il 13  (R&D Systems)

Journal: Journal of Cellular and Molecular Medicine

Article Title: Longchai Decoction Treated the Fibrosis of Liver Induced by CCl 4 Regulates Nrf2/ GPX4 Pathway to Suppress Ferroptosis

doi: 10.1111/jcmm.71151

Figure Lengend Snippet: The effect of LCD on suppressing inflammation in CCl 4 ‐induced mice. (A) The levels of serum IL‐1β were measured. (B) The concentrations of serum IL‐6 were evaluated. (C) The serum TNF‐α levels were determined. (D) Serum IL‐10 was analysed. (E) The IL‐1β/IL‐10 ratio was calculated. (F) The IL‐6/IL‐10 ratio was derived. (G) The TNF‐α/IL‐10 ratio was established.

Article Snippet: Mouse interleukin‐1β (IL‐1β, EK201B), IL‐6 (EK206), and TNF‐α (EK282) were bought from Lianke Bioengineering Institute (Shanghai, China).

Techniques: Derivative Assay

Journal: Redox Biology

Article Title: Nrf2 pathway mediates copper oxide nanoparticle-induced exacerbation of allergic asthma

doi: 10.1016/j.redox.2026.104180

Figure Lengend Snippet: Effects of CuONPs on allergic inflammation and oxidative stress in OVA-induced asthmatic mice. (A) Airway hyperresponsiveness assessed as total respiratory system resistance in response to methacholine challenge (10, 20, and 40 mg/mL). (B–F) Total and differential inflammatory cell counts in BALF. (G–L) IL-1β, IL-6, TNF-α, IL-4, IL-5, and IL-13 levels in BALF, measured by ELISA. (M and N) Total IgE and OVA specific IgE levels in serum. (O and P) MDA levels and SOD activity in lung tissue. Data are presented as means ± SD (n = 6 mice/group). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 versus NC group. # p < 0.05, ## p < 0.01, and ### p < 0.001 versus OVA group.

Article Snippet: BALF was centrifuged at 300× g for 10 min at 4 °C, and the supernatant was stored for cytokine analysis using commercially available enzyme-linked immunosorbent assay (ELISA) kits to quantify IL-1β, IL-6, tumor necrosis factor (TNF)-α, IL-4, IL-5, and IL-13 (R&D Systems, Minneapolis, MN, USA; Cat. No. MLB00C, M6000B, MTA00B, M4000B, M5000, and M1300CB, respectively).

Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Redox Biology

Article Title: Nrf2 pathway mediates copper oxide nanoparticle-induced exacerbation of allergic asthma

doi: 10.1016/j.redox.2026.104180

Figure Lengend Snippet: Effects of Nrf2 overexpression on allergic inflammation and oxidative stress in CuONP-exposed asthmatic mice. (A) Immunofluorescence analysis of lung tissue from mice administered PBS or AAV2/8-GFP via intratracheal instillation. (B) Airway hyperresponsiveness assessed as total respiratory system resistance in response to methacholine challenge (10, 20, and 40 mg/mL). (C–G) Total and differential inflammatory cell counts in BALF. (H–M) IL-1β, IL-6, TNF-α, IL-4, IL-5, and IL-13 levels in BALF, measured by ELISA. (N and O) Total IgE and OVA specific IgE levels in serum. (P and Q) MDA levels and SOD activity in lung tissue. Data are presented as means ± SD (n = 3 mice/group for panels A; n = 6 mice/group for panels B–Q). In panel B, ## p < 0.01 indicates significant differences between GFP-NC and GFP-OVA, and ∗∗ p < 0.01 indicates significant differences between GFP-OVA + CuONPs and Nrf2-OVA + CuONPs. For panels C–Q, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 indicate significant differences between AAV-GFP and AAV-Nrf2 within each condition.

Article Snippet: BALF was centrifuged at 300× g for 10 min at 4 °C, and the supernatant was stored for cytokine analysis using commercially available enzyme-linked immunosorbent assay (ELISA) kits to quantify IL-1β, IL-6, tumor necrosis factor (TNF)-α, IL-4, IL-5, and IL-13 (R&D Systems, Minneapolis, MN, USA; Cat. No. MLB00C, M6000B, MTA00B, M4000B, M5000, and M1300CB, respectively).

