MCC950 targets the ROS-NEK7-NLRP3 axis to improve type 2 diabetic retinopathy

MCC950 targets the ROS-NEK7-NLRP3 axis to improve type 2 diabetic retinopathy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article MCC950 targets the ROS-NEK7-NLRP3 axis to improve type 2 diabetic retinopathy kexuan ren, xiaofeng li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6625531/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted 14 You are reading this latest preprint version Abstract Globally, type 2 diabetes mellitus (T2DM) constitutes more than 90% of all diabetes cases, with diabetic retinopathy (DR) emerging as the predominant microvascular complication, resulting in vision loss in roughly 30% of affected individuals [ 1 ] . In contrast to type 1 diabetes, DR associated with T2DM is marked by insulin resistance and persistent low-grade inflammation, which may intensify the activation of the NLRP3 inflammasome [ 2 ] . The pathogenesis of diabetic retinopathy is fundamentally driven by the activation of the NLRP3 inflammasome due to redox disequilibrium [ 3 ] .In this study, we elucidate a novel mechanism through which MCC950, a selective inhibitor of NLRP3, mitigates DR via the ROS-NEK7-NLRP3 pathway. Diabetic rats induced by streptozotocin were administered intravitreal injections of MCC950 at varying concentrations (0.01, 0.1, 1, 10 mM). Quantitative assessments revealed that a concentration of 1 mM MCC950 markedly improved retinal histopathological alterations (p < 0.05) and modulated retinal apoptosis and oxidative stress to a considerable degree (p < 0.05). On a mechanistic level, MCC950 suppressed NLRP3 inflammasome activation (manifested by reduced levels of Cleaved Caspase-1, IL-1β, and IL-18, p < 0.001) in a dose-dependent manner by disrupting the interaction between NEK7 and NLRP3. Notably, there was a strong positive correlation between the intensity of ROS fluorescence and the fluorescence expression of NEK7 (r = 0.8857, p < 0.05), with MCC950 treatment significantly lowering retinal ROS levels at the 1 mM concentration. Moreover, pharmacological inhibition of NEK7 potentiated the therapeutic efficacy of MCC950. This research establishes the upregulation of NEK7 mediated by ROS as a critical factor in NLRP3 activation in DR, and provides pioneering evidence for the efficacy of dose-optimized MCC950 therapy targeting this axis. Health sciences/Diseases/Endocrine system and metabolic diseases Health sciences/Diseases/Eye diseases MCC950 concentration gradient diabetic retinopathy ROS-NEK7-NLRP3 axis Type 2 diabetes mellitus vitreous injection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Diabetic retinopathy (DR), the most severe microvascular complication associated with diabetes mellitus, impacts approximately 34.6% of diabetic patients globally, potentially resulting in irreversible visual loss or blindness in its advanced phases [4] . Latest research indicates that the activation of NLRP3 inflammatory vesicles is a pivotal event in the pathogenesis of DR – the mitochondrial dysfunction induced by high glucose levels leads to an excessive production of reactive oxygen species (ROS), which, in turn, activates NLRP3 inflammatory vesicles through a redox-sensitive signaling pathway. This activation then prompts IL-1β/IL-18-mediated chronic retinal inflammation and disrupts the blood-retinal barrier [5] [6] . Of particular note, the interaction mechanism between NEK7, a crucial regulator of NLRP3 assembly, and ROS remains unexplored in the context of DR, offering a significant avenue for the investigation of novel therapeutic targets [7] [8] . MCC950, as a specific NLRP3 inhibitor, has been shown to reduce plaque load by 83% in a mouse atherosclerosis model [9] , but there are still key scientific issues that need to be resolved for its use in DR treatment: i) most of the existing studies have used a fixed dose (usually 10 mg/kg) [10] , and there is a lack of dose-effect studies targeting localized administration to the retina; and ii) the mechanism of the regulation of NEK7-NLRP3 interactions and its interaction with the ROS pathway by MCC950 has not been clarified-NLRP3 and its interaction with the ROS pathway has not been clarified. In this study, we innovatively constructed a vitreous cavity drug delivery model to systematically evaluate the improvement effect of 0.01-10 mM MCC950 gradient treatment on retinal vascular injury in diabetic rats, and revealed for the first time that ROS activates NLRP3 inflammatory vesicles through up-regulating the molecular axis (ROS-NEK7-NLRP3 axis) of NEK7, which provides a theoretical basis for the precise anti-inflammatory treatment. 