 pathway analysis, BV‐2 microglia were incubated with 10 ng/ml anti‐mouse IL‐6 neutralizing antibody (α‐IL‐6; R&D systems, #MAB406) or anti‐mouse IL‐6Rα blocking antibody (α‐IL‐6R; R&D systems, #AF1830) in CM‐containing medium from bEnd.3 cultures.

Techniques: Activation Assay, RNA Sequencing, Western Blot, Immunofluorescence, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Control, Transfection, Two Tailed Test

Journal: Alzheimer's & Dementia

Article Title: Endothelial NAD + depletion drives vascular senescence and neuroinflammation via mtDNA‐cGAS/STING‐CD38 signaling in Alzheimer's disease

doi: 10.1002/alz.71423

Figure Lengend Snippet: NAD + supplementation suppresses cGAS/STING activation by enhancing mitochondrial function and preventing cytosolic mtDNA leakage. (A) Quantification of mitochondrial membrane potential using JC‐1 staining in bEnd.3 endothelial cells treated with Aβ, Aβ + NR, or control conditions; representative images are shown in Figure ( n = 5 per group). (B, C) Flow cytometric analysis of intracellular ROS levels in bEnd.3 cells under indicated treatments ( n = 4 per group). (D) qPCR quantification of cytosolic mitochondrial DNA (mtDNA; D‐loop , Non‐Numt , Cox1 ) and nuclear DNA (nDNA; Tert , B2m ) in cerebral vessel‐enriched fractions isolated from APPwt, APPwt + NR, APPtg, and APPtg + NR mice ( n ≥5 per group). (E, F) Representative immunofluorescence images (E) and quantification (F) of co‐localization of CD31 (green) and oxidative DNA damage marker 8‐OHdG (red) in hippocampal and cortex of APPtg and APPtg + NR mice; nuclei were counterstained with DAPI (blue) ( n ≥5 mice per group). (G) Quantification of cytosolic mtDNA and nDNA levels in bEnd.3 cells transfected with siRNA targeting control (si‐Ctrl), Cgas (si‐ Cgas ), or Sting1 (si‐ Sting ) followed by Aβ treatment ( n = 4 per group). (H) Quantification of cytosolic mtDNA and nDNA levels in bEnd.3 cells treated with Aβ, Aβ + mtDNA depletion (ddC), or Aβ + ddC + NR ( n = 4 per group). (I) Relative mRNA expression of SASP‐related cytokines (IL‐6, TNF‐α, IL‐1β, CXCL10, CXCL2) under the same treatment conditions as in (H) ( n = 4 per group). (J, K) Western blot analysis (J) and quantification (K) of cGAS/STING pathway components (cGAS, STING, p‐TBK1, p‐IRF3) and tight junction proteins (ZO‐1, Occludin) in bEnd.3 cells under treatments with Aβ, Aβ + ddC, and Aβ + ddC + NR ( n = 4 per group). Data are presented as mean ± SEM. Statistical significance was assessed using one‐way ANOVA followed by Tukey's multiple comparisons test. P ‐values are indicated in the figure.

Article Snippet: For IL‐6 pathway analysis, BV‐2 microglia were incubated with 10 ng/ml anti‐mouse IL‐6 neutralizing antibody (α‐IL‐6; R&D systems, #MAB406) or anti‐mouse IL‐6Rα blocking antibody (α‐IL‐6R; R&D systems, #AF1830) in CM‐containing medium from bEnd.3 cultures.

Techniques: Activation Assay, Membrane, Staining, Control, Isolation, Immunofluorescence, Marker, Transfection, Expressing, Western Blot

