Journal: Scientific Reports

Article Title: Selective antagonism of adenosine A2A receptor reduces hypobaric hypoxia-induced neuroinflammation by inhibiting cGAS-STING pathway

doi: 10.1038/s41598-025-30717-8

Figure Lengend Snippet: KW6002 prevented mtDNA release by inhibiting EPAC1/VDAC1 pathway-mediated opening of MPTP. ( a ) Representative TEM images showing mitochondrial ultrastructure and quantitative analysis of lesions mitochondria in the hippocampal tissues, n = 6/group. ( b ) Cytosolic mtDNA levels of D-loop3, COX1 and mt- ND1 in the hippocampal tissues, n = 4/group. ( c ) Representative images of immunofluorescence staining and quantitative analysis of dsDNA (red)/DAPI (blue), n = 4/group. ( d ) The concentration of cAMP in brain, n = 12/group. ( e , f ) Representative western blot images showing EPAC1 and VDAC1 protein levels in the hippocampal tissues, n = 6/group. ( g ) The levels of inflammatory factors secreted by HY-treated BV2 microglial cells, n = 12/group. ( h ) Representative immunofluorescence staining images showing mitochondrial EPAC1 and VDAC1 levels in HY-induced BV2 cell model, quantitative analysis of levels of EPAC1 and VDAC1, quantitative analysis of colocalization between EPAC1 and VDAC1, n = 8 or 12/group. *** p < 0.001, ** p < 0.01 vs. control group. ### p < 0.001, ## p < 0.01, # p < 0.05 vs. HY group.

Article Snippet: After blocked with 5% non-fat milk, the membranes were incubated successively with specific primary overnight at 4 °C.and secondary antibodies at room temperature for 1 h. (ADORA1 antibody[ R23384 , 1:1000 dilution, Chengdu Zen-Bioscience], ADORA2A antibody[ab79714, 1:1000 dilution, Abcam], ADORA2B antibody[21071-1-AP, 1:1000 dilution, Proteintech], ADORA3 antibody[820199, 1:1000 dilution, Chengdu Zen-Bioscience], ZO-1 antibody[511417, 1:1000 dilution, Chengdu Zen-Bioscience], Occludin antibody [27260-1-AP, 1:1000 dilution, Proteintech], Claudin 1 antibody[13050-1-AP, 1:1000 dilution, Proteintech], PSD95 antibody[20665-1-AP, 1:1000 dilution, Proteintech], SYN1 antibody[20258-1-AP, 1:1000 dilution, Proteintech], ARC antibody[16290-1-AP, 1:1000 dilution, Proteintech], BDNF antibody[66292-1-Ig, 1:1000 dilution, Proteintech], HO-1 antibody[ R22808 , 1:1000 dilution, Chengdu Zen-Bioscience], Nrf2 antibody[12721 S, 1:1000 dilution, Cell Signaling Technology], cGAS antibody[31659, 1:1000 dilution, Cell Signaling Technology], STING antibody[19851-1-AP, 1:2000 dilution, Proteintech], p-STING antibody[PA5-105674, 1:500 dilution, Thermo Fisher Scientific], p-IRF3 antibody[530857, 1:1000 dilution, Chengdu Zen-Bioscience], EPAC1 antibody[12572-1-AP, 1:1000 dilution, Proteintech], VDAC1 antibody[ab154856, 1:1000 dilution, Abcam], GAPDH antibody[ R24404 , 1:5000 dilution, Chengdu Zen-Bioscience], ACTB antibody[R380624, 1:5000 dilution, Chengdu Zen-Bioscience], Tubulin antibody[80713-1-RR, 1:1000 dilution, Proteintech], TOM20 antibody[11802-1-AP, 1:5000 dilution, Proteintech]. secondary antibody Goat Anti-Rabbit IgG H&L(HRP)[511203, 1:5000 dilution, Chengdu Zen-Bioscience], secondary antibody Goat Anti-Mouse IgG H&L(HRP)[511103, 1:5000 dilution, Chengdu Zen-Bioscience].