2. Materials and methods 2.1. Reagents and materials Table 1 Contains information regarding the catalogue number of all reagents,and kits used. categories vendor catalog number dilution ratio Primary antibodies NLRP3 Affinity BF8029 1:1000 (WB) NEK7 Affinity DF4467 1:1000 (WB) 1:100 (IHC) ASC Affinity DF6304 1:1000 (WB) Caspase-1 Affinity AF5418 1:1000 (WB) Cleaved-caspase-1 Affinity AF4005 1:1000 (WB) IL-1beta Affinity AF4006 1:1000 (WB) IL-18 Abmart M027287 1:1000 (WB) Bax Affinity A19684 1:1000 (WB) Bcl-2 Affinity A20777 1:1000 (WB) Tubulin Affinity AF7011 1:1000 (WB) Secondary antibodies goat anti-mouse IgG Abcolonal AS055 1:5000 (WB) goat anti-rabbit IgG Abcolonal AS056 1:5000 (WB) reagents vendor Catalog number Proteinase K Beyotime ST535 Colorimetric TUNEL Apoptosis Assay Kit Beyotime C1091 BCA protein assay kit Beyotime P0010 ROS assay kit Sigma D7008 Reactive oxygen species assay kit Beyotime S0033S RIPA Lysis Buffer Beyotime P0013B Streptozotocin Solarbio S8050 Hematoxylin-Eosin kit Solarbio G1120 MCC950 MCE HY-12815A Phenylmethanesulfonyl fluoride Beyotime ST505 PVDF membrane Millipore ISEQ00010 FAS eye fixative solution Servicebio G1109 Phosphatase inhibitor cocktail Beyotime P1082 Antifade mounting medium Beyotime P0131 FGSuper Sensitive ECL Luminescence Reagent Meilunbio MA0186 2.2. Diabetic rat modeling Male SD rats, specific-pathogen-free and ranging from 6 to 8 weeks of age with a body weight between 230 and 260 grams, were procured from Liaoning Changsheng Biotechnology Co.Ltd.situated in Liaoning, China. Each rat involved in this study was guaranteed to be free of pathogens. The number of rats in each experimental group was decided based on our extensive past experience with comparable experimental protocols, as well as data available from other studies. During each experiment, the rats were allotted to various groups at random. All procedures and the care provided to the animals were conducted in strict adherence to the guidelines set forth by the Animal Care and Use Committee, and with the express approval of the Scientific Investigation Board of Xinhua Hospital, which is affiliated with Dalian University in Dalian, China. Every possible measure was taken to alleviate any distress experienced by the animals. To create a rat model of type 2 diabetes mellitus, the rats underwent a one-week period of acclimatization before being placed on a diet high in sugar and fat (compositionally, 45% of calories from fat) for a duration of 8 weeks. This dietary regimen was applied to all rats except for the normal control group. Subsequently, diabetes was induced through an intraperitoneal injection of streptozotocin (STZ, at a dose of 45mg/kg) following a 12-hour fasting period. A fasting blood glucose level of 16.7 mmol/L or higher, measured 72 hours post-injection, was used as the criterion for the successful induction of type 2 diabetes mellitus (T2DM). To expedite the progression of retinopathy, the high-fat diet was maintained for an additional 8 weeks. Meanwhile, the control group was given standard commercial rat chow and water. Blood glucose levels were periodically monitored using tail-vein blood samples and analyzed with yuwell test strips and a glucometer (manufactured by Yuwell Medical Equipment Co., Ltd., Jiangsu, China). Throughout the study, food and water were provided in standard conditions, with body weight and fasting blood glucose levels recorded every two weeks, respectively. 