Dopamine Alleviated Diabetic Retinal Neurodegeneration through Protecting Retinal Ganglion Cells from Ferroptosis via the Nrf2/HO‑1 Signaling Pathway.

C57BL/6 mice (20–25 g, 6 w) were purchased from GemPharmatech Co., Ltd. (Guangdong, China, license No. SCXK (yue) 2020-0054). The animals were housed in a specific pathogen-free environment and maintained on a 12 h light and 12 h dark cycle with unrestricted access to food and water. The experimental protocols were approved by the Tongji Hospital Experimental Animal Ethics Committee (approval No. 287-20230927) with the ARVO statement. Diabetes was induced in mice through a single intraperitoneal injection of streptozotocin (STZ) (S0130, Sigma-Aldrich, St. Louis, MO, USA) at a dose of 100 mg/kg, dissolved in freshly prepared citrate buffer (pH 4.5). Control animals received an equivalent volume of citrate buffer without STZ. Blood glucose levels were measured 3 days postinjection using a hand-held glucose meter (OneTouch VerioVue, Johnson Medical Co., Ltd., China) with samples from the tail vein. A diabetic model was considered to be successfully established in mice with blood glucose concentrations higher than 16.7 mmol/L. Body weight and blood glucose levels were measured biweekly throughout the study. At 8 weeks post-STZ injection, diabetic mice received daily intraperitoneal injections of l -DOPA (10 mg/kg) (HY-N0304, MedchemExpress), dissolved in 0.1% ascorbic acid (ST1434, Beyotime) in saline, lasting for 2 weeks. Control diabetic mice were administered an equivalent volume of the vehicle solution (0.1% ascorbic acid in saline) via intraperitoneal injection. Experiment detail was performed as described by Kim. Prior to electroretinography (ERG) recordings (RetiMINER; IRC, Chongqing, China), mice were dark-adapted overnight in a completely light-free environment to ensure maximal retinal sensitivity. On the day of the experiment, mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg) to minimize movement and discomfort during the procedure. Pupil dilation was achieved using topical application of 1% tropicamide. ERG recordings were performed using a corneal gold-ring electrode as the active electrode, gently placed on the corneal surface. Physiological saline was applied to the corneal surface to enhance the electrical conductivity and ensure stable contact. A subcutaneous reference electrode was positioned on the forehead, and a ground electrode was clipped onto the tail. Scotopic ERG responses were recorded under dark-adapted conditions using flash stimuli at 0.1, 0.3, 1.0, and 3.0 log cd·s/m 2 . The amplitudes of the a-wave and b-wave were analyzed quantitatively. The a-wave amplitude was calculated as the difference between the baseline and the negative peak, while the b-wave amplitude was determined as the difference between the negative peak of the a-wave and the subsequent positive peak. Oscillatory potentials (OPs) were also extracted and quantified for further analysis. Paraffin-embedded retinal sections were first deparaffinized by immersing the slides in xylene, followed by rehydration through a graded ethanol series (100%, 95%, and 70%). After rehydration, the sections were stained with hematoxylin for 5 min to visualize the nuclei. To differentiate the hematoxylin, the slides were briefly treated with 1% acid alcohol for approximately 5 s, followed by counterstaining with eosin for 2 min to highlight the cytoplasm. Excess stain was removed by rinsing the slides with distilled water. Finally, the stained sections were mounted by using a resin mounting medium to preserve the tissue and prevent dehydration. The HE-stained slides were then visualized and imaged under a light microscope, providing clear images of retinal structures for further analysis. SH-SY5Y cells were cultured under standard conditions at 37 °C under a humidified atmosphere of 5% CO 2 . Cells were cultured in DMEM/F12 media (PM150322, Procell) supplemented with an addition of 1% penicillin–streptomycin (G4003, Servicebio) and 10% fetal bovine serum (FBS-CS500, NEWZERUM, Ltd.). These cells were exposed for 24 h to either 5.5 mM d -glucose (normal glucose), 30 mM d -glucose (high glucose) (ST491, Beyotime), 30 mM d -glucose +100 μM dopamine (DA) (D103111, Shanghai Aladdin Biochemical Technology Co., Ltd.), or 30 mM d -glucose +100 μM dopamine +5 μM ML385 (HY-100523, MedchemExpress) for 24 h. Eyes were fixed in FAS Eye fixative (G1109, Servicebio) for 24 h, dehydrated, and embedded in paraffin. Sections (4 μm) were deparaffinized in xylene, rehydrated through graded ethanol, and subjected to antigen retrieval in a citrate buffer (pH 6.0). Permeabilization and blocking were performed with 0.5% Triton X-100 and 10% normal donkey serum. Primary antibodies, diluted in PBS, were applied overnight at 4 °C. Rabbit TH polyclonal antibody (25859-1-AP, Proteintech, 1:100), rabbit RBPMS polyclonal antibody (ab152101, Abcam, 1:200), rabbit GFAP polyclonal antibody (16825-1-AP, Proteintech, 1:200), and rabbit Iba1 polyclonal antibody (ab178846, Abcam, 1:500) were used. After 24 h, sections were washed with PBS 3 times, incubated with Cy3 conjugated Donkey Anti-Rabbit IgG (GB21403, Servicebio), counterstained with DAPI (G1012, Servicebio), and mounted in antifade medium. Fluorescence images were captured by using a fluorescence microscope under standardized