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Overexpression of Annexin A1 Inhibits Pyroptosis and Improves Dry Eye Signs by Regulating the TRIM72/Nrf2/HO-1 Signaling Pathway

Li Zhang

Li Zhang

  • Department of Ophthalmology, Shanxi Eye Hospital Affiliated to Shanxi Medical UniversityTaiyuan, China
  • Department of Ophthalmology, Aier Eye Hospital (Taiyuan)Taiyuan, China
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Zhang, Li
, 
Peng Chen

Peng Chen

Department of Ophthalmology, Shanxi Aier Eye Hospital (Shanxi)Taiyuan, China
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Chen, Peng
, 
Pengfei Han

Pengfei Han

Department of Ophthalmology, Shanxi Aier Eye Hospital (Shanxi)Taiyuan, China
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Han, Pengfei
, 
Huizhe Fu

Huizhe Fu

Department of Ophthalmology, Aier Eye Hospital (Taiyuan)Taiyuan, China
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Fu, Huizhe
 oraz 
Bin Sun

Bin Sun

Department of Ophthalmology, Shanxi Eye Hospital Affiliated to Shanxi Medical UniversityTaiyuan, China
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Sun, Bin
  
06 sie 2025
Overexpression of Annexin A1 Inhibits Pyroptosis and Improves Dry Eye Signs by Regulating the TRIM72/Nrf2/HO-1 Signaling Pathway's Cover Image
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Kategoria artykułu: Original
Data publikacji: 06 sie 2025
Otrzymano: 06 mar 2025
Przyjęty: 18 cze 2025
DOI: https://doi.org/10.2478/aite-2025-0022
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© 2025 Li Zhang et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig 1.

ANXA1 expression was reduced in the murine DED model. (A) qPCR analysis showing ANXA1 mRNA levels in the eyes of mice from the Sham and DED groups. (B) Immunoblot analysis illustrating ANXA1 protein expression in the eyes of Sham and DED mice. (C) qPCR analysis of ANXA1 mRNA levels in HCE-T cells following treatment with control or BAC for 24 h. (D) Immunoblot analysis of ANXA1 protein expression in HCE-T cells treated with control or BAC for 24 h. **p < 0.01, ***p < 0.001, DED or BAC vs Sham or control. ANXA1, annexin A1; BAC, benzalkonium chloride; DED, dry eye disease; HCE-T, human corneal epithelial; qPCR, quantitative polymerase chain reaction.
ANXA1 expression was reduced in the murine DED model. (A) qPCR analysis showing ANXA1 mRNA levels in the eyes of mice from the Sham and DED groups. (B) Immunoblot analysis illustrating ANXA1 protein expression in the eyes of Sham and DED mice. (C) qPCR analysis of ANXA1 mRNA levels in HCE-T cells following treatment with control or BAC for 24 h. (D) Immunoblot analysis of ANXA1 protein expression in HCE-T cells treated with control or BAC for 24 h. **p < 0.01, ***p < 0.001, DED or BAC vs Sham or control. ANXA1, annexin A1; BAC, benzalkonium chloride; DED, dry eye disease; HCE-T, human corneal epithelial; qPCR, quantitative polymerase chain reaction.

Fig 2.

ANXA1 overexpression in vivo improved tear function and attenuated pyroptosis. (A) Immunoblot analysis showing ANXA1 protein expression in the eyes of Sham and DED mice following infection with Ad-NC or Ad-ANXA1. (B) Tear film stability and secretion were assessed using fluorescein break-up time (TBUT, left) and Schirmer test (wetting length, right) in Sham and DED groups after infection with Ad-NC or Ad-ANXA1. (C) Immunoblot analysis demonstrating the expression levels of NLRP1, C-caspase-1, IL-1β, and GSDMD-1 in ocular tissues of Sham and DED mice following Ad-NC or Ad-ANXA1 infection. ***p < 0.001, DED vs Sham, ^^p < 0.01, ^^^p < 0.001, DED + Ad-ANXA1 vs DED + Ad-NC. Ad-ANXA1, ANXA1-overexpressing adenoviral vectors; ANXA1, annexin A1; C-caspase-1, cleaved caspase-1; DED, dry eye disease; GSDMD, gasdermin D; IL, interleukin; NC, negative control; NLRP, NOD-like receptor family pyrin domain containing; TBUT, tear break-up time.
ANXA1 overexpression in vivo improved tear function and attenuated pyroptosis. (A) Immunoblot analysis showing ANXA1 protein expression in the eyes of Sham and DED mice following infection with Ad-NC or Ad-ANXA1. (B) Tear film stability and secretion were assessed using fluorescein break-up time (TBUT, left) and Schirmer test (wetting length, right) in Sham and DED groups after infection with Ad-NC or Ad-ANXA1. (C) Immunoblot analysis demonstrating the expression levels of NLRP1, C-caspase-1, IL-1β, and GSDMD-1 in ocular tissues of Sham and DED mice following Ad-NC or Ad-ANXA1 infection. ***p < 0.001, DED vs Sham, ^^p < 0.01, ^^^p < 0.001, DED + Ad-ANXA1 vs DED + Ad-NC. Ad-ANXA1, ANXA1-overexpressing adenoviral vectors; ANXA1, annexin A1; C-caspase-1, cleaved caspase-1; DED, dry eye disease; GSDMD, gasdermin D; IL, interleukin; NC, negative control; NLRP, NOD-like receptor family pyrin domain containing; TBUT, tear break-up time.