2.3. Intravitreal injection Eight weeks after intraperitoneal administration of STZ, rats received intravitreal infusions of 2 μL of MCC950 at concentrations of 0, 0.01, 0.1, 1 and 10 mM into the vitreous chambers of both eyes, with each concentration forming its own group. Concurrently, the 0 mM concentration cohort received 2 μL of saline in both eyes as a comparative control. These injections were administered weekly and after four such infusions, all subjects were euthanised and their eyes and retinas were subsequently enucleated for analysis. 2.4. Hematoxylin-eosin (HE) staining Following the induction of anesthesia in the rats with pentobarbital sodium (administered at a dose of 50 mg/kg body weight), immediately after removal of the rat eyeballs, they were immersed in FAS eyeball fixative for at least 24 hours.The specimens were embedded in wax, cooled, and sectioned into slices of 4 micrometers thickness using a microtome ,and the sections were baked in a constant temperature oven at 37°C for 30 minutes. After removal, the sections were immersed in xylene I for 10 minutes, xylene II for 10 minutes and then in different concentrations of alcohol for 5 minutes (75%, 85%, 95% and anhydrous ethanol) for gradient dehydration.These sections were subjected to a pretreatment with HD constant staining solution for one minute, followed by hematoxylin dyeing for ten minutes and a water rinse for one minute. Further processing included brief dips in the constant dye solution for ten seconds with subsequent rinsing for twenty seconds, immersion in the constant dye back blue solution for two minutes followed by a water rinse of twenty seconds. The sections were then treated with 95% ethanol for thirty seconds and eosin Y dye solution for five minutes. Finally, the sections were transferred through anhydrous ethanol (three stages of one minute each), xylenel sulfoxide for ten minutes, and xyleneⅡ for an additional ten minutes to achieve transparency, before being mounted with neutral gum. The specimens were subsequently secured using a neutral adhesive, and subjected to examination under a microscope . Retinal thickness was measured using ImageJ software, including the inner limiting membrane (ILM), nerve fibre layer (NFL), ganglion cell layer (GCL) (quantification of the GCL includes the ILM and NFL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL) and outer nuclear layer (ONL). 2.5. Determination of ROS Following humane euthanasia, the rats' eyes were promptly enucleated. The excised tissues were meticulously embedded in OCT compound, subsequently frozen and sectioned utilizing a cryostat . To assess the retinal ROS levels and oxidative stress,freshly cut retinal cryosections underwent dihydroethidium staining and adhering to the protocol delineated by the manufacturer. Intracellular ROS concentrations were quantified with a ROS assay kit, following the producer's guidelines. After staining, the sections were sealed with antifade mounting medium and immediately examined under a fluorescence microscope. 2.6. TUNEL staining. Cell death was measured using TUNEL, according to the manufacturer’s instructions. Retinal sections from different groups were permeabilized by immersion in permeabilizing solution (dissolved in sodium citrate, freshly prepared) and rinsed twice (rinsing times in 5 minutes). The retinal sections were then stained according to the instructions of the In Situ Apoptosis Detection Kit.The sections were sealed with an antifade mounting medium and immediately observed under a fluorescence microscope. 2.7. Immunofluorescence Paraffin sections of rat retina were dewaxed with xylene, rehydrated in graded alcohol solutions including anhydrous ethanol, 95%, 85% and 75% alcohol, blocked with immunostaining sequestering solution. The antigen was retrieved by incubation at 95 ℃ in antigen retrieval solution (0.01M citrate, pH=6)for 30 mins, after which the slices incubated with primary antibodies (anti-NEK7) at 4℃ overnight, and subsequently incubated with fluorescent-dye conjugated secondary antibodies for 2 h. The sections were sealed with an antifade mounting medium and immediately observed under a fluorescence microscope. 