Techniques: Immunofluorescence, Staining, Concentration Assay, Western Blot, Control

Journal: Scientific Reports

Article Title: Selective antagonism of adenosine A2A receptor reduces hypobaric hypoxia-induced neuroinflammation by inhibiting cGAS-STING pathway

doi: 10.1038/s41598-025-30717-8

Figure Lengend Snippet: Schematic illustration depicting that selective antagonism on ADORA2A reduces HY-induced neuroinflammation by inhibiting cGAS-STING pathway. HY exposure enhanced the level of ADORA2A, which in turn promoted the production of cAMP and cascading mitochondrial levels of EPAC1 and VDAC1. The interaction between EPAC1 and VDAC1 regulated MPTP opening and promoted mtDNA release to cytoplasm, which subsequently initiated cGAS-STING inflammatory pathway. Selective antagonism on ADORA2A restores HY-induced neuroinflammation through negatively regulating above biological process. The illustrative picture was created in BioRender. (yehui, G. (2025) https://BioRender.com/e23u690 ).

Article Snippet: After blocked with 5% non-fat milk, the membranes were incubated successively with specific primary overnight at 4 °C.and secondary antibodies at room temperature for 1 h. (ADORA1 antibody[ R23384 , 1:1000 dilution, Chengdu Zen-Bioscience], ADORA2A antibody[ab79714, 1:1000 dilution, Abcam], ADORA2B antibody[21071-1-AP, 1:1000 dilution, Proteintech], ADORA3 antibody[820199, 1:1000 dilution, Chengdu Zen-Bioscience], ZO-1 antibody[511417, 1:1000 dilution, Chengdu Zen-Bioscience], Occludin antibody [27260-1-AP, 1:1000 dilution, Proteintech], Claudin 1 antibody[13050-1-AP, 1:1000 dilution, Proteintech], PSD95 antibody[20665-1-AP, 1:1000 dilution, Proteintech], SYN1 antibody[20258-1-AP, 1:1000 dilution, Proteintech], ARC antibody[16290-1-AP, 1:1000 dilution, Proteintech], BDNF antibody[66292-1-Ig, 1:1000 dilution, Proteintech], HO-1 antibody[ R22808 , 1:1000 dilution, Chengdu Zen-Bioscience], Nrf2 antibody[12721 S, 1:1000 dilution, Cell Signaling Technology], cGAS antibody[31659, 1:1000 dilution, Cell Signaling Technology], STING antibody[19851-1-AP, 1:2000 dilution, Proteintech], p-STING antibody[PA5-105674, 1:500 dilution, Thermo Fisher Scientific], p-IRF3 antibody[530857, 1:1000 dilution, Chengdu Zen-Bioscience], EPAC1 antibody[12572-1-AP, 1:1000 dilution, Proteintech], VDAC1 antibody[ab154856, 1:1000 dilution, Abcam], GAPDH antibody[ R24404 , 1:5000 dilution, Chengdu Zen-Bioscience], ACTB antibody[R380624, 1:5000 dilution, Chengdu Zen-Bioscience], Tubulin antibody[80713-1-RR, 1:1000 dilution, Proteintech], TOM20 antibody[11802-1-AP, 1:5000 dilution, Proteintech]. secondary antibody Goat Anti-Rabbit IgG H&L(HRP)[511203, 1:5000 dilution, Chengdu Zen-Bioscience], secondary antibody Goat Anti-Mouse IgG H&L(HRP)[511103, 1:5000 dilution, Chengdu Zen-Bioscience].