Fig 3.

ANXA1 overexpression promoted proliferation and inhibited pyroptosis in HCE-T cells. (A) Immunoblot analysis showing ANXA1 protein expression in HCE-T cells treated with control or BAC, and after infection with Ad-NC or Ad-ANXA1 for 24 h. (B) CCK-8 assay demonstrating the proliferative capacity of HCE-T cells after control or BAC treatment, and following Ad-NC or Ad-ANXA1 infection for 24 h, with OD450 values measured. (C) Immunoblot analysis of NLRP1, C-caspase-1, IL-1β, and GSDMD-1 expression in HCE-T cells treated with control or BAC and infected with Ad-NC or Ad-ANXA1 for 24 h. (D) TUNEL staining to assess apoptosis levels in HCE-T cells treated as indicated. Red fluorescence indicates TUNEL-positive apoptotic cells. Scale bar, 100 mm. **p < 0.01, ***p < 0.001, BAC vs control, ^p < 0.05, ^^p < 0.01, ^^^p < 0.001, BAC + Ad-ANXA1 vs BAC + Ad-NC. Ad-ANXA1, ANXA1-overexpressing adenoviral vectors; ANXA1, annexin A1; BAC, benzalkonium chloride; C-caspase-1, cleaved caspase-1; GSDMD, gasdermin D; HCE-T, human corneal epithelial; IL, interleukin; NC, negative control; NLRP, NOD-like receptor family pyrin domain containing.
ANXA1 overexpression promoted proliferation and inhibited pyroptosis in HCE-T cells. (A) Immunoblot analysis showing ANXA1 protein expression in HCE-T cells treated with control or BAC, and after infection with Ad-NC or Ad-ANXA1 for 24 h. (B) CCK-8 assay demonstrating the proliferative capacity of HCE-T cells after control or BAC treatment, and following Ad-NC or Ad-ANXA1 infection for 24 h, with OD450 values measured. (C) Immunoblot analysis of NLRP1, C-caspase-1, IL-1β, and GSDMD-1 expression in HCE-T cells treated with control or BAC and infected with Ad-NC or Ad-ANXA1 for 24 h. (D) TUNEL staining to assess apoptosis levels in HCE-T cells treated as indicated. Red fluorescence indicates TUNEL-positive apoptotic cells. Scale bar, 100 mm. **p < 0.01, ***p < 0.001, BAC vs control, ^p < 0.05, ^^p < 0.01, ^^^p < 0.001, BAC + Ad-ANXA1 vs BAC + Ad-NC. Ad-ANXA1, ANXA1-overexpressing adenoviral vectors; ANXA1, annexin A1; BAC, benzalkonium chloride; C-caspase-1, cleaved caspase-1; GSDMD, gasdermin D; HCE-T, human corneal epithelial; IL, interleukin; NC, negative control; NLRP, NOD-like receptor family pyrin domain containing.

Fig 4.