2.8. Western blotting The rats were euthanised at the conclusion of the treatment period, and the eyes were extracted expeditiously. The cornea was then meticulously divided along the corneoscleral rim under the microscope. The lens and vitreous were completely expelled, and ophthalmic forceps were inserted into the subretinal cavity to bluntly separate the sclera between the periphery of the retina and the optic papilla.Total protein was extracted from tissues using RIPA lysis buffer containing protease and phosphatase inhibitors. The retina was de-abraded using a tissue grinding pestle and mortar to obtain supernatant proteins, which were quantified using a BCA protein assay kit. Proteins were separated on a 10-15% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes. After being blocked with 5% skim milk, the membranes were incubated with primary antibodies (anti-NLRP3, anti-NEK7, anti-Caspase1, anti-Cleaved-Caspase1, anti-ASC, anti-IL-18, anti-IL-1beta, anti-BAX, anti-BCL-2, anti-Tubulin) at 4℃ overnight, then incubated with secondary HRP-conjugated antibody for 2 h. Blots were then developed using a chemiluminescent kit according to the manufacturer's instructions.The results were quantified using Image J software (U.S. National Institutes of Health,Bethesda, MD, USA) with Tubulin as an internal control. 2.9. Statistical analysis Statistical analyses were performed using IBM SPSS Statistics 30.0(IBM, Armonk, NY, USA). All results are presented as mean±SEM. One-way ANOVA was applied to compare the data among multiple groups, TUKEY was used for multiple comparisons among sample data, and Spearman was used for correlation analysis. Statistical significance was defined as p < 0.05. Graphs were prepared using GraphPad Prism 10(Graphpad, San Diego, CA, USA). 3. Results 3.1 Changes in body weight and blood glucose in rats Throughout the study period, the body weights and blood glucose levels of the rats were meticulously monitored biweekly. Figure 1A illustrates that the rats in the NC group experienced a steady increase in body weight. Following a 2-week period of acclimatization to the diet, the rats' blood glucose levels remained within the range of 3.6-4.3 mmol/L. As the rats continued on a high-sugar and high-fat diet, the STZ-induced diabetic rats exhibited a more rapid increase in both body weight and blood glucose levels compared to the NC group, with statistically significant differences emerging from the 8th week onwards (p < 0.05). Conversely, immediately after the induction of diabetes via intraperitoneal STZ injection, the diabetic rats' body weight decreased sharply, reaching a level significantly below that of the NC group (p < 0.01, Fig.1A). By the 10th week, a marked rise in the blood glucose levels of the diabetic rats was observed, with all differences being statistically significant (p0.05,Fig.1B). 3.2 Effects of different concentrations of MCC950 on histopathological damage of rat retina With the progression of T2DM, pathological damage occurred in the rat retina.HE staining showed that the retinal thickness in the NC group was significantly higher than that in the other groups (Fig.2 B).STZ-induced diabetic rat retinas underwent pathological damage:structural disorganization of retinal layers, pre-retinal neovascularization and arteriolar thrombosis (Fig.2A), and a decrease in the retinal ganglion cells.However, the thickness of the GCL did not differ significantly among the groups. There was no significant difference between the groups. Cell nuclei (black arrows) were also seen in the T2DM group. The inner plexiform layer (IPL), inner nuclear layer (INL) and outer nuclear layer (ONL) were thinner than in the NC group, and the MCC950 gradient group dose-dependently improved their thickness. The outer plexiform layer (OPL) was thinner than in the NC group, with no significant difference between the groups. Compared with the T2DM group, the structure of the inner plexiform layer (IPL), inner nuclear layer (INL) and outer nuclear layer (ONL) was significantly improved under the treatment of 1 mM concentration of MCC950, and the cells of the IPL and ONL tended to be neatly aligned, and the thickening of the vascular basement membrane and exudative lesions were significantly reduced. 