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. 2025 Mar 1;48(5):3194–3205. doi: 10.1007/s10753-025-02259-8

Inhibition of Cullin3 Neddylation Alleviates Diabetic Retinopathy by Activating Nrf2 Signaling to Combat ROS-Induced Oxidative Stress and Inflammation

Yueqin Chen 1,#, Cong Liu 1,#, Jun Tong 1,#, Chang He 1, Xinru Ling 1, Jinjin Xiang 2, Chunyan Xue 3,#, Genhong Yao 4,#, Lingyun Sun 4,#, Zhenggao Xie 1,✉,#
PMCID: PMC12596376  PMID: 40021543

Abstract

Oxidative stress and inflammation induced by reactive oxygen species (ROS) play important roles in the development of diabetic retinopathy (DR). Nuclear factor erythroid 2-related factor 2 (Nrf2) signaling, which is negatively controlled by Cullin3-RING E3 ligase (CRL3) and controls ROS levels, is compromised in DR. CRL3 activity is regulated by Cullin3 neddylation. Nonetheless, the relationship between Cullin3 neddylation and DR remains uncertain. The goal of this study was to evaluate the effect of Cullin3 neddylation on DR and its underlying mechanisms by utilizing MLN4924, a neddylation inhibitor. Cullin3 neddylation was elevated in diabetic rats’ retinas as well as in advanced glycation end products (AGEs)-induced endothelial cells. Inhibiting neddylation of Cullin3 with MLN4924 downregulated Nrf2 ubiquitination, promoted Nrf2 accumulation, suppressed ROS-induced oxidative stress and inflammation, and attenuated blood-retinal barrier (BRB) breakdown in both diabetic vivo and vitro models. However, the beneficial impact of MLN4924 was compromised when Nrf2 was suppressed with siRNA in vitro. This study showed that inhibition of Cullin3 neddylation with MLN4924 exerted protective effect on DR by activating Nrf2 signaling to inhibit ROS-induced retinal injury, which indicated that targeting Cullin3 neddylation could be a promising treatment option for DR.

Graphical Abstract

Schematic diagram of the role of Cullin3 neddylation in DR and the effect of inhibition of Cullin3 neddylation with MLN4924 on DR.

graphic file with name 10753_2025_2259_Figa_HTML.jpg

Keywords: Diabetic retinopathy, Neddylation, Nrf2, Oxidative stress, Inflammation

Introduction

As a microvascular complication of diabetes, diabetic retinopathy (DR) is the main cause of visual loss in the working population [1]. By 2030, the estimated number of patients with DR will increase to 130 million [2]. A recent therapeutic advancement for DR that targets VEGF-A has produced favorable results. However, anti-VEGF therapy requires multiple intra-vitreous injections, may inadvertently cause microvascular occlusion and other adverse effects, and does not produce favorable outcomes for all patients [3, 4]. Therefore, it is imperative to identify new potential therapeutic targets for DR.

Reactive oxygen species (ROS) accumulation is crucial for the development of DR [5, 6]. Persistent hyperglycemia-induced metabolic abnormalities cause the generation of ROS. Conversely, increased ROS exacerbates the metabolic abnormalities. Furthermore, the suppressed antioxidant defense system also results in the imbalance between the elimination and generation of ROS in DR [5]. ROS-induced inflammation and oxidative stress are critical factors in blood-retinal barrier (BRB) disruption, a characteristic of DR [6]. Nuclear factor erythroid 2-related factor 2 (Nrf2) activates the transcription of antioxidant genes to regulate ROS levels [7]. In DR, Nrf2 signaling is compromised [7, 8]. However, the mechanisms underlying the compromised Nrf2 signaling in DR is incompletely understood.

Cullin3-RING E3 ligase (CRL3) inactivates Nrf2 signaling by facilitating its ubiquitination and subsequent degradation through the ubiquitin proteasome system (UPS) [9]. The function of CRL3 is regulated by Cullin3 neddylation [10]. Neddylation is a post-translational protein modification, in which neural precursor cell expressed developmentally downregulated gene 8 (NEDD8) is covalently conjugated to target proteins. Neddylation is involved in a variety of diseases, such as fibrotic diseases, cancer, and oxidative stress [11, 12]. Nevertheless, the role of Cullin3 neddylation in DR and its relationship with compromised Nrf2 signaling in DR are uncertain.

Therefore, this study was to evaluate the role of Cullin3 neddylation in DR and determine whether the suppressed Nrf2 signaling in DR can be ascribed to Cullin3 neddylation by using of MLN4924, an inhibitor of neddylation.