ANXA1 overexpression enhanced the TRIM72/Nrf2/HO-1 signaling axis. (A) Immunoblot analysis showing the expression of TRIM72, Nrf2, and HO-1 in the eyes of Sham and DED mice infected with Ad-NC or Ad-ANXA1. (B) Immunoblot analysis showing TRIM72, Nrf2, and HO-1 expression in HCE-T cells treated with control or BAC and infected with Ad-NC or Ad-ANXA1 for 24 h. *** p < 0.001, DED or BAC vs Sham or control, ^^^p < 0.001, Ad-ANXA1 vs Ad-NC. Ad-ANXA1, ANXA1-overexpressing adenoviral vectors; ANXA1, annexin A1; BAC, benzalkonium chloride; DED, dry eye disease; HCE-T, human corneal epithelial; HO-1, heme oxygenase-1; NC, negative control; Nrf2, nuclear factor erythroid 2-related factor 2; TRIM72, tripartite motif-containing protein 72.
ANXA1 overexpression enhanced the TRIM72/Nrf2/HO-1 signaling axis. (A) Immunoblot analysis showing the expression of TRIM72, Nrf2, and HO-1 in the eyes of Sham and DED mice infected with Ad-NC or Ad-ANXA1. (B) Immunoblot analysis showing TRIM72, Nrf2, and HO-1 expression in HCE-T cells treated with control or BAC and infected with Ad-NC or Ad-ANXA1 for 24 h. *** p < 0.001, DED or BAC vs Sham or control, ^^^p < 0.001, Ad-ANXA1 vs Ad-NC. Ad-ANXA1, ANXA1-overexpressing adenoviral vectors; ANXA1, annexin A1; BAC, benzalkonium chloride; DED, dry eye disease; HCE-T, human corneal epithelial; HO-1, heme oxygenase-1; NC, negative control; Nrf2, nuclear factor erythroid 2-related factor 2; TRIM72, tripartite motif-containing protein 72.

Fig 5.

TRIM72 inhibition reversed ANXA1-mediated improvements in DED parameters. (A) Tear film stability and secretion were evaluated using fluorescein break-up time (TBUT, left) and Schirmer test (wetting length, right) in the indicated groups. (B) CCK-8 assay showing the proliferative capacity of HCE-T cells under the indicated treatment conditions. (C) Immunoblot analysis demonstrating the expression of NLRP1, C-caspase-1, IL-1β, and GSDMD-1 in ocular tissues of mice in the indicated groups. (D) Immunoblot analysis showing NLRP1, C-caspase-1, IL-1β, and GSDMD-1 expression in HCE-T cells under the indicated treatments. (E) Immunoblot analysis of TRIM72, Nrf2, and HO-1 expression in the eyes of mice in the indicated groups. (F) Immunoblot analysis of TRIM72, Nrf2, and HO-1 expression in HCE-T cells under the indicated treatments. **p < 0.01, ***p < 0.001, DED or BAC vs Sham or control, ^^p < 0.01, ^^^p < 0.001, si-TRIM72 + Ad-ANXA1 vs si-NC + Ad-ANXA1. Ad-ANXA1, ANXA1-overexpressing adenoviral vectors; ANXA1, annexin A1; BAC, benzalkonium chloride; C-caspase-1, cleaved caspase-1; DED, dry eye disease; GSDMD, gasdermin D; HCE-T, human corneal epithelial; HO-1, heme oxygenase-1; IL, interleukin; NC, negative control; NLRP, NOD-like receptor family pyrin domain containing; Nrf2, nuclear factor erythroid 2-related factor 2; TBUT, tear break-up time; TRIM72, tripartite motif-containing protein 72.
TRIM72 inhibition reversed ANXA1-mediated improvements in DED parameters. (A) Tear film stability and secretion were evaluated using fluorescein break-up time (TBUT, left) and Schirmer test (wetting length, right) in the indicated groups. (B) CCK-8 assay showing the proliferative capacity of HCE-T cells under the indicated treatment conditions. (C) Immunoblot analysis demonstrating the expression of NLRP1, C-caspase-1, IL-1β, and GSDMD-1 in ocular tissues of mice in the indicated groups. (D) Immunoblot analysis showing NLRP1, C-caspase-1, IL-1β, and GSDMD-1 expression in HCE-T cells under the indicated treatments. (E) Immunoblot analysis of TRIM72, Nrf2, and HO-1 expression in the eyes of mice in the indicated groups. (F) Immunoblot analysis of TRIM72, Nrf2, and HO-1 expression in HCE-T cells under the indicated treatments. **p < 0.01, ***p < 0.001, DED or BAC vs Sham or control, ^^p < 0.01, ^^^p < 0.001, si-TRIM72 + Ad-ANXA1 vs si-NC + Ad-ANXA1. Ad-ANXA1, ANXA1-overexpressing adenoviral vectors; ANXA1, annexin A1; BAC, benzalkonium chloride; C-caspase-1, cleaved caspase-1; DED, dry eye disease; GSDMD, gasdermin D; HCE-T, human corneal epithelial; HO-1, heme oxygenase-1; IL, interleukin; NC, negative control; NLRP, NOD-like receptor family pyrin domain containing; Nrf2, nuclear factor erythroid 2-related factor 2; TBUT, tear break-up time; TRIM72, tripartite motif-containing protein 72.
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Angielski
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1 razy w roku
Dziedziny czasopisma:
Medycyna, Podstawowe nauki medyczne, Biochemia, Immunologia, Medycyna kliniczna, Medycyna kliniczna, inne, Chemia kliniczna
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