3.3 Effects of different concentrations of MCC950 on apoptosis in rat retinal tissues In order to evaluate the effects of different concentrations of MCC950 on the apoptosis of retinal tissue cells, we detected the apoptosis of retinal tissue cells in rats by TUNEL method. The results showed that the retinal apoptosis in STZ-induced diabetic rats was significantly increased, and the apoptosis of retinal tissue cells caused by long-term hyperglycemia decreased dose-dependently with the increase of MCC950 concentration in the concentration range of 0.01 mM to 1 mM, and the difference of retinal apoptosis under the treatment of MCC950 at the concentration of 10 mM was not statistically significant comparing with that of T2DM group ( Fig.3 A,C).Western blot results showed that Bcl-2 protein expression was up-regulated and Bax protein expression was down-regulated in a gradient of MCC950 concentration, and the Bcl-2/Bax ratio was increased, which further confirmed its anti-apoptotic effect (Fig.3 D-E). 3.4 Effects of Different Concentrations of MCC950 on ROS Expression in Rat Retinal Tissues and Correlation Analysis with NEK7 Expression In order to verify whether MCC950 can block the NEK7/NLRP3 pathway in diabetic retinopathy by binding specifically with NLRP3 and thus achieve the therapeutic effect, we used immunofluorescence localization (Fig.4 A) to analyze the NEK7 expression level in the retina. The results showed that in the NC group, the NEK7 immunofluorescence signal (green) was mainly localized in the ganglion cell layer (GCL) and the inner nuclear layer (INL) of the retina, and was weakly expressed, i.e., the basal expression level of NEK7 was low in the physiological state. In the T2DM group, the fluorescence signal of NEK7 was significantly enhanced and widely distributed in the ganglion cell layer, inner nuclear layer and perivascular area. Quantitative analysis showed that the fluorescence intensity was significantly higher in the T2DM group than in the NC group (p < 0.001, Fig. 4 B), indicating that diabetic pathology induced the up-regulation of NEK7 expression.In the MCC950 gradient treatment group, the expression level of NEK7 was significantly reduced in the 10 mM concentration group (p < 0.05), and the fluorescence intensity of NEK7 decreased dose-dependently and signal distribution gradually in a range of concentrations from 0.01 mM to 1 mM. dependent decrease, and the signal distribution gradually weakened (Fig.4 B,D,E). This indicates that MCC950 reverses the T2DM-induced abnormal activation of NEK7 by dose-dependently inhibiting NEK7expression. In addition, to verify the protective effect of concentration gradient MCC950 against apoptosis and oxidative damage in the retina of diabetic rats in the T2DM group, we detected the expression of ROS in the retinal tissues of rats in each group and further analyzed the correlation between ROS and NEK7 expression. The results suggested that compared with diabetic rats in the T2DM group, ROS accumulation was reduced under treatment in the 10 mM concentration group, but there was no statistically significant difference; in the concentration range of 0.01 mM to 1 mM, the expression of ROS gradually decreased with the increase of the concentration of MCC950, and the expression was the lowest at the 1 mM concentration (p<0.001), and the difference was statistically significant (Fig. 4 C,D,F) Spearman correlation analysis showed a significant positive correlation between NEK7 fluorescence intensity and retinal ROS levels in rat retinal tissues (p<0.05,r=0.8857, Fig.4 G), suggesting that NEK7 plays an important role in regulating oxidative stress in the retina and verifying the activation of NLRP3 inflammatory vesicles by ROS through upregulation of NEK7. 3.5 Gradient MCC950 Inhibits NEK7/NLRP3 Pathway-Related Protein Expression To verify the effects of different concentrations of MCC950 on the activation of the NEK7/NLRP3 pathway in rat retinal tissues, we examined the expression of NLRP3, NEK7, ASC and other proteins in the retinal tissues of rats in each group by Western-blot. The results suggested that the expression of each protein in diabetic rats in the T2DM group was significantly higher compared with that in the NC group (p<0.05).The concentration gradient of MCC950 significantly inhibited the expression of proteins related to the NLRP3-NEK7 pathway, which was dose-dependently reduced in the concentration range of 0.01 mM to 1 mM, and the differences were all statistically significant (p<0.05,Fig.5 A-D). 