Techniques: Over Expression, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Materials Today Bio

Article Title: Prodrug-integrated multifunctional liposome encapsulation enhances probiotic-based bacteriotherapy for inflammatory bowel disease

doi: 10.1016/j.mtbio.2026.103110

Figure Lengend Snippet: Preventative efficacy of DTI@EcN against DSS-induced colitis. ( A ) Schematic of the experimental schedule. Mice received daily oral gavage of various bacterial formulations or drugs throughout the experimental period (bacteria: 5 × 10 8 CFU; IDE: 65 mg/kg). 3% DSS was given via drinking water from day 4 to day 8 to induce colitis. ( B ) Quantitative assessment of body weight variation in colitis mice across treatment groups. ( C ) Macroscopic appearance of colon tissues and ( D ) corresponding length measurements. ( E ) Histopathological evaluation by H&E staining and ( F ) histopathological scores of colon tissues based on H&E images. Scale bar: 100 μm. ( G ) Immunofluorescence localization of tight junction proteins ZO-1 (red) and Occludin (green) in colonic epithelia. Cell nuclei were stained with DAPI (blue). Scale bar: 100 μm. ( H ) Quantified ZO-1 and ( I ) occludin fluorescence intensities in colon tissues shown in (G) through ImageJ. ( J ) LSCM images showing DCF fluorescence in colon tissues incubated with DCFH-DA, representing the relative ROS levels across different treatment groups. Scale bar: 100 μm. ( K ) Quantified DCF fluorescence intensities from (J), analyzed with ImageJ. ( L ) Relative colonic MPO activities in colon tissues in different treatment groups. ( M - P ) Levels of pro-inflammatory cytokines, including IL-6 ( M ), IL-1β ( N ), TNF-α (O), and IFN-γ ( P ), in colon tissues, determined by ELISA kits. Data represent the mean ± SEM (n = 5). Statistical analysis was performed using one-way ANOVA. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The selected chemicals and biological materials used in this research are as follows: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, Macklin), 4-bromobutyric acid (Bidepharm), thiourea (Macklin), sodium hydroxide (NaOH, Macklin), hydrochloric acid (HCl, Macklin), trifluoroacetic acid (Bidepharm), idebenone (Bidepharm), hexadecyltrimethylammonium bromide (Macklin), 4-dimethylaminopyridine (DMAP, Macklin), N-hydroxysuccinimide (NHS, Macklin), 2′,7′-dichlorofluorescin diacetate (DCFH-DA, Aladdin), DSPE-PEG2000-NH 2 (Yusi), DSPE-PEG2000-FITC (Yusi), cholesterol (Yuanye), trypsin (Macklin), pepsin powder (Bidepharm), bile salts (Macklin), ampicillin (Bidepharm), lysin (Bidepharm), dextran sulfate sodium salt (DSS, MW 40,000, MP Bio), LB nutrient agar (Hopebiol), Cell Counting Kit-8 (CCK-8, Beyotime), mouse IL-6, IFN-γ, IL-1β, and TNF-α uncoated ELISA kits (Thermo Fisher Scientific), and antigen retrieval solution (Servicebio).

Techniques: Bacteria, Staining, Immunofluorescence, Fluorescence, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Materials Today Bio

Article Title: Prodrug-integrated multifunctional liposome encapsulation enhances probiotic-based bacteriotherapy for inflammatory bowel disease