Journal: Alzheimer's & Dementia

Article Title: Endothelial NAD + depletion drives vascular senescence and neuroinflammation via mtDNA‐cGAS/STING‐CD38 signaling in Alzheimer's disease

doi: 10.1002/alz.71423

Figure Lengend Snippet: NAD + supplementation disrupts IL‐6‐mediated endothelial‐microglial inflammatory crosstalk in AD. (A) Representative immunofluorescence images showing co‐staining of microglial marker Iba1 (red) and endothelial marker CD31 (green) in the cortex and hippocampus of APP/PS1 mice; white arrows indicate perivascular microglia closely associated with cerebral vessels. (B) Quantification of the proportion of perivascular microglia relative to total microglia ( n ≥ 5 per group). (C) Triple immunofluorescence staining of Iba1 (red), CD31 (green), and IL‐6R (gray) to visualize IL‐6R expression in perivascular microglia; yellow arrows indicate IL‐6R‐positive perivascular microglia. (D) Quantification of IL‐6R fluorescence intensity in vessel‐associated microglia ( n ≥5 per group). (E–F) Western blot analysis (E) and densitometric quantification (F) of IL‐6R, JAK1, and phosphorylation levels of STAT3 and NF‐κB p65 in microglia stimulated with conditioned media from bEnd.3 cells treated with vehicle (Con), NR, Aβ, or Aβ + NR ( n = 6 per group). (G–H) Western blot analysis (G) and quantification (H) of IL‐6R, JAK1, and p‐STAT3/p‐NF‐κB p65 in microglia co‐treated with Aβ‐challenged endothelial conditioned medium and isotype IgG, IL‐6‐neutralizing antibody (α‐IL‐6), or IL‐6R‐neutralizing antibody (α‐IL‐6R) ( n = 4 per group). Data are presented as mean ± SEM. Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test. P ‐values are indicated in the figure.

Article Snippet: For IL‐6 pathway analysis, BV‐2 microglia were incubated with 10 ng/ml anti‐mouse IL‐6 neutralizing antibody (α‐IL‐6; R&D systems, #MAB406) or anti‐mouse IL‐6Rα blocking antibody (α‐IL‐6R; R&D systems, #AF1830) in CM‐containing medium from bEnd.3 cultures.

Techniques: Immunofluorescence, Staining, Marker, Expressing, Fluorescence, Western Blot, Phospho-proteomics

Journal: Frontiers in Aging Neuroscience

Article Title: Unraveling the anti-neuroinflammatory mechanisms of Cervus cucumis polypeptide injection in Alzheimer’s disease: insights from network pharmacology, molecular docking, molecular dynamics simulation, and experimental validation

doi: 10.3389/fnagi.2026.1797302

Figure Lengend Snippet: Molecular docking analysis of LA with IL-6 in both 3D and 2D frameworks. (A) Surface-form molecular docking. (B) Ribbon-form molecular docking. (C) 2D intermolecular interactions. (D) 3D intermolecular interactions.

Article Snippet: After being blocked in 5% skim milk for 2 h at room temperature, the membranes were incubated overnight with the following primary antibodies: inducible nitric oxide synthase (iNOS) mouse antibody (CAS No. IC259554, Abmart, China, 1:1,000), CD206 mouse antibody (CAS No. ZY-5843R, Abmart, China, 1:1,000), IL-6 mouse antibody (CAS No. 66146-2, Abmart, China, 1:1,000), STAT3 mouse antibody (CAS No. YA056, Abmart, China, 1:1,000), STAT3 phosphorylation mouse antibody (CAS No. 05-485, Sigma, United States, 1:1,000), VEGF rabbit antibody (CAS No. AF1309, Abmart, China, 1:1,000) and GAPDH rabbit antibody (CAS No. 10494-1-AP, Abmart, China, 1:1,000).

Techniques:

Journal: Frontiers in Aging Neuroscience

Article Title: Unraveling the anti-neuroinflammatory mechanisms of Cervus cucumis polypeptide injection in Alzheimer’s disease: insights from network pharmacology, molecular docking, molecular dynamics simulation, and experimental validation

doi: 10.3389/fnagi.2026.1797302

Figure Lengend Snippet: The statistical data of LA and IL-6 simulated by MD simulation. (A) The dynamic stability of the complexes assessment by employing the RMSD analysis. (B) RMSF analysis evaluated the mobility of amino acid residues during the simulation. (C) The stability of the IL-6-LA complex biological system was analyzed along MD trajectories by calculating the structural compactness of biomolecules using the Rg. (D) The surface area of the protein exposed to the solvent was analyzed during the simulation using the SASA plot. (E) Number of hydrogen bonds of the IL-6-LA complex formed during the computational simulations. (F) The conformational behavior of the IL-6-LA complex was employed by Gibbs FEL.