conditions. Mice were euthanized, and their retinas were carefully isolated for dopamine quantification. The retinas were homogenized in 0.01 M PBS at pH 7.4, supplemented with a protease inhibitor to prevent protein degradation. The homogenate was centrifuged at 10,000 g for 10 min at 4 °C to separate the supernatant from cell debris and other insoluble components. The supernatant was carefully collected for dopamine quantification. Dopamine concentrations were measured using a Mouse Dopamine (DA) ELISA Kit (MU30456, Bioswamp) according to the manufacturer’s protocol. The assay involved adding the supernatant to ELISA plate wells precoated with antibodies specific for dopamine. After washing steps and substrate addition, the absorbance was measured at 450 nm using a microplate reader. A calibration curve, ranging from 50 to 4000 pmol/L, was used to determine the dopamine levels in the retinal samples. Results were expressed as picomoles of dopamine per retina (pmol/retina), providing insight into the retinal dopamine content. Total proteins were extracted with RIPA lysis buffer (P0013B, Beyotime) and supplemented with protease and phosphatase inhibitors. The lysates were incubated on ice for 30 min, followed by centrifugation at 12,000 g for 15 min at 4 °C to collect the supernatant containing the soluble proteins. The protein concentration was determined using the BCA protein assay (P0012, Beyotime) according to the manufacturer’s instructions. Equal amounts of protein (typically 20–50 μg) were mixed with loading buffer, and the samples were denatured at 100 °C for 8 min. Protein was subjected to electrophoresis on a 10% SDS-PAGE gel and transferred onto 0.45 μm PVDF membranes (Millipore). Membranes were blocked with 5% nonfat milk for 1 h at room temperature and primary antibodies were incubated overnight at 4 °C. The following primary antibodies were used: antibeta-actin mouse monoclonal antibody (GB12001-100, 1:2000) and anti-NRF2 rabbit polyclonal antibody ( GB113808 , 1:1000) were purchased from Servicebio. Antiferritin rabbit monoclonal antibody (AF2104, 1:1000) and anti-SLC7A11 rabbit polyclonal antibody (AF7992, 1:1000) were purchased from Beyotime. Anti-GPX4 rabbit polyclonal antibody (ab125066, 1:1000) was purchased from Abcam. Anti-TH rabbit polyclonal antibody (25859-1-AP, 1:1000), anti-HO-1 rabbit polyclonal antibody (10701-1-AP, 1:1000), and anti-NCOA4 rabbit polyclonal antibody (10968-1-AP, 1:1000) were purchased from Proteintech. After primary antibody incubation, the membranes were washed three times for 10 min each in TBST to remove any unbound antibody. The membranes were then incubated for 1 h at room temperature with HRP-conjugated secondary antibodies (1:10000, Abclonal). Then the membranes were washed three times for 10 min each in TBST to remove excess secondary antibody. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection reagent (Servicebio) and captured with an imaging system. Total mRNA was extracted using TRIzol (G3013-100 ML, Servicebio) according to the manufacturer’s instructions. Reverse transcription is performed by HiScript Q RT SuperMix for qPCR (R223-01, Vazyme). Real-time quantitative PCR (qPCR) was then performed by using Taq Pro Universal SYBR qPCR Master Mix (R712-02, Vazyme). Primer sequences are as follows: Tyrosine hydroxylase 5′-GGAAGGCCGTGCTAAACCT-3′ (forward) and 5′-GGATTTTGGCTTCAAACGTCTC-3′ (reverse); beta actin 5′- GGAAGGCCGTGCTAAACCT-3′ (forward) and 5′- AGTGGGGTGGCTTTTAGGATG-3′ (reverse). Cell viability was assessed using the Cell Counting Kit-8 (CCK-8, A311-01, Vazyme) according to the manufacturer’s instructions. Cells were seeded into a 96-well plate at a density suitable for the experiment, with each well containing 100 μL of culture medium. After the treatment or incubation period, 10 μL of the CCK-8 solution was added to each well. After incubation, the absorbance of each well was measured at 450 nm by using a microplate reader. The cell viability was evaluated by comparing the absorbance of treated cells with that of control cells, and the results were expressed as a percentage of cell viability. MDA and SOD levels were measured using the Lipid Peroxidation MDA Content Assay Kit (BC0025, Solarbio) and the SOD Activity Assay Kit (BC5165, Solarbio), respectively, following the manufacturer’s protocols. For sample preparation, we added 1 mL of extraction buffer per 5 × 10 6 cells, and cells were lysed using ultrasonic disruption with ice cold, followed by centrifugation at 8000 g for 10 min at 4 °C to collect the supernatant. Then appropriate reagents were added according to the manufacturer’s instructions. After incubation, the absorbance of the resulting complex was measured using a 96-well plate reader. MDA was detected at 532 and 600 nm, and SOD was detected at 450 nm. Concentrations were calculated and expressed as nmol per 10 4 cells and U per 10 4 cells. All statistical analyses were performed using GraphPad Prism (version 9.5.0). All data are expressed as the mean ± standard error of mean (SEM). Student’s t -test was conducted for comparisons between two groups. One-way analysis of variance (ANOVA) was used to evaluate the differences between three or more groups, and Tukey’s multiple comparison was preformed to find possible contrast. p < 0.05 was regarded as indicating a statistically significant difference.