Methods and Methods

Experimental Animals

Male Sprague–Dawley rats weighing 180 ± 20 g and aged 7–8 weeks were purchased from Lingchang Biotechnology Corporation (Shanghai, China). Animal experiments were complied with ethical standards of the Animal Care and Welfare Committee of Nanjing Drum Tower Hospital. A high-fat diet and intraperitoneal injection of streptozotocin (STZ; 60 mg/kg) were utilized to establish the diabetic rat model [13]. Three days following STZ injection, rats were classified as diabetics if their blood glucose were higher than 16.7 mmol/L. The diabetic rats were randomly allocated to the DM (diabetes mellitus) group or the DM + MLN4924 group. MLN4924 (HY-70062, MedChemExpress, Shanghai, China) was dissolved in SBE-β-CD (HY-17031, MedChemExpress, China) at a final concentration of 2 mg/ml in 20% SBE-β-CD, and was injected intraperitoneally at a dosage of 10 mg/kg 3 times per week for 4 weeks starting from twelve weeks following STZ administration [14, 15]. Rats in the DM group were administered saline solution containing 20% SBE-β-CD. Finally, the rats were sacrificed for further analysis.

Cell Culture and Treatment

Procell Life Science (Shanghai, China) provided rat retinal microvascular endothelial cells (RMEC), which were grown in DMEM with 10% FBS. Endothelial cells, with and without siNrf2 transfection, were exposed to 300 µg/ml of advanced glycation end products (AGEs) (ab51995, abcam, Cambridge, UK) to mimic the microenvironment of diabetes and treated with 0.3 µM MLN4924 (HY-70062, MedChemExpress, China) for 48 h in vitro [8]. The sequences of Nrf2 siRNA were sense, 5′-CCCUGUGUAAAGCUUUCAATT-3′, and antisense, 5′-UUGAAAGCUUUACACAGGGAC-3′, and siNrf2 were transfected into endothelial cells using Lipofectamine 3000 (L3000015, Invitrogen, USA).

Evans Blue Assay

Under anesthesia, 30 mg/mL Evans blue (EB) dye (Sigma-Aldrich, USA) was injected into rats’ femoral vein and allowed to circulate for 2 h. Following the removal and fixation of the eyeballs for 2 h, the retinas were isolated, mounted on glass slides, and analyzed using a confocal microscope (FluoView FV3000, Olympus Corporation). In addition, the leakage of Evans blue was quantified using the method described previously, and the results were presented as ng Evans blue/mg retinal weight [16].

Immunoprecipitation

Immunoprecipitation of Nrf2 was conducted using anti-Nrf2 antibody (#33,649, CST, MA, USA) that was conjugated to protein A magnetic beads (#73,778, CST). The complex of magnetic beads and antibody was incubated with protein lysate overnight at 4 °C. The beads underwent five rounds of washing and were subsequently boiled with sample buffer for western blot assay.

Western Blot

As described previously, western blot was used to test the levels of proteins in cells or retinas [13]. Primary antibodies: Nrf2 (1:1000, A0674, ABclonal), NLRP3 (1:1000, ab263899, abcam), NQO1 (1:2000, ab80588, abcam), IL-1β (1:500, ab254360, abcam), caspase-1 (1:500, ab179515, abcam), occludin (1:1000, ab216327, abcam), NEDD8 (1:2000, ab81264, abcam), ubiquitin (1:1000, ab134953, abcam), and Cullin3 (1:500, ab75851, abcam). GAPDH was used for internal reference.

Permeability Assay

The paracellular permeability of endothelial monolayers was evaluated using FITC-dextran leakage and transwell inserts with a pore size of 0.4 µm (Labselect, China) [8]. Transwell inserts were used to culture endothelial cells. Following the specified stimulation, FITC-dextran (100 µg/ml, #46,945, Sigma) was added to the transwell filters. Following a duration of 3 h, a volume of 200 µl of medium was obtained from the bottom wells. The fluorescence was subsequently read at 485 nm excitation/530 nm emission.

Assessment of Oxidative Stress

ROS levels were detected using a fluorescent probe dihydroethidium (DHE) [12]. Retinal cryosections were air-dried and subjected to 30 min of dark staining with DHE (3.5 µM). After being washed, the slides were examined using confocal microscopy. ImageJ was used to calculate the relative fluorescence intensity of DHE red. In vitro, after indicated stimulation, endothelial cells were rinsed and incubated in the dark for 20 min with DHE at a concentration of 10 µM. Following washing, the DHE fluorescence was examined with fluorescence microscope and flow cytometry.