Article Snippet: After being blocked in 5% skim milk for 2 h at room temperature, the membranes were incubated overnight with the following primary antibodies: inducible nitric oxide synthase (iNOS) mouse antibody (CAS No. IC259554, Abmart, China, 1:1,000), CD206 mouse antibody (CAS No. ZY-5843R, Abmart, China, 1:1,000), IL-6 mouse antibody (CAS No. 66146-2, Abmart, China, 1:1,000), STAT3 mouse antibody (CAS No. YA056, Abmart, China, 1:1,000), STAT3 phosphorylation mouse antibody (CAS No. 05-485, Sigma, United States, 1:1,000), VEGF rabbit antibody (CAS No. AF1309, Abmart, China, 1:1,000) and GAPDH rabbit antibody (CAS No. 10494-1-AP, Abmart, China, 1:1,000).

Techniques: Solvent

Journal: Frontiers in Aging Neuroscience

Article Title: Unraveling the anti-neuroinflammatory mechanisms of Cervus cucumis polypeptide injection in Alzheimer’s disease: insights from network pharmacology, molecular docking, molecular dynamics simulation, and experimental validation

doi: 10.3389/fnagi.2026.1797302

Figure Lengend Snippet: Effect of CCPI on pro-inflammatory cytokines in the AD model cells. (A) CCPI down-regulated the levels of IL-6. (B) CCPI down-regulated the levels of IL-1β. (C) CCPI down-regulated the levels of TNF-α. Data are presented as mean ± SD ( n = 6). ### p < 0.001, ## p < 0.01, # p < 0.05 compared with the Control group. *** p < 0.001, ** p < 0.01, * p < 0.05 compared with the AD group.

Article Snippet: After being blocked in 5% skim milk for 2 h at room temperature, the membranes were incubated overnight with the following primary antibodies: inducible nitric oxide synthase (iNOS) mouse antibody (CAS No. IC259554, Abmart, China, 1:1,000), CD206 mouse antibody (CAS No. ZY-5843R, Abmart, China, 1:1,000), IL-6 mouse antibody (CAS No. 66146-2, Abmart, China, 1:1,000), STAT3 mouse antibody (CAS No. YA056, Abmart, China, 1:1,000), STAT3 phosphorylation mouse antibody (CAS No. 05-485, Sigma, United States, 1:1,000), VEGF rabbit antibody (CAS No. AF1309, Abmart, China, 1:1,000) and GAPDH rabbit antibody (CAS No. 10494-1-AP, Abmart, China, 1:1,000).

Techniques: Control

Journal: Frontiers in Aging Neuroscience

Article Title: Unraveling the anti-neuroinflammatory mechanisms of Cervus cucumis polypeptide injection in Alzheimer’s disease: insights from network pharmacology, molecular docking, molecular dynamics simulation, and experimental validation

doi: 10.3389/fnagi.2026.1797302

Figure Lengend Snippet: Effects of CCPI on the expression of IL-6/STAT3/VEGF signaling pathway. (A) Western blot showed the expression of the IL-6/STAT3/VEGF pathway after CCPI treatment. (B–D) Quantitative analysis of the IL-6/GAPDH, STAT3/GAPDH, and VEGF/GAPDH ratios in the CCPI-treated groups, respectively. Data are presented as mean ± SD ( n = 3). ### p < 0.001 vs. ontrol group; *** p < 0.001, ** p < 0.01 and * p < 0.05 vs. AD model group.

Article Snippet: After being blocked in 5% skim milk for 2 h at room temperature, the membranes were incubated overnight with the following primary antibodies: inducible nitric oxide synthase (iNOS) mouse antibody (CAS No. IC259554, Abmart, China, 1:1,000), CD206 mouse antibody (CAS No. ZY-5843R, Abmart, China, 1:1,000), IL-6 mouse antibody (CAS No. 66146-2, Abmart, China, 1:1,000), STAT3 mouse antibody (CAS No. YA056, Abmart, China, 1:1,000), STAT3 phosphorylation mouse antibody (CAS No. 05-485, Sigma, United States, 1:1,000), VEGF rabbit antibody (CAS No. AF1309, Abmart, China, 1:1,000) and GAPDH rabbit antibody (CAS No. 10494-1-AP, Abmart, China, 1:1,000).