To explore alterations in dopaminergic neurons within the retina, we first examined the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis and a well-established marker for dopaminergic neurons. Immunofluorescence staining of retinal tissues from diabetic mice revealed a marked reduction in TH expression compared to the control group ( Figure A). Quantitative analysis further corroborated this finding, showing a significant decrease in TH fluorescence intensity in the diabetic group ( Figure B), thereby confirming diminished TH expression in the diabetic model. Subsequently, we directly measured dopamine concentration in the retina, which were significantly lower in the diabetic group, consistent with the observed reduction in TH expression ( Figure C). To investigate the effects of high-glucose conditions in vitro, SH-SY5Y cells were cultured with 30 mM d -glucose to mimic hyperglycemia. Under these conditions, we observed substantial downregulation of TH protein levels, accompanied by a parallel decrease in TH mRNA expression ( Figure D,E). In conclusion, these findings demonstrate that both diabetic conditions in vivo and high glucose environments in vitro lead to a reduction in TH expression and a consequent decrease in dopamine levels, suggesting that hyperglycemia may impair dopaminergic neurons in the retina. Dopaminergic retinal neurons lose in the diabetic model. (A) Immunofluorescence stained retina sections with antibodies against TH (red); nucleus were stained with DAPI (blue). Scale bar, 100 μm. (B) Quantitative analysis of the TH fluorescence intensity. (C) Quantification of dopamine concentration in mouse retina. (D) SH-SY5Y cells were collected for TH determination by Western blot analysis in CON (5 mM glucose) and HG (30 mM glucose) groups. (E) SH-SY5Y cells were collected for mRNA determination by RT-PCR in CON and HG groups. Data are presented as the mean ± SEM, p < 0.05, ** p < 0.01. To establish a diabetic mouse model, a single high-dose (100 mg/kg) STZ injection was administered on the first day, effectively inducing a hyperglycemic state characterized by blood glucose levels consistently exceeding 16.7 mmol/L. This hyperglycemia was maintained for a period of 10 weeks, confirming the successful induction and stability of the diabetic model. Subsequently, l -DOPA was administered daily during the final 2 weeks to assess its therapeutic effects. At the study end point, ocular tissues and plasma samples were collected for detailed biochemical and histological analysis ( Figure A). Control mice group exhibited normal weight gain over the study duration, while STZ-treated mice showed significantly reduced weight gain. Blood glucose levels in STZ-treated mice remained markedly elevated throughout the first 8 weeks, demonstrating the reliability of the diabetic model. l -DOPA treatment during the last 2 weeks resulted in a mild but measurable reduction in blood glucose levels, suggesting a partial amelioration of hyperglycemia, potentially indicating a beneficial metabolic effect of l -DOPA treatment ( Figure B,C). Retinal physiological function in mice was assessed by electroretinography (ERG). The amplitude of a-wave and b-wave in diabetic mice was significantly reduced, which indicates dysfunction of photoreceptor and bipolar cells within the retina , ( Figure D,F,G). Specifically, l -DOPA treatment markedly restored the diminished b-wave amplitudes at a scotopic flash intensity of 3.0 cd·s/m 2 ( p < 0.001) ( Figure G). Additionally, retinal oscillatory potentials (OPs), which reflect the synchronous activity of inner retinal neurons including amacrine cells, bipolar cells, and retinal ganglion cells (RGC), were significantly decreased in diabetic mice and had partial amplitude recovery following l -DOPA treatment ( Figure E,H). These results show that l -DOPA may enhance dopaminergic signaling to mitigate inner retinal dysfunction and preserve the overall retinal function in DRN. l -DOPA restored diabetic mice retinal function. (A) Schematic illustration of the established experimental model. Drawn by MedPeer (medpeer.cn). (B) Blood glucose levels and (C) body weights of mice were examined at the indicated time points ( n = 5 for each group). (D) Representative electroretinography (ERG) waveforms at a scotopic flash intensity of 1.0 cd·s/m2 of control (CON), diabetic (STZ), and l -DOPA group. (E) Representative of oscillatory potentials (OPs) waveforms at a scotopic flash intensity of 3.0 cd·s/m2 of CON, STZ, and l -DOPA group. Quantitative analysis of ERG parameters: (F) scotopic a-wave amplitudes, (G) scotopic b-wave amplitudes, and (H) OPs amplitudes. Data are presented as the mean ± SEM. The ANOVA test was used to evaluate the differences between groups, and Tukey’s multiple comparison was preformed to find possible contrast. * indicates the difference between CON and STZ groups, # indicates the difference between STZ and l -DOPA groups. */# p < 0.05, **/## p < 0.01, ***/### p < 0.001, and ****/#### p < 0.0001. HE staining demonstrated a significant reduction in overall retinal thickness in the diabetic group compared to the control group, as evidenced by thinning across multiple retinal layers, including the ganglion cell layer (GCL), inner plexiform layer (IPL), and outer nuclear layer (ONL). Following 2 weeks of intraperitoneal l -DOPA administration, a partial restoration of retinal thickness was observed, particularly within the GCL and ONL ( P < 0.05), indicating the