Journal: Bioactive Materials

Article Title: Immunomodulatory supramolecular hydrogel for rheumatoid arthritis management via adenosine A2A receptor-mediated macrophage remodeling

doi: 10.1016/j.bioactmat.2025.11.031

Figure Lengend Snippet: The design of an inflammatory macrophages-targeted, acid-sensitive PNSH for delivery MTX and its role in promoting A2AR activation on M2 macrophages for arthritis therapy. (A)The structure of acid-sensitive polymer-nanomedicine supramolecular hydrogels, composed of drug-loaded MTX NPs. (B) Schematic illustration of arthritis therapy targeting inflammatory joint network via mPECN NPs-mediated release of MTX. This approach leverages MTX targeting the A2AR and repolarization of macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. Activation of A2AR and macrophage repolarization by mPECN-MTX NPs synergistically enhanced the anti-inflammatory effect of PNSH-mediated nanomedicine in an arthritis rat model.

Article Snippet: Subsequently, 10 mg of protein was loaded into 10 % SDS-polyacrylamide gel electrophoresis for detection of adenosine A2AR (sc-32261, Santa Cruz), IRF4 (#62834S, Cell Signaling Technology, MA, USA), IRF8 (#5628T, Cell Signaling Technology, MA, USA) and GAPDH (TA-08, ZSGB-BIO, Beijing, China).

Techniques: Activation Assay, Polymer

Journal: Bioactive Materials

Article Title: Immunomodulatory supramolecular hydrogel for rheumatoid arthritis management via adenosine A2A receptor-mediated macrophage remodeling

doi: 10.1016/j.bioactmat.2025.11.031

Figure Lengend Snippet: MTX@PNSH promotes anti-inflammatory M2 macrophages through the A2AR signaling pathway. (A) The intracellular cAMP levels in Raw 264.7 cells were determined by homogeneous time-resolved fluorescence (HTRF) following different treatments. (B) qRT-PCR analysis of relative mRNA of A2ar with different treatments. (C) Immunostaining of A2AR expression in Raw 264.7 cells with different treatments. (D) Flow cytometry analysis of A2AR + cells in Raw 264.7 cells with different treatments and the percentage of A2AR + cells in Raw 264.7 cells with different treatments. (E) qRT-PCR analysis of relative mRNA expression of Pdl1 without LPS treatment. (F) qRT-PCR analysis of relative mRNA expression of Ido1 without LPS treatment. (G) qRT-PCR analysis of relative mRNA expression of Pdl1 with LPS treatment. (H) qRT-PCR analysis of relative mRNA expression of Ido1 with LPS treatments. (I) Western blotting analysis of A2AR protein level in macrophages after 24 h of different treatment with or without A2AR inhibitor. (J) Quantitative analysis of A2AR protein level in different treatments with or without A2AR inhibitor. (K) Flow cytometry analysis of CD11b and CD39 in Raw 264.7 cells with different treatments and the percentage of CD11b + CD39 + macrophages. (L) Flow cytometry analysis of CD11b and CD73 in Raw 264.7 cells with different treatments and the percentage of CD11b + CD73 + macrophages. (M) Flow cytometry analysis of CD11b and PD-L1 in Raw 264.7 cells after 24 h of different treatments and the percentage of CD11b + PD-L1 + macrophages in Raw 264.7 cells after 24 h of different treatments. Data are presented as mean ± SD, n = 3. Statistical significance was determined using one-way ANOVA, followed by Dunnett's post hoc test for comparisons between groups.

Article Snippet: Subsequently, 10 mg of protein was loaded into 10 % SDS-polyacrylamide gel electrophoresis for detection of adenosine A2AR (sc-32261, Santa Cruz), IRF4 (#62834S, Cell Signaling Technology, MA, USA), IRF8 (#5628T, Cell Signaling Technology, MA, USA) and GAPDH (TA-08, ZSGB-BIO, Beijing, China).

Techniques: Fluorescence, Quantitative RT-PCR, Immunostaining, Expressing, Flow Cytometry, Western Blot

Journal: Nature Communications

Article Title: Timosaponin AIII enhances CAR-T cell potency and prevents relapse through impairing CAR-Tregs