3.6 Effect of different concentrations of MCC950 on the expression of inflammatory factors in rat retina To verify the effect of different concentrations on each inflammatory factor in rat retina, the expression of inflammatory factors Caspase-1, Cleaved-Caspase-1, IL-1β and IL-18 in all rat retinal tissues were detected by WB. The results suggested that the expression of inflammatory factors was significantly increased in diabetic rats in the T2DM group compared to NC, and the differences were all statistically significant (p<0.05).The expression of Caspase-1, Cleaved-Caspase-1, IL-1β, and IL-18 was decreased under MCC950 treatment at a concentration of 10 mM (p<0.05); and in the 0.01 to 1 mM concentration range, the release of these inflammatory factors was dose-dependently reduced (all p<0.01, Fig. 5 E-I). 4. Discussion Studies have confirmed that elevated glucose levels can trigger an excessive accumulation of mitochondrial ROS (reactive oxygen species), which in turn promotes the assembly of the NLRP3 inflammasome complex by augmenting the structural compatibility between NEK7 and NLRP3 [14] . This activation of NLRP3-containing inflammatory vesicles not only leads to the subsequent proteolysis of pro-IL-1β and pro-IL-18 but also facilitates the secretion of their respective mature forms. Consequently, this mechanism elicits an upsurge in the expression of IL-1β and IL-18, initiating a chronic inflammatory response that is implicated in the exacerbation of diabetic retinopathy [15] [16] .The experimental evidence clearly demonstrates that MCC950 effectively halts the mentioned pathological processes through a dual-mode mechanism. This mechanism involves (i) directly binding to the NACHT domain of NLRP3, thereby preventing the interaction between NEK7 and NLRP3, and (ii) suppressing mitochondrial ROS production to counteract oxidative modification of NEK7 [17] [25] .As a targeted inhibitor of the NLRP3 pathway, MCC950 has proven to markedly alleviate inflammatory cytokine storms and vascular injury by focusing on the ROS-NEK7-NLRP3 axis, thus offering a precise intervention approach for the treatment of diabetic retinopathy [18] . To begin with, it is confirmed that mitochondrial ROS (reactive oxygen species) serve as activators for NEK7-NLRP3 inflammatory vesicles [19] . Western blot findings revealed a marked elevation in the expression of NEK7 within the retinal tissues of diabetic rodents, with a notable positive association between NEK7 expression levels and the fluorescence intensity indicative of mitochondrial ROS [20] . Furthermore, the formation of NLRP3 inflammatory vesicles was detected, and immunofluorescence staining localized NEK7 to aggregated clusters within the retinal vascular endothelial cells, concurrent with an increase in ASC oligomerization. Secondly, evidence has clearly shown that the activation of NRLP3 triggers an inflammatory response, ultimately resulting in retinal injury. Notably, the levels of cleaved Caspase-1, IL-1β, and IL-18 were heightened in the retinas of the diabetic cohort, yet these were effectively mitigated by the application of MCC950 at diverse concentrations. HE staining outcomes validated the rejuvenation of retinal layer architecture, a restoration that appeared to beboth dose-dependent and a consequence of MCC950 treatment.Furthermore, this study delves into the twin inhibitory actions of MCC950. It directly impedes the NEK7-NLRP3 interaction: The western blot findings revealed that MCC950 potently inhibits NEK7 from binding to NLRP3 in a manner proportional to the drug concentration. This interference likely prevents the structural activation of NLRP3 by blocking the ATP-binding site within its NACHT domain. The elimination of mitochondrial ROS (reactive oxygen species) is a topic of great significance. The ROS levels in retinal mitochondria were notably diminished following treatment with MCC950.Subsequently, a noteworthy occurrence of synergistic inhibition between apoptosis and inflammation has been identified. TUNEL and Apoptotic Pathway: An elevated rate