potential neuroprotective and therapeutic effects of l -DOPA against diabetes-induced retinal neuron degeneration. Quantitative analysis of retinal layer thickness further confirmed these observations, with statistically significant improvements in the l -DOPA-treated group compared to the untreated diabetic group ( Figure A,B). Immunofluorescence staining for RBPMS, a specific marker for RGC, revealed a marked reduction in RGC density in the diabetic group compared to the control group. Quantitative analysis further confirmed this significant decrease in RGC counts. However, intraperitoneal administration of l -DOPA for 2 weeks significantly restored RGC density, as evidenced by both immunofluorescence staining and quantitative assessments ( P < 0.01) ( Figure C,D). This recovery suggests a neuroprotective role of l -DOPA in mitigating diabetes-induced damage to retinal neurons, specifically RGCs. l -DOPA ameliorate diabetic mice retinal impairment. (A) Hematoxylin and eosin (HE) staining showing the retinal structure in the CON, STZ, and l -DOPA groups. Scale bar, 100 μm. (B) Quantitative analysis of retinal layer thickness, including the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (IPL), outer nuclear layer (ONL), and total retinal thickness. (C) Immunofluorescence staining with antibodies against RBPMS (red); nuclei are stained with DAPI (blue). Scale bar, 100 μm. (D) Quantification of RGC cell counts. (E) Transmission electron microscopy (TEM) images of retinal ultrastructure in the CON, STZ, and l -DOPA groups. Mitochondria indicated by asterisk, solid triangle, and hollow triangle. Scale bar, 10 μm. Data are presented as the mean ± SEM, * p < 0.05, ** p < 0.01, and *** p < 0.001. Transmission electron microscopy (TEM) revealed that diabetic mice exhibited ferroptosis like mitochondrial morphological alterations, including mitochondrial condensation, increased membrane density, and altered cristae architecture, indicated by a solid triangle. These features were distinguished from apoptosis and necroptosis by the absence of apoptotic bodies, nuclear condensation, and cytoplasmic leakage. Notably, in the l -DOPA group, the mitochondrial cristae increased, accompanied by an enlargement in mitochondrial volume as shown by the hollow triangle ( Figure E). Integrating all these results above, we conclude that the potential neuroprotective and therapeutic effects of l -DOPA against diabetes induced retinal neuron degeneration and mitochondrial abnormalities. Retinal sections were stained for GFAP and IBA1 to evaluate the activation of astrocytes and microglia, respectively ( Figure A,B). Immunofluorescence staining revealed a significant increase in astrocyte activation within the ganglion cell layer (GCL) and heightened microglial activation in the inner plexiform layer (IPL) of the STZ-induced diabetic retina. These changes are consistent with the characteristic glial reactivity and neuroinflammation associated with diabetic retinopathy. However, daily administration of l -DOPA for 2 weeks significantly reduced GFAP expression levels in the GCL, as shown by fluorescence imaging ( Figure A). Quantitative analysis confirmed a marked decrease in GFAP fluorescence intensity in the l -DOPA-treated group compared to the STZ group ( p < 0.05). Similarly, IBA1 expression, an indicator of microglial activation, was substantially attenuated by l -DOPA treatment, indicating suppressed microglial activity in the diabetic retina ( p < 0.05) ( Figure B). These results suggest that l -DOPA effectively mitigates the activation of both glial and microglial cells in the diabetic retina, highlighting its potential for reducing retinal inflammation and protecting against diabetic retinopathy. Immunofluorescence staining of GFAP and IBA1. Immunofluorescence staining images of GFAP (A) and IBA1 (B) in retinal sections of CON, STZ, and l -DOPA-treated groups. Nucleus are stained with DAPI (blue). Scale bar, 100 μm. Bar graph shows fluorescence intensity. Data are presented as the mean ± SEM, * p < 0.05, and ** p < 0.01. In vitro, the CCK8 test showed that high glucose reduced the viability of SH-SY5Y cells and led to increased cell death, effects that were notably counteracted by the addition dopamine in a concentration-dependent way ( Figure A). Since 100 μM dopamine exhibited the most pronounced protective effect, we selected this concentration for further investigation. We assessed antioxidant capacity by measuring MDA and SOD levels. MDA, a product of lipid peroxidation and functioning as a significant biomarker for oxidative stress, was found to be elevated under high glucose conditions and reduced following dopamine treatment ( Figure B). SOD protects cells against ROS under redox imbalance, high glucose decreases SOD activity in SH-SY5Y cells, and dopamine helps to reverse the influence ( Figure C). The oxidation level was further confirmed by BODIPY fluorescent probes. Similarly, dopamine can inhibit lipid peroxidation accumulation caused by high glucose ( Figure D,E). Collectively, we know that high glucose leads to redox imbalance, and dopamine can help restore the antioxidant capacity under high glucose stress. Lipid peroxidation is the important cause to trigger ferroptosis, characterized by iron accumulation. Therefore, we decided to test the iron ion concentration in the cell using FerroOrange fluorescent probes; the result demonstrated that iron ion concentration increases in high glucose and decreases