Procyanidin C1 modulates microglia cellular iron homeostasis via targeting HuR/ACSL4 pathway and alleviates high-glucose-induced ferroptosis in diabetic retinopathy.
Investigating the potential role of the RNA-binding protein human antigen R (HuR) in mediating ferroptosis during DR progression, as well as the protective effects of Procyanidin C1 (PC1), demonstrated that PC1 significantly alleviated oxidative stress and ferroptosis in both diabetic mice and high-glucose-treated BV2s.
Abstract
Diabetic retinopathy (DR), one of the most prevalent microvascular complications of diabetes, is a vision-threatening ocular disease. Although ferroptosis has been implicated in DR development, research on its underlying mechanisms and targeted therapeutics remains limited. Procyanidins, a class of polyphenolic compounds, exhibit robust anti-inflammatory properties and regulatory effects on various pathological processes. Herein, we investigated the potential role of the RNA-binding protein human antigen R (HuR) in mediating ferroptosis during DR progression, as well as the protective effects of Procyanidin C1 (PC1). Streptozotocin-induced diabetic mice and high-glucose-exposed BV2 microglial cells (BV2s) were treated with PC1 to evaluate oxidative stress and the expression of ferroptosis markers. The interaction between HuR and ACSL4 was characterized using ribonucleoprotein (RNP) immunoprecipitation (IP) and mRNA stability assays. Our results demonstrated that PC1 significantly alleviated oxidative stress and ferroptosis in both diabetic mice and high-glucose-treated BV2s. Mechanistically, post-transcriptional regulation by HuR via stabilizing Acsl4 mRNA contributes to the acceleration of ferroptosis during DR progression. Ultimately, PC1 suppresses DR advancement by targeting the HuR-ACSL4 signaling axis, highlighting its potential as a therapeutic intervention.
Diabetic retinopathy (DR) is characterized by progressive retinal microvascular injury, with oxidative stress and ferroptosis increasingly recognized as key pathogenic contributors. This study investigated whether ubiquitin C-terminal hydrolase L1 (UCHL1) regulates retinal endothelial ferroptosis through stabilization of nuclear factor erythroid 2-related factor 2 (NRF2). Analysis of the GSE102485 dataset identified UCHL1 as a downregulated deubiquitinating enzyme in DR. Streptozotocin-induced type 1 diabetic mice and high glucose (HG)-induced human retinal capillary endothelial cells (HRCECs) were used, together with UCHL1 inhibition/knockdown, AAV-mediated UCHL1 overexpression, and NRF2 knockdown. UCHL1 expression was reduced in diabetic retinas and HG-induced HRCECs, accompanied by ferroptosis activation, mitochondrial injury, and endothelial dysfunction. Pharmacological inhibition or siRNA-mediated depletion of UCHL1 intensified oxidative stress, Fe2⁺ accumulation, lipid peroxidation, mitochondrial fragmentation, and cristae disruption, while impairing endothelial barrier integrity, migration, and tube formation. In vivo, LDN57444 aggravated retinal vascular leakage and fundus vascular abnormalities, whereas AAV-UCHL1 preserved retinal architecture and reduced vascular permeability. Mechanistically, co-immunoprecipitation and ubiquitination assays demonstrated that UCHL1 interacted with NRF2 and stabilized NRF2 by removing K48-linked polyubiquitin chains. Nuclear-cytoplasmic fractionation further showed that UCHL1 overexpression restored NRF2 abundance and increased nuclear NRF2 accumulation under HG conditions. Ferrostatin-1 rescued UCHL1 depletion-induced ferroptotic injury, whereas NRF2 knockdown abolished the protection conferred by UCHL1 overexpression. These findings highlight the UCHL1/NRF2 axis may represent a therapeutic target in DR.
Sanhua Xu, Jun Huang, Yicang Wang et al.· Biochemical Pharmacology· 0 citations
PURPOSE
Diabetic retinopathy (DR) is the leading cause of preventable blindness among working-age adults. Sodium-glucose cotransporter 2 inhibitors (SGLT2is), first-line treatments for diabetes mellitus (DM), have demonstrated efficacy in decelerating DR progression. However, the mechanisms remain unclear. This study investigates how canagliflozin (CANA), an SGLT2 inhibitor, exerts neuroprotective effects in DR by suppressing microglial proinflammatory cytokine release.
MATERIALS AND METHODS
Using high-fat diet (HFD) and streptozotocin (STZ)-induced diabetic mice and BV-2 microglial cells under high glucose (HG) conditions, we elucidate the mechanistic link between CANA and neuroinflammation suppression in DR. Retinal structure and function were assessed using optical coherence tomography and electroretinography. Protein was evaluated via Western blotting, immunostaining, and enzyme-linked immunosorbent assay. Cell viability was measured using the Cell Counting Kit-8 assay.
RESULTS
CANA demonstrated significant neuroprotective effects in diabetic retinopathy (DR) by attenuating microglia-mediated neuroinflammation. In HFD and STZ-induced diabetic mice, the ganglion cell complex thickness and a-wave and b-wave amplitudes decreased (p < 0.05). CANA upregulated the ganglion cell complex thickness (p < 0.05) and TSPO expression (p < 0.05), and tended to increase a-wave and b-wave amplitudes (p > 0.05) in retinas in DM mice. In BV-2 cells, HG activated BV-2 cells, downregulated TSPO expression, and promoted proinflammatory cytokine release, toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) expression, and nuclear translocation of p65 and phosphorylated p65 (p < 0.05). CANA reduced BV-2 cell viability, upregulated TSPO, while suppressing proinflammatory cytokine release, TLR4 and NLRP3 expression, and nuclear translocation of p65 and phosphorylated p65 (p < 0.05). Notably, CANA inhibited the TLR4/NF-κB/NLRP3 pathway, evidenced by reduced TLR4/NLRP3 protein levels and diminished nuclear translocation of p65 and phosphorylated p65 in microglia.