Techniques: Expressing, Western Blot

Journal: Frontiers in Aging Neuroscience

Article Title: Unraveling the anti-neuroinflammatory mechanisms of Cervus cucumis polypeptide injection in Alzheimer’s disease: insights from network pharmacology, molecular docking, molecular dynamics simulation, and experimental validation

doi: 10.3389/fnagi.2026.1797302

Figure Lengend Snippet: Effect of LA on IL-6 levels in the AD model cells as determined by ELISA. (A) Chemical structure of LA. (B) Dose–response graphs showing cell viability after treatment with 0, 10, 20, 40, 80, and 160 μM LA for 24 h. (C) Effects of 10, 40, and 80 μM LA on cell viability in the AD model cells. (D) LA suppresses IL-6 levels in the AD model cells. Data are presented as mean ± SD ( n = 6). ### p < 0.001 vs. the Control group; *** p < 0.001, ** p < 0.01, and ns p > 0.05 compared with the AD group; compared with the multiple-group, ns p > 0.05.

Article Snippet: After being blocked in 5% skim milk for 2 h at room temperature, the membranes were incubated overnight with the following primary antibodies: inducible nitric oxide synthase (iNOS) mouse antibody (CAS No. IC259554, Abmart, China, 1:1,000), CD206 mouse antibody (CAS No. ZY-5843R, Abmart, China, 1:1,000), IL-6 mouse antibody (CAS No. 66146-2, Abmart, China, 1:1,000), STAT3 mouse antibody (CAS No. YA056, Abmart, China, 1:1,000), STAT3 phosphorylation mouse antibody (CAS No. 05-485, Sigma, United States, 1:1,000), VEGF rabbit antibody (CAS No. AF1309, Abmart, China, 1:1,000) and GAPDH rabbit antibody (CAS No. 10494-1-AP, Abmart, China, 1:1,000).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Aging Neuroscience

Article Title: Unraveling the anti-neuroinflammatory mechanisms of Cervus cucumis polypeptide injection in Alzheimer’s disease: insights from network pharmacology, molecular docking, molecular dynamics simulation, and experimental validation

doi: 10.3389/fnagi.2026.1797302

Figure Lengend Snippet: Comparison of the effects of CCPI and LA on IL-6 secretion, STAT3 phosphorylation, and the expression of markers associated with pro-inflammation (iNOS) and anti-inflammation/repair (CD206) in AD model cells. (A) IL-6 levels in the AD model cells were measured by ELISA. (B) Western blot showed the expression of iNOS, CD206, STAT3 phosphorylation, and STAT3 after CCPI and LA treatment. (C–E) Quantitative analysis of the iNOS/GAPDH, CD206/GAPDH, and STAT3 phosphorylation/STAT3 ratios in the CCPI and LA-treated groups, respectively. Data are presented as mean ± SD ( n = 3). ### p < 0.001 vs. control group; *** p < 0.001, ** p < 0.01 and * p < 0.05 vs. AD model group; ns p > 0.05 compared with the CCPI group; compared with the LA group, ns p > 0.05.

Article Snippet: After being blocked in 5% skim milk for 2 h at room temperature, the membranes were incubated overnight with the following primary antibodies: inducible nitric oxide synthase (iNOS) mouse antibody (CAS No. IC259554, Abmart, China, 1:1,000), CD206 mouse antibody (CAS No. ZY-5843R, Abmart, China, 1:1,000), IL-6 mouse antibody (CAS No. 66146-2, Abmart, China, 1:1,000), STAT3 mouse antibody (CAS No. YA056, Abmart, China, 1:1,000), STAT3 phosphorylation mouse antibody (CAS No. 05-485, Sigma, United States, 1:1,000), VEGF rabbit antibody (CAS No. AF1309, Abmart, China, 1:1,000) and GAPDH rabbit antibody (CAS No. 10494-1-AP, Abmart, China, 1:1,000).

Techniques: Comparison, Phospho-proteomics, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Control