with the addition of dopamine ( Figure F,G). Dopamine alleviates SH-SY5Y cell ferroptosis. (A) Cell viability under different dopamine concentrations in HG (30 mM) conditions. MDA (B) and SOD (C) levels in cells treated with normal glucose (5 mM), high glucose (30 mM), and high glucose + dopamine (100 μM). (D) BODIPY fluorescent images and (E) quantitative analysis, representing the lipid peroxidation in cells. (F) FerroOrange fluorescent images and (G) quantitative analysis, indicating the iron ion concentration in cells. (H) Western blot analysis of NCOA4, SLC7A11, ferritin, and GPX4 protein levels with quantitative analysis shown on the right. Beta actin was used as the control. Data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, and *** p < 0.001. WB analysis further confirmed the occurrence of ferroptosis under high glucose conditions. GPX4, a critical antioxidant enzyme essential for defending against ferroptosis, was significantly decreased in high glucose environments. Similarly, SLC7A11, an upstream mediator of GPX4 that is vital for maintaining glutathione levels, exhibited a comparable decrease in protein expression. Given the imbalance of iron ions in cells under high glucose, we examined the expression of iron-related proteins, ferritin, and NCOA4. Ferritin, an iron storage protein critical for maintaining intracellular iron homeostasis, was found to be reduced under high glucose conditions. NCOA4, a selective cargo receptor responsible for ferritinophagy that mediates the degradation of ferritin and promotes the release of stored iron, was notably upregulated under high glucose conditions. Moreover, dopamine treatment was observed to elevate the expression of GPX4, SLC7A11, and ferritin and suppress NCOA4 expression, thereby inhibiting ferroptosis ( Figure H). Given the oxidative stress and ferroptosis induced by high glucose and considering the pivotal role of the Nrf2/HO-1 signaling pathway in regulating oxidative stress responses, we aimed to investigate whether dopamine could activate this pathway to counteract the oxidative damage and ferroptosis triggered by high glucose. To address this hypothesis, we examined the involvement of Nrf2 and HO-1 in mouse retinas through immunofluorescence staining. The results show elevated levels of Nrf2 in the l -DOPA group compared to the diabetic group; this finding indicates the activation of the Nrf2 pathway under l -DOPA treatment. Consistently, HO-1, a downstream antioxidant enzyme transcriptionally regulated by Nrf2, also exhibited a marked increase in the l -DOPA group ( Figure A,B). Additionally, in vitro, Western blot analysis confirmed the significant upregulation of Nrf2 and HO-1 expression in dopamine-treated group compared to the high glucose group ( P < 0.05), the result was consistent with the immunofluorescence staining analysis and provide further evidence that dopamine treatment can activate the Nrf2/HO-1 pathway ( Figure C). Dopamine promotes the expression of Nrf2 and HO-1. Immunofluorescence staining stained retina sections with antibodies against (A) Nrf2 and (B) HO-1, nuclei were stained with DAPI. (C) Western blot of NRF2 and HO-1 protein expression with quantitative analysis shown on the right. Beta actin was used as the control. Data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. To further confirm the involvement of the Nrf2/HO-1 pathway, we treated SH-SY5Y cells with ML385, a selective Nrf2 inhibitor. Western blot results showed that ML385 treatment abolished dopamine-induced upregulation of both Nrf2 and HO-1. Moreover, the protective effects of dopamine were reversed by ML385, as evidenced by decreased expression of ferroptosis-related proteins GPX4, SLC7A11, and ferritin and an increase in NCOA4 expression ( Figure A). Additionally, BODIPY fluorescence staining demonstrated that ML385 treatment significantly elevated lipid peroxidation levels, effectively abolishing the inhibitory effect of dopamine ( Figure B,D). Similarly, FerroOrange staining showed that intracellular free iron ions, which were reduced by dopamine, were markedly increased after ML385 treatment, suggesting that NRF2 inhibition exacerbates ferroptosis ( Figure C,E). Furthermore, the ML385 treatment reversed the dopamine-induced reduction in MDA levels, leading to a marked increase in lipid peroxidation ( Figure F). And SOD activity, which was restored by dopamine, significantly decreased following ML385 treatment ( Figure G). Inhibition of Nrf2 attenuates the protective effects of dopamine. (A) Western blot analysis of NRF2, HO-1, and ferroptosis-related proteins (NCOA4, ferritin, SLC7A11, and GPX4) in SH-SY5Y cells treated with NRF2 inhibitor ML385. (B,D) BODIPY fluorescent images and corresponding quantitative analysis, representing the lipid peroxidation in cells. (C,E) FerroOrange fluorescent images and quantitative analysis, indicating the iron ion concentration in cells. MDA (F) and SOD (G) levels in cells treated with normal glucose (5 mM), high glucose (30 mM), high glucose + dopamine (100 μM), and high glucose + dopamine + ML385 (10uM). Data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