doi: 10.1038/s41467-026-70867-5

Figure Lengend Snippet: a Photosensitive biotinylated-TAIII (TAIII-P) was generated via click chemistry and used for proximity labeling. b Human T cells isolated from PBMCs were treated with TAIII-P (0–20 μM) for 4 h. Association with A2AR was assessed by avidin-biotin pulldown and western blot (upper), and quantified using ImageJ (lower). Data are presented as mean ± SD from four independent experiments ( n = 4 biological replicates); two-way ANOVA with Dunnett’s test. c – d Competition assay in cell lysates pretreated with TAIII, followed by TAIII-P pulldown. Relative A2AR levels were quantified. Data are presented as mean ± SD ( n = 3 biological replicates); two-way ANOVA with Dunnett’s test. e 293 T cells were co-transfected with A2AR and a CREB luciferase reporter, followed by treatment with the indicated compounds. CREB transcriptional activity was measured using a luciferase assay. NECA was not included, as A2AR overexpression alone was sufficient to activate the CREB reporter. f 293 T cells transfected with A2AR were incubated with NECA (0.1 μM) and TAIII or AZD4635 for 30 min. cAMP levels were determined by LANCE assay; fluorescence ratios (615/665 nm) were proportional to cAMP. Blank wells were negative controls. Data are presented as mean ± SD from four independent experiments ( n = 4 biological replicates); ordinary one-way ANOVA with Dunnett’s test. g 293 T cells transfected with or without A2AR were treated with indicated compounds; cAMP levels were measured by LANCE assay. Data are presented as mean ± SD ( n = 3 biological replicates); two-way ANOVA with Dunnett’s test. h Predicted mode of binding of TAIII to A2AR based upon molecular modeling (PDB ID: 4EIY). i – j CREB-Fluc reporters with wild-type or mutant A2AR residues were used to assess the contribution of three residues to TAIII binding ( i ) and dose-dependent effects ( j ). Data are presented as mean ± SD ( n = 3 biological replicates); two-way ANOVA with Dunnett’s test.

Article Snippet: For immunoblotting, the following primary antibodies were used: β-Actin (Yeason, 30101ES60, 1:5000), CREB (Cell Signaling Technology, 9197 T, 1:1000), phospho-CREB (Ser133; Cell Signaling Technology, 9198 T, 1:1000), A2AR (Santa Cruz Biotechnology, sc-32261, 1:1000), and GAPDH (Cell Signaling Technology, #2118, 1:5000).

Techniques: Generated, Labeling, Isolation, Avidin-Biotin Assay, Western Blot, Competitive Binding Assay, Transfection, Luciferase, Activity Assay, Over Expression, Incubation, Fluorescence, Binding Assay, Mutagenesis

Journal: Nature Communications

Article Title: Timosaponin AIII enhances CAR-T cell potency and prevents relapse through impairing CAR-Tregs

doi: 10.1038/s41467-026-70867-5

Figure Lengend Snippet: a CD19 CAR-T cells were generated as in (Fig. ) and co-cultured with Raji-Luc or Nalm6-Luc cells under indicated conditions for 24 h; the CD19 levels were assessed by measuring the luciferase signal. Data represent pooled biological replicates; Data are presented as SD (n = 3 biological replicates); two-way ANOVA with Tukey’s test. b – c CAR-T cells from different donors were co-cultured with Raji-Luc or Nalm6-Luc cells under indicated treatments. IFN-γ, IL-2, and TNF-α in supernatants were measured by ELISA. Data are presented as mean ± SD ( n = 3 biological replicates); ordinary one-way ANOVA or two-way ANOVA with Dunnett’s test. d CD19 CAR-T cells were co-cultured with Raji cells; FoxP3 mRNA levels were determined by qRT-PCR. Data are presented as mean ± SD ( n = 3 biological replicates); ordinary one-way ANOVA with Dunnett’s test. e Effects of TAIII on central memory T cells (CD62L⁺CD45RO⁺, Tcm) were assessed after 48 h co-culture with Raji cells at E:T ratios of 1:1, 1:2, or 1:4 by flow cytometry. Data are presented as mean ± SD ( n = 3 biological replicates); two-way ANOVA with Dunnett’s test. f A2AR knockdown (KD) CD19-CAR-T cells were generated via shRNA transduction (GFP⁺), and knockdown efficiency was confirmed by qRT-PCR. Data are presented as mean ± SD ( n = 3 biological replicates); two-sided unpaired t -test. g Intracellular cAMP levels in A2AR-KD CAR-T cells were measured to assess TAIII-mediated inhibition of A2AR signaling; Two-way ANOVA with Šídák’s multiple comparisons test. h – i A2AR-KD, parental CD19-CAR-T, and UNT cells were co-cultured with Nalm6-Luc cells for 24 h. Cytotoxicity was measured by residual luciferase activity at E:T ratios of 1:1 and 1:2 (normalized to Nalm6-only controls), and IL-2 secretion was quantified by ELISA at E:T = 1:1. Data are presented as mean ± SD ( n = 3 biological replicates); two-way ANOVA or ordinary one-way ANOVA with Tukey’s test.

Article Snippet: For immunoblotting, the following primary antibodies were used: β-Actin (Yeason, 30101ES60, 1:5000), CREB (Cell Signaling Technology, 9197 T, 1:1000), phospho-CREB (Ser133; Cell Signaling Technology, 9198 T, 1:1000), A2AR (Santa Cruz Biotechnology, sc-32261, 1:1000), and GAPDH (Cell Signaling Technology, #2118, 1:5000).

