Journal: PLOS One

Article Title: Inhibition of neutrophil infiltration and NETs formation ameliorates neuropsychiatric and renal dysfunction in MRL/lpr mice with lupus

doi: 10.1371/journal.pone.0348011

Figure Lengend Snippet: A) Kidney function assessment of various mouse groups at week 20, evaluated by measuring the urine albumin-to-creatinine ratio. B) Upper half: PAS staining of kidney tissue was used to visualize renal tissue architecture and structural injury. Immune cell infiltration was assessed by multiplex immunofluorescence staining for CD11b and CD16; lower half: Multiplex immunofluorescence staining for CD11b, CD16, NE, MPO with localized positive areas enlarged. C) and D) Statistical count of CD11b positive and CD11b/CD16 double-positive cells across different groups. E) and F) Semi-quantitative analysis of fluorescence intensity for NE and MPO in different groups; * indicates comparisons of NE fluorescence intensity, # indicates comparisons of MPO fluorescence intensity. * p < 0.05, ** p < 0.01 compared to the MRL/MpJ group; # p < 0.05, ## p < 0.01 compared to the MRL/lpr+Avacopan group. G) Western blot bands for IL-6, IL-17, TNF-α, and β-actin in different groups. H and I) Semi-quantitative analysis of Western blot results for brain and kidney tissues respectively; * p < 0.05, ** p < 0.01.

Article Snippet: Membranes were incubated with the following primary antibodies overnight at 4°C: IL-6 (1:5000, GB11117-100, Servicebio), IL-17 (1:5000, GB11110-1-100, Servicebio), TNF-α (1:5000, GB115726-100, Servicebio), β-Actin (1:5000, GB15003, Servicebio).

Techniques: Staining, Multiplex Assay, Immunofluorescence, Fluorescence, Western Blot

Journal: Journal of Traditional and Complementary Medicine

Article Title: Chemical characterization of Jing Guan Fang and its application in alleviating coronavirus envelope protein-induced proinflammatory responses in vitro and in vivo

doi: 10.1016/j.jtcme.2025.12.003

Figure Lengend Snippet: JGF inhibits NO, IL-6, and TNF-α production in RAW264.7 and MH-S cells. The cells were treated with JGF (50, 100, 150, 300, 600 μg/mL), 2-E (0.1 μM), DXT (10 μM), or LPS (0.1 μg/mL) for 24 h. ( A ) Cell viability was evaluated using crystal violet. ( B ) NO production was measured using the Griess assay. ( C-D ) IL-6 ( C ) and TNF-α ( D ) levels were determined by ELISA. EC 50 was calculated by CompuSyn software. Data was presented as mean ± standard deviation (SD) for groups (n = 3). Significant differences are denoted as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Primary antibodies against IL-6 (Bioss, BS0379R, 1:500), TNF-α (Bioworld, BS1857, 1:300), and IL-1β (Bioss, BS6319R, 1:500) were applied overnight at room temperature.

Techniques: Griess Assay, Enzyme-linked Immunosorbent Assay, Software, Standard Deviation

Journal: Journal of Traditional and Complementary Medicine

Article Title: Chemical characterization of Jing Guan Fang and its application in alleviating coronavirus envelope protein-induced proinflammatory responses in vitro and in vivo

doi: 10.1016/j.jtcme.2025.12.003

Figure Lengend Snippet: Components of JGF inhibit 2-E-induced inflammation. The RAW264.7 and MH-S cells were co-treated with JGF compounds and 2-E for 24 h. ( A ) The 3D-HPLC fingerprint of JGF. Compound structures were sourced from the PubChem database. The detection wavelength ranged from 200 to 400 nm, and the injection volume was 20 μL. ( B ) Cell viability was evaluated using crystal violet. ( C ) NO production was measured using the Griess assay. ( D-E ) IL-6 ( D ) and TNF-α ( E ) levels were determined by ELISA. Data are presented as mean ± SD (n = 3). Statistical significance was determined relative to the 2-E group. Significant differences are denoted as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Primary antibodies against IL-6 (Bioss, BS0379R, 1:500), TNF-α (Bioworld, BS1857, 1:300), and IL-1β (Bioss, BS6319R, 1:500) were applied overnight at room temperature.

Techniques: Injection, Griess Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Traditional and Complementary Medicine

Article Title: Chemical characterization of Jing Guan Fang and its application in alleviating coronavirus envelope protein-induced proinflammatory responses in vitro and in vivo

doi: 10.1016/j.jtcme.2025.12.003

Figure Lengend Snippet: JGF reduces the 2-E-induced proinflammatory cytokines in vivo . ( A ) The experimental scheme for mouse exposure. ( B-F ) Levels of IL-6 ( B ), TNF-α ( C ), IFN-γ ( D ), IL-1β ( E ), and IL-12 ( F ) in lung tissue and serum were measured by ELISA. Data are presented as mean ± SD (n = 9 for serum, except DXT group n = 6; n = 6 for lung tissue, except DXT group n = 3) ( G ) Representative histological images of lung tissue stained with H&E and IHC images for IL-6, TNF-α, and IL-1β expression. ( H-J ) Quantification of IL-6 ( H ), TNF-α ( I ), and IL-1β ( J ) positive areas using ImageJ (n = 3). Significant differences between the control (CTL) group and other groups are denoted by ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Significant differences between the 2-E group and 2-E + JGF group are indicated by #p < 0.05, ##p < 0.01, ###p < 0.001.

Article Snippet: Primary antibodies against IL-6 (Bioss, BS0379R, 1:500), TNF-α (Bioworld, BS1857, 1:300), and IL-1β (Bioss, BS6319R, 1:500) were applied overnight at room temperature.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Control

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