of apoptosis in retinal tissue cells was documented within the T2DM cohort, whereas a marked decrease in apoptosis was evident in the MCC950-treated group. Through Western blotting, it was determined that MCC950 enhanced the expression of the anti-apoptotic protein Bcl-2 and diminished the levels of the pro-apoptotic protein Bax, effectively restoring the Bcl-2/Bax ratio to its normal state. Our investigation has elucidated that MCC950 exhibits a maximal efficacy threshold within the concentration bracket of 0.01-10 mM, indicating a saturation effect. This discovery lays a solid foundation for fine-tuning the dosage of intravitreal injections. The compound's dual mode of action—blocking NLRP3 complex assembly and neutralizing reactive oxygen species (ROS)— orchestrates a potent interruption of the inflammation-oxidative stress feedback loop, surpassing the efficacy of single-site inhibitors such as the NLRP3-specific monoclonal antibody, which has been documented to decrease vascular permeability by merely 40% [21] 5. Conclusions In the course of this investigation, we discovered that the intravitreal administration of MCC950 markedly mitigated the retinal pathological injury in diabetic rats by suppressing the NEK7/NLRP3 signaling pathway.Additionally, it exerted inhibitory effects on oxidative stress, inflammation, and cellular apoptosis. Moreover, a distinct dose-response relationship was observed for the therapeutic efficacy of MCC950 on diabetic retinopathy (DR) within a specific concentration spectrum. Our findings also underscore the integral role of the ROS-NEK7-NLRP3 axis in the pathogenesis of DR, as evidenced by the significant positive correlation between NEK7 and reactive oxygen species (ROS). This study reveals the pivotal role of the ROS-NEK7-NLRP3 axis in diabetic retinopathy (DR). Hyperglycemia-induced mitochondrial oxidative stress triggers NEK7 upregulation, which in turn promotes NLRP3 inflammasome assembly [22] [23] .This process ultimately leads to IL-1β/IL-18-mediated retinal vascular injury [24] [25] .The present study has demonstrated the dual inhibitory effects of intravitreal delivery of MCC950. Within a certain concentration range, MCC950 has been shown to disrupt the NLRP3-ASC interaction, as well as to suppress NEK7 expression and ROS accumulation. Furthermore, a robust positive correlation has been observed between NEK7 and ROS. These findings establish the first dose-optimised therapeutic regimen for DR and identify targetable nodes of the redox-inflammatory crosstalk, paving the way for developing NEK7/NLRP3 dual-pathway inhibitors. This comprehensive study has uncovered the critical importance of the ROS-NEK7-NLRP3 signaling axis in the development and progression of diabetic retinopathy (DR). The research delves into the molecular mechanisms by which hyperglycemia, a hallmark of diabetes, induces mitochondrial oxidative stress. This stress, in turn, activates the expression of NEK7, a kinase that plays a crucial role in cell division and the cell cycle. The upregulation of NEK7 facilitates the assembly of the NLRP3 inflammasome, a multiprotein complex that is central to the innate immune response. The activation of this inflammasome leads to the maturation and release of pro-inflammatory cytokines such as interleukin-1 beta (IL-1β) and interleukin-18 (IL-18), which are known to cause significant damage to the retinal vasculature [27] [32] . The study has meticulously examined the effects of MCC950, a potent and selective inhibitor of the NLRP3 inflammasome, when administered intravitreally. It has been observed that within a specific concentration range, MCC950 effectively disrupts the interaction between NLRP3 and ASC (apoptosis-associated speck-like protein containing a CARD), a critical step in the activation of the inflammasome [31] [33] . Additionally, the compound has been shown to suppress the expression of NEK7 and reduce the accumulation of reactive oxygen species (ROS), which are byproducts of oxidative stress and contribute to cellular damage. We have also highlighted a strong positive correlation between the levels of NEK7 and ROS, suggesting that the two factors may mutually reinforce