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 is downregulated in orbital tissues of patients with TAO . ( A – E ) The collected orbital tissue samples from patients with TAO and healthy donors were assessed for histopathological features using H&E and Masson stainings ( A , B ); Epac1 level and distribution using IHC staining ( C ); Epac1 mRNA expression using qRT-PCR ( D ); and Epac1 and vimentin protein levels using immunoblotting ( E ) ( n = 6 or 5). The Student's t -test was conducted to assess comparisons between the two groups, ** P < 0.01 versus the healthy group. TAO, thyroid-associated orbitopathy; H&E, hematoxylin and eosin staining; IHC, immunohistochemical staining; OAT, orbital adipose tissues.

Article Snippet: Afterward, the sections were incubated (30 minutes) with 5% normal goat serum in 1% BSA-containing PBS to block the nonspecific immunoglobulin binding sites, followed by incubation (overnight at 4°C) with primary antibodies against Epac1 (DF6922; Affinity Bioscience, Changzhou, China), CD40 (AF5336; Affinity Bioscience), collagen I (14695-1-AP; Proteintech, Wuhan, China), and alpha-smooth muscle actin (α-SMA; 55135-1-AP; Proteintech).

Techniques: Immunohistochemistry, Expressing, Quantitative RT-PCR, Western Blot, Staining, Immunohistochemical staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 is downregulated in OAT and OMT in a TAO mouse model. Mouse OAT and OMT samples were evaluated for histopathological features using H&E and Masson stainings ( A , B ); Epac1 level and distribution using IHC staining ( C ); Epac1 mRNA expression using qRT-PCR ( D ); and Epac1 and vimentin protein levels using immunoblotting ( E ) ( n = 5). The Student's t -test was conducted to assess comparisons between the two groups, ** P < 0.01 versus the Ad-NC group. H&E, hematoxylin and eosin staining; IHC, immunohistochemical staining; OAT, orbital adipose tissues; OMT, orbital muscle tissues; Ad, adenovirus; NC, negative control.

Article Snippet: Afterward, the sections were incubated (30 minutes) with 5% normal goat serum in 1% BSA-containing PBS to block the nonspecific immunoglobulin binding sites, followed by incubation (overnight at 4°C) with primary antibodies against Epac1 (DF6922; Affinity Bioscience, Changzhou, China), CD40 (AF5336; Affinity Bioscience), collagen I (14695-1-AP; Proteintech, Wuhan, China), and alpha-smooth muscle actin (α-SMA; 55135-1-AP; Proteintech).

Techniques: Immunohistochemistry, Expressing, Quantitative RT-PCR, Western Blot, Staining, Immunohistochemical staining, Negative Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 overexpression attenuates the effects of TGFβ1 on normal and TAO OFs. ( A – G ) Healthy or TAO OFs were transfected with Epac1 -overexpressing plasmid ( Epac1 ), treated with TGFβ1 (10 ng/mL), and examined for cell viability using CCK-8 ( A ); cell migration using scratch wound healing and Transwell assays ( B , C ); relative α-SMA and fibronectin expressions using IF staining ( D ); collagen I content in the cell culture supernatant using ELISA ( E ); the mRNA expression of Epac1 , α-SMA, vimentin, fibronectin, collagen I, and collagen III using qRT-PCR ( F ); and the protein level of Epac1, α-SMA, vimentin, fibronectin, collagen I, and collagen III using immunoblotting ( G ) ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. ** P < 0.01 versus healthy OFs; # P < 0.05, ## P < 0.01 versus TAO OFs; && P < 0.01 versus healthy OFs + TGFβ1 + NC; $ P < 0.05, $$ P < 0.01 versus TAO OFs + TGFβ1 + NC. TAO, thyroid-associated orbitopathy; sh, short-hairpin; NC, negative control; IF staining, immunofluorescent staining.

Article Snippet: Afterward, the sections were incubated (30 minutes) with 5% normal goat serum in 1% BSA-containing PBS to block the nonspecific immunoglobulin binding sites, followed by incubation (overnight at 4°C) with primary antibodies against Epac1 (DF6922; Affinity Bioscience, Changzhou, China), CD40 (AF5336; Affinity Bioscience), collagen I (14695-1-AP; Proteintech, Wuhan, China), and alpha-smooth muscle actin (α-SMA; 55135-1-AP; Proteintech).