In the present study, we sought to investigate the neuroprotective effects of dopamine in alleviating DRN and to elucidate its underlying mechanisms. Our findings demonstrate that dopamine effectively attenuates retinal damage in DRN and alleviates ferroptosis in retinal RGCs and SH-SY5Y cells. Furthermore, Nrf2 inhibitor ML385 was able to reverse the protective effects of dopamine, verifying that dopamine exerts its effects through the Nrf2 signaling pathway. These results not only highlight the detrimental impact of diabetes on the retinal dopaminergic system but also underscore the potential of dopamine-based interventions as therapeutic strategies targeting the DRN. Dopamine is well-known for its role as a neurotransmitter in the central nervous system; however, emerging evidence suggests that it also exerts protective effects outside the brain, including antineovascularization in endometriosis, inhibition of allergic inflammation in lung, maintenance of blood perfusion in kidney, etc. , , Additionally, previous studies have reported a reduction of dopamine in diabetic mouse retinas, supplementation with dopamine improved ERG deficits, and decreased vascular leakage in the retina. , However, the underlying mechanisms by which dopamine confers its protective effect, particularly during the early stages of neurodegeneration in DR, remain poorly understood. The STZ injection successfully induced diabetes in C57BL/6 mice, marked by significant weight loss and sustained hyperglycemia, consistent with diabetic pathophysiology. We found that diabetes impairs dopaminergic neurons in the retina, consequently leading to a decrease in the dopamine concentration. In this study, functional impairments were evident through ERG examination, where diabetic mice exhibited reduced amplitudes of a-wave, b-wave, and oscillatory potentials, reflecting compromised photoreceptor activity and dysfunction in inner retinal layers, including amacrine cells and RGCs. Notably, l -DOPA supplementation led to a marked recovery in OP wave amplitudes, highlighting its selective enhancement of inner retinal function and support of amacrine cell activity, as well as RGC function ( Figure D,H). The retinal thickness of diabetic mice was significantly decreased, and administration of l -DOPA partially restored retinal morphology thinning, suggesting its neuroprotective role in preserving retinal structure. Additionally, retinal immunofluorescence staining demonstrated that l -DOPA treatment restored RGC numbers, addressing a critical aspect of retinal neurodegeneration in DRN and suggesting that l -DOPA may protect RGCs from death ( Figure C,D). Collectively, these findings indicate that l -DOPA exerts multifaceted protective effects on both the structural and functional integrity of the retina in diabetic models, positioning it as a promising therapeutic agent for mitigating the DRN. Previous studies have demonstrated a close association between neurodegeneration disease and ferroptosis, , , a regulated form of cell death which depends on iron accumulation and characterized by excessive lipid peroxidation. Researchers found that dopamine could inhibit ferroptosis in both normal and malignant cells, as well as Alzheimer’s disease and lung fibrosis. , , In our study, we aim to figure out whether dopamine participates in regulating the ferroptosis of DRN. TEM revealed mitochondrial ultrastructural abnormalities in the retinas of diabetic mice, characteristic of ferroptosis-like damage, including mitochondrial shrinkage and disrupted cristae, as solid triangles shown in Figure . These pathological changes were notably alleviated following l -DOPA administration, suggesting that DA restores mitochondrial integrity and reduces ferroptosis susceptibility. The conclusion aligns with previous reports demonstrating dopamine’s role in attenuating ferroptosis in Alzheimer’s disease and lung fibrosis. , To further investigate the role of dopamine in mitigating neuronal ferroptosis in DRN, we employed the SH-SY5Y cell line to simulate neuronal injury under high glucose conditions. In vitro, our findings demonstrate that high glucose conditions induce oxidative stress and ferroptosis in SH-SY5Y cells, and dopamine exerts a protective effect by mitigating these detrimental processes. High glucose disrupts redox homeostasis, as evidenced by the reduction in SOD activity and the elevation of MDA levels, accompanied by increased intracellular lipid peroxides ( Figure B–D). These oxidative alterations not only compromise cellular viability but also create a susceptible environment for ferroptosis. With dopamine added, it exerts a protective effect by enhancing antioxidant defenses to counter oxidative stress. Additionally, intracellular iron homeostasis was profoundly disrupted under high glucose conditions, characterized by increasing iron fluorescence staining intensity and dopamine significantly diminishing iron accumulation ( Figure E). Under high glucose conditions, a reduction in ferritin levels and upregulation of NCOA4 promotes ferritinophagy, releasing iron from ferritin and leading to an increase in the labile iron pool. This iron accumulation facilitates the Fenton reaction, exacerbating lipid peroxidation and amplifying oxidative damage. Dopamine effectively reversed these changes, as it suppressed NCOA4 levels and maintained the ferritin content, thus mitigating iron-mediated oxidative stress. Furthermore, the depletion of GPX4 and SLC7A11 under high glucose conditions impaired antioxidant systems in ferroptosis. GPX4 plays a critical role in detoxifying lipid peroxides, and its upstream regulator, , SLC7A11, is essential for glutathione synthesis, which provides electrons for the antioxidant function of GPX4. , The downregulation of these proteins under high glucose stress