doi: 10.1016/j.mtbio.2026.103110

Figure Lengend Snippet: Therapeutic efficacy of DTI@EcN against DSS-induced colitis. ( A ) Schematic of the experimental schedule. Colitis was induced by administering 3% DSS in drinking water for the first 5 days. Subsequently, different treatments were administered daily for 5 days to evaluate their therapeutic efficacy. ( B ) Body weight dynamics of colitis mice under different treatments. ( C ) Macroscopic colon appearance and ( D ) corresponding length measurements across groups. ( E) Histopathological assessment by H&E staining. Scale bar: 100 μm. ( F ) Histopathology scores of colon tissues based on the H&E images. ( G ) Localization of tight junction proteins ZO-1 (red) and occludin (green) in colonic epithelium across treatment groups. Cell nuclei were stained with DAPI (blue). Scale bar: 100 μm. ( H ) Quantification of ZO-1 and ( I ) Occludin fluorescence intensities from (G), analyzed using ImageJ. ( J ) LSCM images showing DCF fluorescence in colon tissues incubated with DCFH-DA, representing the relative ROS levels across different treatment groups. Scale bar: 100 μm. ( K ) Quantitative analysis of DCF fluorescence intensities from (J). ( L ) Relative MPO activity in colon tissues across various treatment groups. ( M - P ) Levels of pro-inflammatory cytokines, including IL-6 ( M ), IL-1β ( N ), TNF-α ( O ), and IFN-γ ( P ), in colon tissues, measured by ELISA kits. Data represented the mean ± SEM (n = 5 or 6). Statistical analysis was performed using one-way ANOVA. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The selected chemicals and biological materials used in this research are as follows: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, Macklin), 4-bromobutyric acid (Bidepharm), thiourea (Macklin), sodium hydroxide (NaOH, Macklin), hydrochloric acid (HCl, Macklin), trifluoroacetic acid (Bidepharm), idebenone (Bidepharm), hexadecyltrimethylammonium bromide (Macklin), 4-dimethylaminopyridine (DMAP, Macklin), N-hydroxysuccinimide (NHS, Macklin), 2′,7′-dichlorofluorescin diacetate (DCFH-DA, Aladdin), DSPE-PEG2000-NH 2 (Yusi), DSPE-PEG2000-FITC (Yusi), cholesterol (Yuanye), trypsin (Macklin), pepsin powder (Bidepharm), bile salts (Macklin), ampicillin (Bidepharm), lysin (Bidepharm), dextran sulfate sodium salt (DSS, MW 40,000, MP Bio), LB nutrient agar (Hopebiol), Cell Counting Kit-8 (CCK-8, Beyotime), mouse IL-6, IFN-γ, IL-1β, and TNF-α uncoated ELISA kits (Thermo Fisher Scientific), and antigen retrieval solution (Servicebio).

Techniques: Drug discovery, Staining, Histopathology, Fluorescence, Incubation, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Materials Today Bio

Article Title: Prodrug-integrated multifunctional liposome encapsulation enhances probiotic-based bacteriotherapy for inflammatory bowel disease

doi: 10.1016/j.mtbio.2026.103110

Figure Lengend Snippet: Therapeutic efficacy of DTI@EcN against STm-induced colitis. ( A ) Schematic of the experimental schedule. C57BL/6J mice were pretreated with streptomycin (100 μL, 200 mg/mL) one day prior to oral administration with STm. Afterward, different bacterial formulations were given to mice for 4 days to evaluate their effectiveness against STm-induced colitis. ( B ) Changes in STm counts in feces during the treatment period. ( C ) Quantification of STm per mg of feces on day 4 post-administration of various EcN formulations. ( D ) STm and ( E ) EcN counts in the cecum after different treatments. ( F ) Histological evaluation of colon damage with H&E staining (Scale bar: 100 μm) and ( G ) corresponding histopathology scores, respectively. ( H ) Neutrophil infiltration measured by colonic MPO activity. ( I - L ) Levels of pro-inflammatory cytokines in colon tissues, including IL-6 ( I ), IL-1β ( J ), TNF-α ( K ), and IFN-γ ( L ), as determined by ELISA kits. Data are presented as means ± SEM (n = 5). Statistical analysis was performed using one-way ANOVA. ns: no significance, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: The selected chemicals and biological materials used in this research are as follows: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, Macklin), 4-bromobutyric acid (Bidepharm), thiourea (Macklin), sodium hydroxide (NaOH, Macklin), hydrochloric acid (HCl, Macklin), trifluoroacetic acid (Bidepharm), idebenone (Bidepharm), hexadecyltrimethylammonium bromide (Macklin), 4-dimethylaminopyridine (DMAP, Macklin), N-hydroxysuccinimide (NHS, Macklin), 2′,7′-dichlorofluorescin diacetate (DCFH-DA, Aladdin), DSPE-PEG2000-NH 2 (Yusi), DSPE-PEG2000-FITC (Yusi), cholesterol (Yuanye), trypsin (Macklin), pepsin powder (Bidepharm), bile salts (Macklin), ampicillin (Bidepharm), lysin (Bidepharm), dextran sulfate sodium salt (DSS, MW 40,000, MP Bio), LB nutrient agar (Hopebiol), Cell Counting Kit-8 (CCK-8, Beyotime), mouse IL-6, IFN-γ, IL-1β, and TNF-α uncoated ELISA kits (Thermo Fisher Scientific), and antigen retrieval solution (Servicebio).