CONCLUSIONS
These findings identify CANA as a promising therapeutic candidate for DR, acting through TLR4/NF-κB/NLRP3-dependent modulation of microglial activation.
Tai Guo, Yue Liu, Mengxiao He et al.· Current Eye Research· 0 citations
AIM
To investigate the protective effects of deferoxamine (DFO) on high glucose (HG)-induced ferroptosis and inflammatory responses in retinal Müller glial cells (MGCs) and offer potential therapeutic targets for early intervention in diabetic retinopathy (DR).
METHODS
Primary MGCs were cultured from C57BL/6J mouse retinas and exposed to normal glucose (NG, 5 mmol/L) or HG (25 mmol/L) conditions for various durations. Ferroptosis-related markers, including intracellular Fe2+ levels, glutathione (GSH), reactive oxygen species (ROS), malondialdehyde (MDA), and key proteins glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), ferritin heavy chain 1 (FTH1), were examined using multiple assays such as immunoblotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), and fluorometric detection. The role of DFO was evaluated through cell viability assessment and analysis of inflammatory cytokines interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α).
RESULTS
HG exposure significantly increased intracellular Fe2+ content, decreased GSH levels, elevated ROS and MDA concentrations, and altered expression profiles of ferroptosis regulators GPX4 and SLC7A11. Immunoblot and IF analyses confirmed downregulation of GPX4 and SLC7A11 alongside accumulation of FTH1 under prolonged HG treatment. DFO administration markedly attenuated these ferroptotic changes while reducing inflammatory cytokine secretion, demonstrating its protective effect against HG-induced damage in MGCs by modulating the nuclear factor erythroid 2-related factor 2 (Nrf2)/thioredoxin reductase 1 (TXNRD1) pathway.
CONCLUSION
Low-dose DFO effectively mitigates HG-induced ferroptosis and inflammatory responses in MGCs through the Nrf2/TXNRD1 signaling axis, providing a theoretical framework for developing novel therapeutic strategies in DR management.
Min Luo, Yi Zhang, Na Li et al.· International Journal of Oph...· 0 citations
Findings suggest that EGCG alleviates RPE damage associated with NF-κB p65-mediated ferroptosis, providing new insights into AMD pathogenesis and a promising therapeutic strategy.
Xiao-Yang Chen, Miao Han, Yi-Wen An et al.· Experimental Eye Research· 0 citations
Diabetic retinopathy (DR), a primary cause of working-age vision loss, is characterized by neuroinflammation, in which microglial polarization toward the proinflammatory M1 phenotype serves as a pivotal driver. Reprogramming microglia toward an anti-inflammatory and reparative phenotype may therefore represent a promising therapeutic strategy. This study investigated the effects of tetrandrine (Tet) on hypoxia-induced microglial activation and explored the underlying mechanisms. We established a chemical hypoxia model using CoCl2 and evaluated inflammatory mediators and associated molecules by qRT-PCR, western blotting, and immunofluorescence. Microglial migratory and phagocytic functions were assessed by scratch wound and phagocytosis assays, respectively. Potential drug targets were identified through an integrative approach combining network pharmacology, molecular docking, and cellular thermal shift assay (CETSA). Tet suppressed CoCl2-induced M1-associated inflammatory gene expression in BV2 cells and attenuated p65 activation and cytotoxic responses in HMC3 cells. Functionally, Tet restored impaired phagocytic capacity, attenuated aberrant migration, and mitigated microglia-mediated injury to 661W photoreceptor-derived cells. Mechanistic analyses showed that Tet reduced p65 phosphorylation and apparent nuclear accumulation, thereby limiting activation of the HIF-1α/NF-κB signaling axis; docking and CETSA findings further supported a potential interaction between Tet and p65. In summary, Tet alleviates hypoxia-induced microglial M1 polarization and microglia-mediated photoreceptor cell injury, at least partly through modulation of NF-κB/p65 signaling. These findings identify Tet as a candidate compound for further preclinical investigation in retinal neuroinflammation associated with DR.
High-glucose (HG) stress induces dysfunction of vascular endothelium, a key factor contributing to diabetic vascular complications, in part through ferroptosis. However, the mechanisms governing ferroptosis under these conditions remain partially understood. Herein, we identified circFBXO7 as a novel circular RNA that promotes ferroptosis and endothelial injury in human umbilical vein endothelial cells (HUVECs) and immortalized human aortic endothelial cells (iHAECs) treated with HG. Transcriptomic profiling revealed that circFBXO7 was markedly upregulated upon HG exposure and positively correlated with ferroptosis-related genes. Functional experiments showed that silencing circFBXO7 alleviated HG-induced cell death, restored migration and tube formation, and reduced oxidative stress, lipid peroxidation, Fe2+ accumulation, and mitochondrial damage. Mechanistically, circFBXO7 interacted with the transcription factor cellular promoter 2 (TFCP2) and promoted its ubiquitination and proteasomal degradation. Rescue experiments demonstrated that TFCP2 inhibition abolished the protective effects of circFBXO7 knockdown and sensitized endothelial cells to HG-induced ferroptosis. Overexpression of TFCP2 inhibited ferroptosis caused by HG treatment. Collectively, these findings identify a novel circFBXO7-TFCP2 regulatory axis linking metabolic stress to endothelial ferroptosis, providing new insight into the molecular basis of diabetic vascular dysfunction.