highlights a key pathway through which ferroptosis is triggered. Dopamine’s ability to restore the expression of GPX4 and SLC7A11 further reinforces its protective effects, as these proteins are vital for maintaining redox balance and preventing ferroptosis. Collectively, these results suggest that high glucose induces ferroptosis in SH-SY5Y cells through a dual mechanism of redox imbalance and iron dysregulation. Dopamine counteracts these processes by enhancing the antioxidant capacity, inhibiting lipid peroxidation, and restoring iron homeostasis. This underscores the potential of dopamine as a therapeutic agent for mitigating oxidative stress and ferroptosis-related neuronal damage in high glucose conditions, such as diabetes associated neurodegeneration. Based on these findings, we further investigated the mechanism through which dopamine exerts its protective effects. Under oxidative stress, the Nrf2 signaling pathway is vital for maintaining cellular homeostasis by activating the transcription of a broad range of antioxidant and detoxifying genes, enhancing cellular resistance to ROS and toxins. , , Beyond its antioxidant role, Nrf2 also regulates metabolism and inflammation, contributing to overall cellular homeostasis. , In neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s disease, Nrf2 activation has been shown to mitigate oxidative damage and inflammation by decreasing NLRP3 and preserving mitochondrial function, underscoring its neuroprotective potential. − Similarly, in cardiovascular diseases, Nrf2-mediated antioxidant responses contribute to the protection of cardiac tissues from ischemia reperfusion injury and atherosclerosis. , These studies collectively clarify the versatility of the Nrf2 pathway in disease modulation and its potential as a therapeutic target to alleviate oxidative damage. In our study, we verified that dopamine activates the Nrf2/HO-1 signaling pathway to modulate the cellular antioxidant response and maintain iron metabolism homeostasis, thereby mitigating oxidative stress and ferroptosis caused by high glucose levels ( Figure ). This mechanism is further supported by reversal experiments using the selective Nrf2 inhibitor ML385, which reversed dopamine’s effects on antioxidant defense and iron homeostasis. Previous studies have demonstrated that increasing ferroptosis contributes to the pathology of diabetic retinopathy. , The Nrf2/HO-1 signaling pathway is well-recognized for its antioxidant and antiferroptosis roles in various metabolic diseases. , Based on these previous studies, our research elucidates the mechanism by which dopamine mitigates DRN through Nrf2 regulation. This finding not only underscores the therapeutic potential of dopamine in DRN but also enhances our understanding of the functional roles of the Nrf2/HO-1 pathway. Mechanism of the dopamine protective effect. Dopamine activates Nrf2, which enhances the transcription of downstream antioxidant genes such as GPX4 and HO-1, thereby defending against ROS. Additionally, dopamine reduces intracellular iron concentration by upregulating NCOA4 and downregulating ferritin, collectively suppressing ferroptosis. Drawn by Figdraw. (Granted ID: UAWSYf4f64). While our findings establish an important role for the Nrf2/HO-1 axis in dopamine’s protective effects, we acknowledge that oxidative stress modulation involves complex crosstalk between multiple pathways. For instance, NF-κB signaling is often activated under neuroinflammation, which can participate with inflammation and immune response. Additionally, MAPK pathway also acts as a sensor and effector of oxidative stress, balancing cell survival, death, and inflammation. , Although our experimental design focused on the Nrf2/HO-1 pathway, these parallel pathways could contribute to the observed neuroprotection and deserve future investigation. This study has certain limitations. First, our experiments were primarily based on animal and cell models, and the results may not fully reflect the actual conditions in clinical patients. Further studies involving human subjects or clinical samples are essential to confirm the translational potential of our findings. Second, peripheral dopa decarboxylase converts a portion of l -DOPA into dopamine, thereby exerting peripheral physiological effects, such as cardiovascular system activation and inhibition of insulin secretion. Subsequent studies may consider combining a dopa decarboxylase inhibitor to reduce the level of peripheral dopamine conversion, thereby improving its bioavailability. Third, although we validated the protective effects of dopamine and its associated pathway, we did not thoroughly investigate the involvement of dopamine receptors or other potentially relevant pathways. Further studies might concentrate on the precise interaction mechanisms between dopamine and its receptors, along with the regulatory specifics of the other possible redox-related signaling pathway, to enhance understanding of its therapeutic potential. Efforts to develop more efficient and stable dopamine analogues could optimize the clinical application. Additionally, combining dopamine with anti-inflammatory or antioxidant drugs may enhance the overall therapeutic effects. In conclusion, we have identified the neuroprotective effect of dopamine against DRN, which involves maintaining oxidative balance and promoting antiferroptosis effects. This protection is mediated through activation of the Nrf2/HO-1 signaling pathway.