Techniques: Generated, Cell Culture, Luciferase, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Co-Culture Assay, Flow Cytometry, Knockdown, shRNA, Transduction, Inhibition, Activity Assay

Journal: Drug Design, Development and Therapy

Article Title: Cordycepin Ameliorates Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice by Inhibiting IL-6/IL-6R-Mediated p38 MAPK and NF-κB Activation Through Adenosine A 2A Receptor Stimulation

doi: 10.2147/DDDT.S575035

Figure Lengend Snippet: Cordycepin abates DSS-induced damage in NCM460 cells by activating adenosine receptor A 2A . ( A ) The cAMP levels in NCM460 cells under treatment with 2% DSS and/or 1 μmol/L and 10 μmol/L COR. ( B ) The cAMP levels in NCM460 cells under treatment with 2% DSS, 10 μmol/L COR, 5 μmol/L SCH58261 (SCH), and / or 5 μmol/L DPCPX for 24 h. The release levels of ( C ) LDH, ( D ) IL-1β, ( E ) IL-6, and ( F ) TNF-α were measured by using Elisa. ( G ) Representative images of PI staining and the percentage of PI-positive cells, scar bar = 100 μm. ( H – J ) The protein expression of ZO-1, A 2A AR and A 1 AR in NCM460 cells was determined by using Western blotting assay. The results were representative of three independent experiments and expressed as mean ± SEM. Data were compared using two-tailed Student’s t tests. vs. control group; # P < 0.05, vs. control group; *P < 0.05, **P < 0.01 and ***P < 0.001 vs. the 2% DSS group.

Article Snippet: Adenosine A 2A receptor Antibody (A 2A AR, sc-32261) was purchased from Santa Cruz Biotechnology (USA).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Expressing, Western Blot, Two Tailed Test, Control

Journal: Drug Design, Development and Therapy

Article Title: Cordycepin Ameliorates Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice by Inhibiting IL-6/IL-6R-Mediated p38 MAPK and NF-κB Activation Through Adenosine A 2A Receptor Stimulation

doi: 10.2147/DDDT.S575035

Figure Lengend Snippet: The results of molecular docking and molecular dynamics simulation of adenosine receptor A 2A -cordycepin. ( A ) 2D (left) and 3D (right) molecular docking diagrams of A 2A AR-COR. The PDB ID of the A 2A AR protein is 5nm4. The yellow stick represents the structure of COR, and the cartoon depicts the protein structure. The green sticks indicate amino acid residues within 4 Å that form hydrogen bonds with COR. Yellow dashed lines represent hydrogen bonds, with adjacent numbers denoting the distance between COR and the hydrogen-bonded amino acids. White sticks represent amino acids involved in hydrophobic interactions with COR. ( B ) The Root Mean Square Deviation (RMSD). ( C ) The number of hydrogen bonds of the complex. ( D ) The Root Mean Square Fluctuation (RMSF) values for the complex. ( E ) The Radius of Gyration (Rg) values of the complex. ( F ) Solvent Accessible Surface Area (SASA) of the complex.

Article Snippet: Adenosine A 2A receptor Antibody (A 2A AR, sc-32261) was purchased from Santa Cruz Biotechnology (USA).

Techniques: Solvent

Journal: Drug Design, Development and Therapy

Article Title: Cordycepin Ameliorates Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice by Inhibiting IL-6/IL-6R-Mediated p38 MAPK and NF-κB Activation Through Adenosine A 2A Receptor Stimulation

doi: 10.2147/DDDT.S575035

Figure Lengend Snippet: Cordycepin inhibits IL-6/IL-6R-mediated p38 MAPK and NF-κB phosphorylation activation relying on activation of adenosine receptor A 2A . ( A – E ) Representative blot bands and relative expression levels for IL-6R, NF-κB P-p65, NF-κB p65, NF-κB P-p65/p65, detected by Western blotting. ( F – I ) Representative blot bands and relative expression levels for P-p38 MAPK, p38 MAPK, P-p38/p38 MAPK. ( J – M ) Representative blot bands and relative expression levels for P-STAT3, STAT3, P-STAT3/STAT3. Data were presented as the means ± SEM of three mice in each group and were compared using two-tailed Student’s t tests. # P < 0.05, ## P < 0.01 vs. control group; *P < 0.05, **P < 0.01 vs. the DSS model (DSS) group.

Article Snippet: Adenosine A 2A receptor Antibody (A 2A AR, sc-32261) was purchased from Santa Cruz Biotechnology (USA).

Techniques: Phospho-proteomics, Activation Assay, Expressing, Western Blot, Two Tailed Test, Control

Journal: Drug Design, Development and Therapy

Article Title: Cordycepin Ameliorates Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice by Inhibiting IL-6/IL-6R-Mediated p38 MAPK and NF-κB Activation Through Adenosine A 2A Receptor Stimulation

doi: 10.2147/DDDT.S575035

Figure Lengend Snippet: The ameliorative effect of cordycepin on DSS-induced colitis is attributed to its activation of A 2A AR, which inhibits IL-6/IL-6R-mediated phosphorylation activation of p38 MAPK and NF-κB.

Article Snippet: Adenosine A 2A receptor Antibody (A 2A AR, sc-32261) was purchased from Santa Cruz Biotechnology (USA).

Techniques: Activation Assay, Phospho-proteomics