The level of MDA (malonaldehyde, a toxic compound resulting from lipid peroxidation) and the ratio of GSH/GSSG was evaluated using corresponding kits (Jiancheng, Nanjing, China).

Statistical Analysis

Statistical analysis was conducted using GraphPad Prism 9.3.1. One-way analysis of variance (ANOVA) followed by Tukey's post-hoc test was used to compare multiple groups. A P value < 0.05 was considered statistically significant. Every experiment was repeated at least three times.

Results

Neddylation Inhibitor MLN4924 Alleviated Retinal Injury in Diabetic Rats

Retinal morphological changes and vascular leakage were utilized to evaluate the effects of MLN4924 on the retinas of diabetic rats. The control group exhibited a regularly and densely packed cells in both the inner nuclear layer (INL) and outer nuclear layer (ONL), as determined by HE staining. Cells in INL and ONL in the DM group were loosely and irregularly arranged. These alterations were alleviated in DM + MLN4924 group (Fig. 1a). The Evans blue assay revealed that the control group had no dye leaking into the retinal parenchyma. However, the DM group demonstrated Evans blue leakage into the retinal parenchyma, indicating the disrupted BRB. Treatment with MLN4924 significantly decreased the dye leakage (Fig. 1b). Quantification of dye leakage confirmed the result (Fig. 1c). The tight junction occludin assessment was utilized to further confirm the beneficial impact of MLN4924 on BRB. Occludin levels were lower in the DM group than the control group; however, MLN4924 treatment elevated occludin levels relative to the DM group (Fig. 1d). This result aligned with the conclusions drawn by Evans Blue assay. These findings collectively demonstrated that treatment with MLN4924 mitigated the retinal histological alterations and BRB disruption induced by diabetes.

Fig. 1.

Fig. 1

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MLN4924 attenuated histological changes and BRB breakdown in the diabetic rats’ retinas (a) HE images of retinas from each group. (b) Fluorescence images of flat-mounted retinas with Evans blue. White arrow: Dye leakage. (c) Quantification of leaked Evans blue. (d) Western blot assay for occludin levels in the retinas. All data were expressed as means ± SD. **P < 0.01, ***P < 0.001, and ****P < 0.0001. NFL: nerve fiber layer; GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; IS/OS: inner segment/outer segment

MLN4924 Attenuated ROS-Induced Oxidative Stress and Inflammation in Diabetic Rats’ Retinas

ROS-induced oxidative stress and inflammation contribute to the development of DR [6]. ROS can induce the activation of NLRP3 inflammasome, leading to the release of IL-1β and the subsequent cascade of inflammation [17]. Thus, the levels of ROS, oxidative stress markers, and NLRP3 inflammasome-related proteins were evaluated. ROS production was greater in the DM group compared to the control group as determined by DHE (Fig. 2a, b). MLN4924 treatment significantly decreased ROS production compared to that in the DM group. Compared to the control, MDA content increased and the GSH/GSSG ratio decreased significantly in the DM group. In contrast, MLN4924 treatment increased the ratio of GSH/GSSG and decreased MDA levels compared to DM group (Fig. 2c, d). Compared to the control, protein levels of NLRP3, cleaved caspase-1, and IL-1β were substantially upregulated in the retinas of the DM group, while these proteins were decreased in DM + MLN4924 group (Fig. 2e-h). These results showed that MLN4924 inhibited ROS-induced oxidative injury and inflammation in the retinas of diabetic rats.

Fig. 2.

Fig. 2

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MLN4924 inhibited ROS-induced oxidative injury and inflammation in the retinas of diabetic rats (a) Representative images of retinal slides stained with DHE (red). (b) Quantification of the relative fluorescence of DHE in the retinas. (c-d) The ratio of GSH/GSSG and the level of MDA in the retinas. (e–h) The protein levels of NLRP3, cleaved caspase-1, and IL-1β in the retinas by western blot. All data were expressed as means ± SD. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

MLN4924 Promoted Nrf2 Accumulation by Downregulating the Neddylation of Cullin3 and Preventing Nrf2 Ubiquitination in the Diabetic Rats’ Retinas

