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<p>Diabetic retinopathy (DR) is a major vision-threatening complication of diabetes. Although cyclin-dependent kinase inhibitor 2&#xa0;A (CDKN2A) is linked to glucose dysregulation and diabetes risk, its role in DR remains unclear. Therefore, this study aimed to investigate whether knockdown of CDKN2A can enhance the therapeutic effects of metformin (Met) and resveratrol (RSV) in ameliorating DR progression. A DR model was established in rats by streptozotocin (STZ) induction, and an in vitro DR model was constructed using high glucose (HG)-treated adult retinal pigment epithelial cell line-19 (ARPE-19) cells. The expression of CDKN2A was examined by Western blotting, immunohistochemistry, and immunofluorescence. Cell injury was assessed via a cell counting kit-8 (CCK-8) assay and flow cytometry. Mitochondrial damage was evaluated via MitoSOX Red staining and JC-1 staining. Retinal histopathological changes were observed through hematoxylin and eosin (H&amp;E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The N6-methyladenosine (m6A) modification level of CDKN2A was detected by chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR). The results showed that in both the DR rat model and HG-treated ARPE-19 cells, the expression of CDKN2A and methyltransferase-like 3 (METTL3) was significantly upregulated. Knockdown of CDKN2A alone did not significantly alter DR progression in rats or HG‑induced cell injury, whereas overexpression of CDKN2A further exacerbated pathological outcomes. Treatment with Met or RSV alleviated retinal damage and HG-induced cell injury, which was associated with improved mitochondrial function, as evidenced by increased mitochondrial membrane potential and adenosine triphosphate (ATP) levels and decreased mitochondrial reactive oxygen species (mROS) levels. Notably, the therapeutic effects of Met and RSV were significantly enhanced when combined with CDKN2A knockdown. Furthermore, Met and RSV act through METTL3 to exert dual regulatory effects. On the one hand, they suppressed the m6A methylation and expression of CDKN2A in a METTL3-dependent manner. On the other hand, the improvement in mitochondrial function induced by Met and RSV also involved METTL3-mediated regulation. Together, these processes formed a reinforcing cycle in which Met and RSV inhibited CDKN2A expression, and the resulting downregulation of CDKN2A further enhanced the therapeutic efficacy of both agents in mitigating DR progression.This study revealed that knockdown of CDKN2A expression significantly enhanced the therapeutic effects of Met and RSV on DR and that these drugs simultaneously regulated CDKN2A expression and improved mitochondrial function through METTL3. The role of CDKN2A in DR is not directly pathogenic; rather, it serves as a key sensitizer for drug treatment.</p>

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Knocking Down CDKN2A Enhances the Therapeutic Effects of Metformin and Resveratrol on Diabetic Retinopathy

  • 期刊论文

  • 发表日期：

    2026年3月17日

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  • Jinghua Li,
  • Junyi Liu,
  • Jiang Li,
  • Yongying Tang,
  • Zailin Xiao,
  • Yan Xu,
  • Xueying Zhao

摘要

Diabetic retinopathy (DR) is a major vision-threatening complication of diabetes. Although cyclin-dependent kinase inhibitor 2 A (CDKN2A) is linked to glucose dysregulation and diabetes risk, its role in DR remains unclear. Therefore, this study aimed to investigate whether knockdown of CDKN2A can enhance the therapeutic effects of metformin (Met) and resveratrol (RSV) in ameliorating DR progression. A DR model was established in rats by streptozotocin (STZ) induction, and an in vitro DR model was constructed using high glucose (HG)-treated adult retinal pigment epithelial cell line-19 (ARPE-19) cells. The expression of CDKN2A was examined by Western blotting, immunohistochemistry, and immunofluorescence. Cell injury was assessed via a cell counting kit-8 (CCK-8) assay and flow cytometry. Mitochondrial damage was evaluated via MitoSOX Red staining and JC-1 staining. Retinal histopathological changes were observed through hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The N6-methyladenosine (m6A) modification level of CDKN2A was detected by chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR). The results showed that in both the DR rat model and HG-treated ARPE-19 cells, the expression of CDKN2A and methyltransferase-like 3 (METTL3) was significantly upregulated. Knockdown of CDKN2A alone did not significantly alter DR progression in rats or HG‑induced cell injury, whereas overexpression of CDKN2A further exacerbated pathological outcomes. Treatment with Met or RSV alleviated retinal damage and HG-induced cell injury, which was associated with improved mitochondrial function, as evidenced by increased mitochondrial membrane potential and adenosine triphosphate (ATP) levels and decreased mitochondrial reactive oxygen species (mROS) levels. Notably, the therapeutic effects of Met and RSV were significantly enhanced when combined with CDKN2A knockdown. Furthermore, Met and RSV act through METTL3 to exert dual regulatory effects. On the one hand, they suppressed the m6A methylation and expression of CDKN2A in a METTL3-dependent manner. On the other hand, the improvement in mitochondrial function induced by Met and RSV also involved METTL3-mediated regulation. Together, these processes formed a reinforcing cycle in which Met and RSV inhibited CDKN2A expression, and the resulting downregulation of CDKN2A further enhanced the therapeutic efficacy of both agents in mitigating DR progression.This study revealed that knockdown of CDKN2A expression significantly enhanced the therapeutic effects of Met and RSV on DR and that these drugs simultaneously regulated CDKN2A expression and improved mitochondrial function through METTL3. The role of CDKN2A in DR is not directly pathogenic; rather, it serves as a key sensitizer for drug treatment.