Techniques: Over Expression, Transfection, Plasmid Preparation, CCK-8 Assay, Migration, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 knockdown amplifies the effects of TGFβ1 on normal and TAO OFs. ( A , B ) Healthy or TAO OFs were transfected with plasmids containing short-hairpin RNA targeting Epac1 (sh- Epac1 #1/#2), treated with TGFβ1 (10 ng/mL), and examined for Epac1 protein levels using immunoblotting; and ( B ) cell viability using CCK-8; sh- Epac1 #1 was selected for the following experiments. ( C – H ) Healthy or TAO OFs were transfected with sh- Epac1 #1, treated with TGFβ1 (10 ng/mL), and examined for cell migration using scratch wound healing and Transwell assays ( C , D ); relative expression of α-SMA and fibronectin using IF staining ( E ); the content of collagen I in cell culture supernatant using ELISA (F); the mRNA expression of Epac1 , α-SMA, vimentin, fibronectin, collagen I, and collagen III using qRT-PCR ( G ); and the protein level of Epac1, α-SMA, vimentin, fibronectin, collagen I, and collagen III using immunoblotting ( H ) ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. * P < 0.05, ** P < 0.01 versus healthy OFs + TGFβ1 + sh-NC; # P < 0.05, ## P < 0.01 versus TAO OFs + TGFβ1 + sh-NC. TAO, thyroid-associated orbitopathy; sh, short hairpin RNA; NC, negative control; IF staining, immunofluorescent staining.

Article Snippet: Afterward, the sections were incubated (30 minutes) with 5% normal goat serum in 1% BSA-containing PBS to block the nonspecific immunoglobulin binding sites, followed by incubation (overnight at 4°C) with primary antibodies against Epac1 (DF6922; Affinity Bioscience, Changzhou, China), CD40 (AF5336; Affinity Bioscience), collagen I (14695-1-AP; Proteintech, Wuhan, China), and alpha-smooth muscle actin (α-SMA; 55135-1-AP; Proteintech).

Techniques: Knockdown, Transfection, shRNA, Western Blot, CCK-8 Assay, Migration, Expressing, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Negative Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 overexpression improves TAO-like features in the mouse model. ( A ) A schematic diagram of the TAO model establishment and Epac1 overexpression administration; ( B ) at the end of the modeling, the appearance of mouse eyes was observed; ( C , D ) the histopathological alterations in mouse OAT and OMT samples were evaluated using H&E and Masson stainings. TAO, thyroid-associated orbitopathy; OAT, orbital adipose tissues; OMT, orbital muscle tissues; AAV, adeno-associated virus; NC, negative control; L, left; R, right; H&E, hematoxylin and eosin staining.

Article Snippet: Afterward, the sections were incubated (30 minutes) with 5% normal goat serum in 1% BSA-containing PBS to block the nonspecific immunoglobulin binding sites, followed by incubation (overnight at 4°C) with primary antibodies against Epac1 (DF6922; Affinity Bioscience, Changzhou, China), CD40 (AF5336; Affinity Bioscience), collagen I (14695-1-AP; Proteintech, Wuhan, China), and alpha-smooth muscle actin (α-SMA; 55135-1-AP; Proteintech).

Techniques: Over Expression, Virus, Negative Control, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 overexpression decreases TAO-associated markers in vivo. ( A , B ) The protein level of Epac1, α-SMA, CD40, and collagen I in mouse OAT was examined using IHC staining; ( C , D ) the protein level of Epac1, α-SMA, CD40, and collagen I in mouse OMT was examined using IHC staining ( n = 5). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. * P < 0.05, ** P < 0.01 versus healthy; ## P < 0.01 versus TAO + AAV-NC. TAO, thyroid-associated orbitopathy; OAT, orbital adipose tissues; OMT, orbital muscle tissues; AAV, adeno-associated virus; NC, negative control; IHC staining, immunohistochemical staining.