Diabetic retinopathy (DR) is a major cause of vision impairment in people with diabetes, impacting over 100 million people globally. , Although much attention has been given to the vascular changes associated with DR, such as microaneurysms, retinal microvascular leakage, retinal arteriolar dilatation, and neovascularization, , increasing evidence suggests that retinal neurovascular unit (NVU) plays a central role in the early stages of the disease. , Retinal neurons, particularly retinal ganglion cells (RGCs), and inner plexiform layer (IPL) are among the first to be damaged by the metabolic disturbances associated with diabetes. , These neurodegenerative changes often precede detectable vascular abnormalities, which can lead to irreversible vision impairment. To fully comprehend the pivotal role of the NVU in the onset of DR, it is crucial to examine the underlying mechanisms that drive retinal neurodegeneration. Pathogenesis of diabetic retinal neurodegeneration (DRN) is multifactorial, involving factors such as hyperglycemia, inflammation, oxidative stress, and mitochondrial dysfunction. Of these, oxidative stress is particularly important, as it leads to the formation of reactive oxygen species (ROS), which can damage cellular structures and disrupt neuronal function. Retina with its high metabolic activity is especially susceptible to oxidative damage. To mitigate this damage, retinal cells rely on a network of antioxidant defenses, the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway is recognized as a critical regulator of oxidative stress and inflammatory responses. , Nrf2, as a nuclear transcriptional regulator, modulates the expression of various antioxidant genes, including HO-1, GSR, TXN, and others. − Among these, HO-1 has been demonstrated to exert cytoprotective, anti-inflammatory, and antiapoptotic effects. , Disruption of the Nrf2 signaling pathway has been linked to the progression of neurodegenerative diseases, including Parkinson disease, Alzheimer disease, and diabetic retinopathy. − Ferroptosis, a recently identified type of regulated cell death, is defined by iron-driven lipid peroxidation, leading to the disruption of cellular membranes. Unlike apoptosis and necrosis, ferroptosis is specifically driven by the accumulation of lipid peroxides, a process that is tightly regulated by iron metabolism, antioxidant defense systems, and key cell signaling pathways such as the Nrf2 pathway, the p53 pathway, and the AMPK pathway. , Recent studies have highlighted the potential involvement of ferroptosis in various neurodegenerative diseases, including Parkinson disease, Alzheimer, and diabetic retinopathy, where oxidative stress and iron homeostasis are disrupted, resulting in neuron function lost. − Due to retina high metabolic activity, there is a pronounced accumulation of oxidative byproducts, which significantly heightens its susceptibility to ferroptosis. While DRN represents an early stage of DR characterized by retinal neuronal dysfunction, the involvement of ferroptosis in DRN and the elucidation of its mechanistic pathways remain to be fully determined. Dopamine (DA), traditionally known for its role as a neurotransmitter in the central nervous system, has recently attracted attention for its potential protective properties beyond classical functions. Recent studies have given that dopamine exhibits antioxidant properties in kidney and improves mitochondrial quality in hippocampal neurons. , Moreover, researchers have found that dopamine acts as a strong inhibitor of ferroptosis in both normal and malignant cells. In addition, some studies have confirmed the potential role of dopamine in preventing or alleviating DR through angiogenesis regulation and inflammation modulation, − while the effects of dopamine on retinal neurons have not been clearly elucidated, and its underlying mechanisms remain underexplored. Understanding the detailed molecular interactions of dopamine in the retina is essential for leveraging its therapeutic potential to effectively prevent and treat diabetic retinopathy. This study aims to explore the therapeutic potential of dopamine in diabetic retinal neurodegeneration with a focus on the Nrf2/HO-1 signaling pathway. We hypothesize that dopamine may alleviate retinal neurodegeneration by activating Nrf2 and subsequently upregulating HO-1, thus reducing oxidative stress and protecting retinal neurons from ferroptosis. The findings of this study could help deepen our understanding of the molecular mechanisms underlying dopamine’s protective effects and provide insights into novel therapeutic strategies for managing diabetic retinal neurodegeneration.