Techniques: Drug discovery, Staining, Histopathology, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Genes & Diseases

Article Title: Targeting MDK alleviates bone loss via dual regulation of osteogenic differentiation and inflammatory cytokine expression

doi: 10.1016/j.gendis.2025.101931

Figure Lengend Snippet: iMDK alleviates bone loss via dual regulation of bone formation and inflammatory cytokines. (A) Representative micro-CT images of distal femora (top) with three-dimensional reconstruction of the region of interest (bottom), with the blue indicating higher-density bone and the red indicating lower-density bone. (B – D) Statistical quantification of trabecular bone microstructural parameters (BMD, BV/TV, and Tb.Th). Inter-group comparisons were analyzed by one-way ANOVA. (E) Representative images of trabecular bone area in distal femur sections stained with hematoxylin and eosin. Scale bar, 200 μm or 50 μm. (F) Quantitative analysis of the trabecular bone area of the distal femur stained with hematoxylin and eosin. Inter-group comparisons were analyzed by one-way ANOVA. (G) Immunohistochemical staining of OCN in distal femurs. Scale bar, 200 μm or 50 μm. (H) Quantitative analysis of OCN-positive area. Inter-group comparisons were analyzed by one-way ANOVA. (I) Western blotting analysis of inflammatory cytokine expression (IL-6, TNF-α, and IL-1β) in mouse bone tissues. (J) Inflammatory cytokine expression (IL-6, TNF-α, and IL-1β) in mouse serum was detected by ELISA. Inter-group comparisons were analyzed by one-way ANOVA. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; “ns” indicates non-significant differences.

Article Snippet: We purchased the Mouse P1NP (Procollagen 1 N-Terminal Propeptide) ELISA Kit (Cat#e-el-m0233), Mouse IL-6 ELISA Kit (Cat#e-el-m0044), Mouse TNF-α ELISA Kit (Cat#e-el-m3063), and Mouse IL-1β ELISA Kit (Cat#e-el-m0037) from Elabscience (Wuhan, China).

Techniques: Micro-CT, Staining, Immunohistochemical staining, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Genes & Diseases

Article Title: Targeting MDK alleviates bone loss via dual regulation of osteogenic differentiation and inflammatory cytokine expression

doi: 10.1016/j.gendis.2025.101931

Figure Lengend Snippet: Recombinant MDK protein triggers the activation of inflammatory cytokines through the NF-κB signaling pathway. (A, B) IL-6, TNFα, and IL-1β expression levels were detected using Western blotting. MC3T3-E1 cells were treated with recombinant MDK protein (600 ng/mL). Osteogenic differentiation was induced for 7 days. Inter-group comparisons were analyzed by a two-tailed unpaired Student's t -test (for normally distributed data with equal variance). (C, D) Western blotting analysis of NF-κB signaling pathway molecules in MC3T3-E1 cells treated with recombinant MDK protein for 7 days during osteoblastic differentiation. Inter-group comparisons were analyzed by two-tailed unpaired Student's t -test (for normally distributed data with equal variance). (E, F) IL-6 and IL-1β expression levels were detected using Western blotting. MC3T3-E1 cells were pretreated with 10 μM BAY 11–7082. Osteogenic differentiation was induced for 7 days. Inter-group comparisons were analyzed by one-way ANOVA. ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: We purchased the Mouse P1NP (Procollagen 1 N-Terminal Propeptide) ELISA Kit (Cat#e-el-m0233), Mouse IL-6 ELISA Kit (Cat#e-el-m0044), Mouse TNF-α ELISA Kit (Cat#e-el-m3063), and Mouse IL-1β ELISA Kit (Cat#e-el-m0037) from Elabscience (Wuhan, China).

