Related Experiment Video
Updated: Sep 30, 2025
Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish
Published on: May 5, 2021
ALKBH5-Mediated m6A Modification of A20 Regulates Microglia Polarization in Diabetic Retinopathy
Tingting Chen1, Wenhui Zhu1, Congyao Wang1
1Department of Ophthalmology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
Background:
To investigate the role of microglia polarization in the pathogenesis of diabetic retinopathy, and study the mechanism of ALKBH5-mediated m6A modification of A20 of retinal microglia polarization.
Methods:
Diabetics rats were constructed and the M1/M2 polarization of retinal microglia was determined using immunofluorescence, flow cytometry, and quantitative real-time PCR (qRT-PCR). Glucose at different concentrations was added to treat the microglia, and the polarization rate was detected. RNA sequencing was performed to identify the differentially expressed gene in glucose treated microglia, and A20 expression was confirmed by qRT-PCR and western blotting. Lentiviruses encoding shRNA for A20 or overexpressing A20 were constructed to clarify the role of A20 in microglia polarization in vitro and vivo. N6-methyladenosine (m6A) modification level and degradation rate of A20 were determined and m6A related proteins were detected.
Results:
Diabetics rats showed a higher M1 polarization rate but lower M2 polarization rate of retinal microglia. With the increase of glucose concentration, microglia tend to polarize into M1 inflammatory type rather than M2 anti-inflammatory type. Shown by RNA sequencing, glucose treated microglia showed a differentially expressed gene profile, which was enriched in kinds of inflammatory categories and pathways. A20 expression was lower in microglia with glucose treatment, which was demonstrated to negatively regulate the M1 polarization. Moreover, intraocular injection of A20-overexpression lentiviruses (OE-A20) rectified the enhanced M1 retinal microglia polarization of diabetes rats. The higher m6A modification level and faster degradation rate of A20 was observed in glucose treated microglia, which was mediated by m6A demethylase ALKBH5.
Conclusion:
Lower expression A20 resulted in the enhanced M1 polarization of retinal microglia in diabetic retinopathy, which was caused by ALKBH5 mediated m6A modification. This study may provide new perspectives on not only the pathogenesis but also the diagnosis and treatment for diabetic retinopathy.
Insights
Diabetic retinopathy involves increased M1 microglia polarization, driven by lower A20 expression. ALKBH5-mediated m6A modification of A20 contributes to this process, offering new therapeutic targets.
Area of Science:
- Ophthalmology
- Immunology
- Molecular Biology
Background:
- Diabetic retinopathy pathogenesis involves microglia polarization.
- Understanding the molecular mechanisms of microglia polarization is crucial for treatment.
Purpose of the Study:
- To investigate the role of microglia polarization in diabetic retinopathy.
- To elucidate the mechanism of ALKBH5-mediated m6A modification of A20 in retinal microglia polarization.
Main Methods:
- Diabetic rat models and in vitro microglia cultures were used.
- Immunofluorescence, flow cytometry, qRT-PCR, western blotting, and RNA sequencing were employed.
- Lentiviruses were used to manipulate A20 expression.
Main Results:
- Diabetic rats exhibited increased M1 and decreased M2 microglia polarization.
- High glucose induced M1 polarization and reduced A20 expression.
- ALKBH5-mediated m6A modification led to increased A20 degradation, promoting M1 polarization.
Conclusions:
- Reduced A20 expression, via ALKBH5-mediated m6A modification, enhances M1 microglia polarization in diabetic retinopathy.
- This pathway offers potential diagnostic and therapeutic targets for diabetic retinopathy.