Nrf2, regulated by CRL3, controls the expression of antioxidant genes that scavenge ROS, such as NQO1. Cullin3 neddylation is critical for the ubiquitin ligase function of CRL3[18]. Therefore, we evaluated Cullin3 neddylation level and the protein expression of Nrf2 and NQO1 in retinas to explore the mechanism by which MLN4924 decreases ROS. Western blot analysis revealed that the expression of Nrf2 and NQO1 proteins was reduced in DM retinas relative to control retinas but increased in response to MLN4924 treatment. Cullins neddylation, Cullin3 neddylation, and Nrf2 ubiquitination were all greater in the retinas of DM rats compared to those of control rats, and these trends were attenuated in DM + MLN4924 group (Fig. 3a-3f). To rule out transcriptional regulation by MLN4924, we also determined the transcriptional levels of Nrf2 mRNA using qRT-PCR. Primer sequences: Nrf2 F: 5'- TGCCCACATTCCCAAACAAG-3' and R: 5'- GCTATCGAGTGACTGAGCCT-3'. Figure 3g showed that there was no significant difference of Nrf2 mRNA level between DM and DM + MLN4924 group. This means that the increased protein level of Nrf2 after MLN4924 treatment is not due to the changes of its transcription. In sum, these results suggested that MLN4924 prevented the neddylation of Cullin3, impeded the ubiquitination of Nrf2, and stabilized Nrf2 in diabetic rat retinas to inhibit ROS-induced oxidative stress and inflammation.

Fig. 3.

Fig. 3

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MLN4924 promoted Nrf2 accumulation by downregulating neddylation of Cullin3 and inhibiting ubiquitination of Nrf2 in the diabetic rats’ retinas (a) Representative bands of Ubi-Nrf2, NEDD8-Culs, NQO1, NEDD8-Cul3, and Nrf2 in the retinas by western blot. (b-f) Quantification of the relative protein expressions. All data were expressed as means ± SD. (g) Relative mRNA level of Nrf2 in the retinas by qRT-PCR. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Culs: Cullins; Cul3: Cullin3; Ubi: ubiquitination

Nrf2 Signaling was Involved in the Protective Impact of MLN4924 on AGEs-Induced BRB Disruption In Vitro

We examined the effect of MLN4924 on BRB function in vitro by stimulating confluent endothelial monolayers to AGEs and treating them with MLN4924. The BRB is formed by the tight junctions between retinal endothelial cells to prevent permeability of substances between the retina and circulating blood. Alterations in tight junctions such as occludin, affect the BRB function. Therefore, the expression of occludin was employed to evaluate the BRB function in vitro. We found that endothelial border staining of tight junction occludin (green) was reduced and discontinued in AGEs group, and MLN4924 attenuated AGEs-induced decrease and discontinuity of occludin staining as shown in confocal images (Fig. 4a). Nevertheless, the protective effect of MLN4924 was undermined when Nrf2 siRNA was transfected into endothelial cells (Fig. 4a). Figure 4b confirmed Nrf2 knockdown via siRNA in endothelial cells using qRT-PCR. Western blot showed that MLN4924 treatment prevented AGEs-induced decrease of occludin in endothelial cells, whereas the effect was compromised when Nrf2 was knocked down in endothelial cells (Fig. 4c). The results of western blot supported the findings of confocal images.

Fig. 4.

Fig. 4

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MLN4924 inhibited AGEs-induced BRB breakdown in vitro via Nrf2 signaling (a) Representative fluorescent pictures of endothelial cells stained with occludin (green). (b) Verification of Nrf2 knockdown via siRNA in endothelial cells using qRT-PCR. (c) Western blot assay for the protein levels of occludin in the cells. (d) The leakage of FITC-dextran in each group. All data were expressed as means ± SD. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

To further confirm the protective effect of MLN4924 on AGEs-induced barrier disruption in endothelial cells, FITC-dextran leakage assay was performed. Figure 4d showed that AGEs induced about threefold increase in the permeability of FITC-dextran compared to the control, and FITC-dextran permeability was reduced in AGEs + MLN4924 group compared to AGEs group. However, the protective effect of MLN4924 was undermined when Nrf2 was knocked down in endothelial cells. These results combined with the results of occludin indicated that MLN4924 attenuated AGEs-induced BRB breakdown in endothelial cells, and that Nrf2 signaling was involved in the protective effects of MLN4924.