Techniques: Recombinant, Activation Assay, Expressing, Western Blot, Two Tailed Test

Journal: Genes & Diseases

Article Title: Targeting MDK alleviates bone loss via dual regulation of osteogenic differentiation and inflammatory cytokine expression

doi: 10.1016/j.gendis.2025.101931

Figure Lengend Snippet: Schematic representation of MDK alleviating bone loss. MDK is significantly elevated in the serum of postmenopausal osteoporotic women and ovariectomized mice. Due to estrogen deficiency, iMDK alleviates bone loss by promoting bone formation and inhibiting inflammatory factors. Recombinant MDK protein inhibits osteogenic differentiation through the PI3K/AKT signaling pathway and up-regulates inflammatory factors IL-6, TNF-α, and IL-1β via the NF-κB signaling pathway.

Article Snippet: We purchased the Mouse P1NP (Procollagen 1 N-Terminal Propeptide) ELISA Kit (Cat#e-el-m0233), Mouse IL-6 ELISA Kit (Cat#e-el-m0044), Mouse TNF-α ELISA Kit (Cat#e-el-m3063), and Mouse IL-1β ELISA Kit (Cat#e-el-m0037) from Elabscience (Wuhan, China).

Techniques: Recombinant

Journal: Redox Biology

Article Title: Hepatic supersulfides attenuate acetaminophen-induced liver injury via enhanced detoxification and anti-inflammatory mechanisms

doi: 10.1016/j.redox.2026.104140

Figure Lengend Snippet: Suppression of proinflammatory cytokine production and M1-type macrophage polarization by a supersulfide donor in livers from APAP-treated mice. (A–C) Levels of IL-1β, IL-6, and IL-10 in liver tissue. Mice were intraperitoneally administered APAP (330 mg/kg), followed by subcutaneous NAC-S2 administration at 30 min and 2 h post-APAP. Livers were harvested 24 h after APAP administration, and cytokine levels were quantified by ELISA. (D) Western blot analysis of hepatic iNOS and Arg-1 expression. (E, F) Quantification of Western blot band intensities. Relative protein levels were normalized to β-actin. Uncropped blots are shown in . Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc multiple comparisons test. Data were expressed as means ± SD (n ≥ 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001; # P < 0.05, # # P < 0.01, # # # P < 0.001 compared to control group.

Article Snippet: Mouse IL-1β/IL-1F2 Quantikine ELISA Kit (MLB00C), Mouse IL-6 Quantikine ELISA Kit (M6000B-1), and Mouse IL-10 Quantikine ELISA Kit (M1000B) were obtained from R&D Systems (Minneapolis, MB, USA).

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

Journal: Redox Biology

Article Title: Hepatic supersulfides attenuate acetaminophen-induced liver injury via enhanced detoxification and anti-inflammatory mechanisms

doi: 10.1016/j.redox.2026.104140

Figure Lengend Snippet: Effects of NAC-S2, oxNAC, and NAC on APAP-induced liver injury. (A) Experimental protocol. Mice were intraperitoneally administered APAP (330 mg/kg), followed by subcutaneous administration of sulfur compounds (NAC-S2, oxNAC, or NAC) at 30 min and 2 h post-APAP. Blood was collected at 8, 12, and 24 h post-APAP, and liver tissue was harvested at 24 h post-APAP. (B, C) Time-course of serum ALT and AST levels. (D) Quantification of necrotic area in liver. Macroscopic appearance and H&E staining of liver tissues are shown in Hepatic cytokine levels: IL-1β (E), IL-6 (F), and IL-10 (G). (H) Western blot analysis of iNOS and Arg-1 expression in liver. (I, J) Quantification of Western blot band intensities. Relative protein levels were normalized to β-actin. Uncropped blots are shown in . Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc multiple comparisons test. Data were expressed as means ± SD (n ≥ 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001; # P < 0.05, # # P < 0.01, # # # P < 0.001 compared to control group.