Article Snippet: Afterward, the sections were incubated (30 minutes) with 5% normal goat serum in 1% BSA-containing PBS to block the nonspecific immunoglobulin binding sites, followed by incubation (overnight at 4°C) with primary antibodies against Epac1 (DF6922; Affinity Bioscience, Changzhou, China), CD40 (AF5336; Affinity Bioscience), collagen I (14695-1-AP; Proteintech, Wuhan, China), and alpha-smooth muscle actin (α-SMA; 55135-1-AP; Proteintech).

Techniques: Over Expression, In Vivo, Immunohistochemistry, Virus, Negative Control, Immunohistochemical staining, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 affects the STAT3 signaling. ( A ) A schematic diagram of Epac1’s role in the JAK/STAT3 signaling pathway during the OF activation process. ( B ) TAO OFs transfected with sh- Epac1 or Epacl overexpression vector, treated with TGFβ1, and determined for the protein level of p-STAT3 and STAT3 using immunoblotting ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. ** P < 0.01 versus NC-transfected OFs; ## P < 0.01 versus sh-NC-transfected OFs. ( C ) The protein level of p-STAT3 and STAT3 in TAO mouse OAT and OMT was determined using immunoblotting ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. ** P < 0.01 versus healthy mice; # P < 0.05, ## P < 0.01 versus TAO + AAV-NC mice. TAO, thyroid-associated orbitopathy; OAT, orbital adipose tissues; OMT, orbital muscle tissues; AAV, adeno-associated virus; sh, short-hairpin RNA; NC, negative control.

Article Snippet: Afterward, the sections were incubated (30 minutes) with 5% normal goat serum in 1% BSA-containing PBS to block the nonspecific immunoglobulin binding sites, followed by incubation (overnight at 4°C) with primary antibodies against Epac1 (DF6922; Affinity Bioscience, Changzhou, China), CD40 (AF5336; Affinity Bioscience), collagen I (14695-1-AP; Proteintech, Wuhan, China), and alpha-smooth muscle actin (α-SMA; 55135-1-AP; Proteintech).

Techniques: Activation Assay, Transfection, Over Expression, Plasmid Preparation, Western Blot, Virus, shRNA, Negative Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: STAT3 mediates the effects of Epac1 on TGFβ1-treated TAO OFs. ( A – G ) Under TGFβ1 treatment, TAO OFs were transfected with sh- Epac1 with or without the JAK-STAT pathway inhibitor Stattic (2 g/mL for 24 hours) and examined for cell viability using CCK-8 ( A ); cell migration using scratch wound healing and Transwell assays ( B , C ); α-SMA and fibronectin expressions using IF staining ( D ); the content of collagen I in cell supernatant using ELISA ( E ); the mRNA expression of Epac1 , α-SMA, vimentin, fibronectin, collagen I, and collagen III using qRT-PCR ( F ); the protein level of α-SMA, vimentin, fibronectin, collagen I, and collagen III using immunoblotting ( G ) ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. ** P < 0.01 versus TGFβ1 + sh-NC; ## P < 0.01 versus TGFβ1 + sh- Epac1 + Stattic. sh, short-hairpin; NC, negative control; IF staining, immunofluorescent staining.

Article Snippet: Afterward, the sections were incubated (30 minutes) with 5% normal goat serum in 1% BSA-containing PBS to block the nonspecific immunoglobulin binding sites, followed by incubation (overnight at 4°C) with primary antibodies against Epac1 (DF6922; Affinity Bioscience, Changzhou, China), CD40 (AF5336; Affinity Bioscience), collagen I (14695-1-AP; Proteintech, Wuhan, China), and alpha-smooth muscle actin (α-SMA; 55135-1-AP; Proteintech).

Techniques: Transfection, CCK-8 Assay, Migration, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot, Negative Control