MLN4924 Inhibited ROS-Induced Oxidative Stress and Inflammation in AGEs-Induced Endothelial Cells Via Nrf2 Signaling In Vitro

As in vivo, the oxidative status and NLRP3 inflammasome activation were accessed to evaluate the protective effect of MLN4924 on AGEs-induced endothelial cells in vitro. As shown in Fig. 5a-c, ROS production was greater in AGEs-induced endothelial cells compared to the control, which was decreased following MLN4924 treatment. In addition, the MDA level and the activation of NLRP3 inflammasome was elevated and the GSH/GSSG ratio was decreased significantly in AGEs-induced endothelial cells relative to the control (Fig. 5d-5i). In contrast, MLN4924 significantly increased the ratio of GSH/GSSG, reduced MDA levels, and suppressed the activation of NLRP3 inflammasome relative to the AGEs group. Furthermore, endothelial cells were transfected with Nrf2 siRNA to validate the critical function of Nrf2 in the regulation of oxidative stress by MLN4924. It was observed that the beneficial effect of MLN4924 was compromised when endothelial cells were transfected with Nrf2 siRNA. These results showed that Nrf2 signaling was involved in the protective effects of MLN4924 on AGEs-induced endothelial cells.

Fig. 5.

Fig. 5

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MLN4924 inhibited ROS-induced oxidative stress and NLRP3 inflammasome activation in AGEs-induced endothelial cells via Nrf2 signaling in vitro (a-c) Representative images and flowcytometry of DHE-stained (red) endothelial cells showing ROS level in each group. Scale bar: 100 µm. (d-e) The ratio of GSH/GSSG and the level of MDA in each group. (f-i) Western blot assay for the protein expressions of NLRP3, cleaved-caspase-1, and IL-1β in each group. All data were expressed as means ± SD. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

MLN4924 Stabilized Nrf2 by Downregulating the Neddylation of Cullin3 to Prevent Ubiquitination of Nrf2 In Vitro

Endothelial cell levels of Cullin3 neddylation, Nrf2 ubiquitination, and Nrf2 signaling proteins were assessed to investigate the mechanism by which MLN4924 regulate Nrf2 signaling to control ROS levels. Figure 6 showed that following MLN4924 treatment, the elevated levels of Cullin3 neddylation and Nrf2 ubiquitination in the AGEs-induced endothelial cells were significantly down-regulated. MLN4924 treatment inhibited ubiquitination-mediated Nrf2 degradation, thus stabilizing Nrf2 and upregulated its target gene NQO1 in endothelial cells. Nrf2 siRNA abrogated MLN4924-mediated elevation of NQO1 in endothelial cells. These findings showed that MLN4924 inhibited Cullin3 neddylation to prevent Nrf2 ubiquitination, thereby stabilizing Nrf2.

Fig. 6.

Fig. 6

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MLN4924 upregulated antioxidant capacity in AGEs-induced endothelial cells by downregulation Cullin3 neddylation to inhibit Nrf2 ubiquitination and promote its accumulation (a) Representative western blot bands of NEDD8-Cul3, Ubi-Nrf2, Nrf2, and NQO1 in each group. (b-e) Quantify the protein expression of NEDD8-Cul3, Ubi-Nrf2, Nrf2, and NQO1 in each group. All data were expressed as means ± SD. ns, not significant, *P < 0.05, ***P < 0.001, and ****P < 0.0001. Culs: Cullins; Cul3: Cullin3; Ubi: ubiquitination

Discussion

This study showed that Cullin3 neddylation was upregulated in the diabetic rats’ retinas and AGEs-induced endothelial cells. Inhibition Cullin3 neddylation with MLN4924 alleviated ROS-induced oxidative injury and inflammation to prevent BRB breakdown in DR by suppressing Nrf2 ubiquitination.

Oxidative stress and inflammation are two major contributors to the development of DR [3]. Chronic hyperglycemia leads to an elevated level of ROS while simultaneously reducing antioxidant capacity [7]. The retina, being an organ with high energy requirements, is particularly susceptible to and prone to injury from elevated ROS. Oxidative stress arises from an imbalance between ROS production and the ability of the retina to counteract them with antioxidants. GSH, the main small molecule antioxidant in cells, is transformed into the oxidized form GSSG upon reacting with ROS [19]. The GSH/GSSG ration and the MDA level are often used as indicators of oxidative stress [19]. Our investigation revealed that both the diabetic rats’ retinas and AGEs-induced endothelial cells exhibited a decreased ratio of GSH/GSSG and an increased level of MDA compared to the control. Meanwhile, elevated ROS activated NLRP3 inflammasome both in diabetic rats’ retinas and AGEs-induced endothelial cells, which leads to caspase-1 dependent release of the pro-inflammatory cytokine IL-1β. The elevated pro-inflammatory cytokines and oxidative stress-induced damages in retinal endothelial cells were reported to contribute to BRB breakdown [5]. We also found that AGEs induced BRB breakdown, indicated by elevated FITC-dextran leakage and decreased tight junction occludin compared to the control. This was aligned with our in vivo data, which indicated increased Evans Blue leakage in DM rats’ retinas compared to the controls.