Article Snippet: Mouse IL-1β/IL-1F2 Quantikine ELISA Kit (MLB00C), Mouse IL-6 Quantikine ELISA Kit (M6000B-1), and Mouse IL-10 Quantikine ELISA Kit (M1000B) were obtained from R&D Systems (Minneapolis, MB, USA).

Techniques: Staining, Western Blot, Expressing, Control

Journal: Redox Biology

Article Title: Hepatic supersulfides attenuate acetaminophen-induced liver injury via enhanced detoxification and anti-inflammatory mechanisms

doi: 10.1016/j.redox.2026.104140

Figure Lengend Snippet: Effects of the supersulfide donor TGS4 on APAP-induced liver injury. (A) Experimental protocol. Mice were intraperitoneally administered APAP (330 mg/kg), followed by subcutaneous injection of TGS4 at 30 min and 2 h post-APAP. Two different doses were used as indicated. Blood was collected at 8, 12, and 24 h post-APAP, and liver tissue was harvested at 24 h post-APAP. (B, C) Time-course of serum ALT and AST levels. (D–F) Hepatic cytokine levels: IL-1β (D), IL-6 (E), and IL-10 (F). Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc multiple comparisons test. Data were expressed as means ± SD (n ≥ 4). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001; # P < 0.05, # # P < 0.01, # # # P < 0.001 compared to control group.

Article Snippet: Mouse IL-1β/IL-1F2 Quantikine ELISA Kit (MLB00C), Mouse IL-6 Quantikine ELISA Kit (M6000B-1), and Mouse IL-10 Quantikine ELISA Kit (M1000B) were obtained from R&D Systems (Minneapolis, MB, USA).

Techniques: Injection, Control

Journal: Bioactive Materials

Article Title: Bioengineered extracellular vesicles escape lysosomal degradation and deliver Tet-PKM2 for macrophage immunometabolic reprogramming and periodontitis treatment

doi: 10.1016/j.bioactmat.2026.01.002

Figure Lengend Snippet: Immunomodulatory effects of the bioengineered LEVs Tet−PKM2 @TA in terms of their ability to modulate macrophage polarization in vitro . The macrophages were treated with 100 ng/mL LPS for 24 h and then treated with PBS (Control), 100 μg/mL LEVs PKM2 , LEVs Tet−PKM2 , or LEVs Tet−PKM2 @TA for another 24 h. ( A ) The relative mRNA expression levels of M1 polarization-related genes ( IL-6 and IL-1β ) and M2 polarization-related genes ( IL-4 and Arg-1 ) in the Control, LEVs PKM2 , LEVs Tet−PKM2 , and LEVs Tet−PKM2 @TA groups (qRT‒PCR) ( n = 3). ( B ) Concentrations of M1-related cytokines (IL-6 and TNF-α) and M2-related cytokines (IL-4 and IL-10) in the supernatants of the Control, LEVs PKM2 , LEVs Tet−PKM2 , and LEVs Tet−PKM2 @TA groups (ELISA) ( n = 3). ( C ) Representative immunofluorescence images and quantification of the expression levels of M1-related proteins (iNOS and CCR7) and M2-related proteins (CD163, CD206, and Arg-1) in the Control, LEVs PKM2 , LEVs Tet−PKM2 , and LEVs Tet−PKM2 @TA groups ( n = 3). The data are expressed as the mean ± SEM. Statistical analysis was performed with one-way ANOVA ( A , B , and C ). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 indicate significant differences between the indicated columns.

Article Snippet: Kits were sourced as follows: TNF-α, IL-4, and IL-10 from Fankew (Shanghai Kexing Trading Co., Ltd., China) and IL-6 from Proteintech.

Techniques: In Vitro, Control, Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence

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