ROS level was regulated by Nrf2 signaling through modulating its downstream antioxidant proteins, such as NQO1 [7]. Numerous studies provide persuasive evidence for the critical role of the Nrf2 signaling in the pathophysiology of DR [8, 20]. Diabetic animals with Nrf2 knockouts had higher levels of ROS and more severe retinal damage than wild-type [20, 21]. In retinal endothelial cells, glucose-induced dysfunction of Nrf2 signaling can be reversed by Nrf2 upregulation after tBHQ (tert-Butylhydroquinone) treatment [22]. There is also a study showing that activation of the Nrf2 pathway enhances vascular integrity, improves endothelial function, and decreases vascular permeability [23]. Here, we showed that Nrf2 signaling was impaired both in the DM rats’ retinas and in AGEs-induced endothelial cells. This is consistent with other studies, which indicated that DR is characterized by impaired Nrf2 pathway and increased oxidative stress within the retina [7].

Prior research of investigating antioxidant medications to mitigate DR directly targets the Nrf2 pathway. However, the potential of regulating its upstream modulation mechanism, Cullin3 neddylation, has not yet been studied. Cullin 3 functions as a scaffolding protein and acts as the substrate adaptor protein that interacts with Nrf2. NEDD8 conjugation to Cullin3 results in the activation of CRL3, which ubiquitinates Nrf2 for degradation [24, 25]. Here we showed that Cullin3 neddylation and Nrf2 ubiquitination was enhanced, while Nrf2 signaling was inactivated in the diabetic rats’ retinas and AGEs-induced endothelial cells, comparing to the control. Inhibiting the neddylation of Cullin3 with MLN4924, a small molecule inhibitor of NEDD8 activating enzyme, decreased Nrf2 ubiquitination and stabilized Nrf2 to combat oxidative stress. As a result, retinal morphology and BRB function was improved. To further confirm that MLN4924's protective impact in DR occurs via the Nrf2 pathway, Nrf2 was knocked down using siRNA in endothelial cells. Our findings indicated that Nrf2 depletion eliminated the protective effects of MLN4924 on BRB, the upregulation of NQO1 expression, the elevation of the GSH/GSSG ratio, and the suppression of ROS and MDA production. Based on these findings, we postulate that MLN4924 safeguards retinas against ROS-induced injury by impeding Cullin3 neddylation to stabilize Nrf2.

Collectively, our study demonstrated that Cullin3 neddylation was upregulated in DR, which promoted Nrf2 ubiquitination and inactivated Nrf2 signaling, thus facilitating ROS-induced BRB breakdown and lead to DR. Inhibiting Cullin3 neddylation with MLN4924 alleviated DR by stabilizing Nrf2 to suppress ROS-induced oxidative damage and inflammation. The findings of this study indicate that targeting Cullin3 neddylation may be a promising treatment option for DR.

Acknowledgements

This work was supported by the Scholarship for Overseas Studies of Jiangsu Province, Youth Cultivation Project of Drum Tower Hospital National Science Foundation (2024-JCYJ-QP-72), The Natural Science Foundation of Jiangsu Province (BK20240245), Jiangsu Funding Program for Excellent Postdoctoral Talent (2022ZB702).

Author Contributions

Y.C. wrote the main manuscript. Y.C., J.T., and C.L. made contributions to the acquisition and interpretation of data. C.H. and X.L. made contributions to the methodology and data curation. J. X. analyzed the data. C.X. and L.S. revised the manuscript and did the investigation of the work. Z.X. and G.Y. designed and administrated the work. All authors reviewed and approved the manuscript.

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval

Animal experiments were complied with ethical standards of the Animal Care and Welfare Committee of Nanjing Drum Tower Hospital.

Competing Interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Yueqin Chen, Cong Liu, and Jun Tong contributed equally to this work and should be considered co-first authors.

Zhenggao Xie, Lingyun Sun, Genhong Yao, and Chunyan Xue contributed equally to this work and should be considered co-corresponding authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

No datasets were generated or analysed during the current study.

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