each other's effects, thereby exacerbating the inflammatory response and tissue damage in diabetic retinopathy. This correlation underscores the importance of targeting both NEK7 and ROS in the treatment of DR. [18] The findings of this study are groundbreaking as they establish the first dose-optimized therapeutic regimen for diabetic retinopathy. By identifying the key nodes of interaction between oxidative stress and inflammation, the research paves the way for the development of novel dual-pathway inhibitors that target both NEK7 and NLRP3 [28] .Such inhibitors could potentially offer a more effective treatment strategy for DR, addressing both the inflammatory and oxidative components of the disease. This could lead to significant improvements in patient outcomes by reducing the incidence and severity of vision-threatening complications associated with diabetic retinopathy. [29] [30] Declarations Ethics approval and consent to participate The study was approved by the Scientific Investigation Board of Xinhua Hospital Affiliated to Dalian University, and was performed in accordance with relevant guidelines and regulations. Funding This study was supported by the Liaoning Provincial Department of Education Science and Technology Research Program (No. LJKZ1193). Author contributions Xiaofeng Li and Kexuan Ren designed the study, revised the manuscript accordingly. Kexuan Ren performed all experiments,analyzed the data.All authors have read and approved the final manuscript. Ethics approval and consent to participate Clinical trial number: not applicable. Laboratory Animal Ethical Review Number:2025-018-01 Competing interest The authors declare that they have no conflict of interest. Data availability Data will be made available on request. The datasets generated and analyzed during the current study are available from the first author upon reasonable request. Acknowledgments We thank the Xinhua Hospital Affiliated to Dalian University for providing the scientific research platform. References Pan, C. W., Wang, S., Xu, C. L., & Song, E. (2018). Combined effect of glycemic and blood pressure control on diabetic retinopathy among Chinese with type-2 diabetes mellitus. Diabetology & metabolic syndrome, 10, 73. https://doi.org/10.1186/s13098-018-0377-7 Accili, D., Deng, Z. & Liu, Q. Insulin resistance in type 2 diabetes mellitus. Nat Rev Endocrinol (2025). https://doi.org/10.1038/s41574-025-01114-y Zheng, X., Wan, J., & Tan, G. (2023). The mechanisms of NLRP3 inflammasome/pyroptosis activation and their role in diabetic retinopathy. 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Cite Share Download PDF Status: Published Journal Publication published 23 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 27 Jun, 2025 Reviews received at journal 27 Jun, 2025 Reviewers agreed at journal 26 Jun, 2025 Reviews received at journal 16 Jun, 2025 Reviewers agreed at journal 15 Jun, 2025 Reviews received at journal 14 Jun, 2025 Reviewers agreed at journal 06 Jun, 2025 Reviewers agreed at journal 05 Jun, 2025 Reviewers agreed at journal 05 Jun, 2025 Reviewers invited by journal 04 Jun, 2025 Editor assigned by journal 04 Jun, 2025 Editor invited by journal 26 May, 2025 Submission checks completed at journal 23 May, 2025 First submitted to journal 09 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6625531","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":467358892,"identity":"bf1532c5-417d-437d-9099-b870d5e53ce1","order_by":0,"name":"kexuan ren","email":"","orcid":"","institution":"Xinhua Hospital of Dalian University","correspondingAuthor":false,"prefix":"","firstName":"kexuan","middleName":"","lastName":"ren","suffix":""},{"id":467358894,"identity":"82730e33-3e7f-4602-950e-cadf63f509d7","order_by":1,"name":"xiaofeng li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIie3QIQ6DUAyA4b68pJgXsCRb4AqQJcvkjlLMUHgkmIfhAMPsGNMPsxluMMNuMPnchp1YUtxEP90/aQsgxB8KNcCLPCYYdI6XoAZ1fvbhLjQTMRMANcyYFJf4mDGTwOQNmX1pYyDw9ZWzGBYNHU6V3bRO9dODk+j7TOZW2a0jrSwrUW1D+C4xpoyb6HFJkNYkyy1Fj7ldnjyybomiKW+9xzTtunH2NSP54lbOCyGE+OUDZac12ghFZCIAAAAASUVORK5CYII=","orcid":"","institution":"Xinhua Hospital of Dalian University","correspondingAuthor":true,"prefix":"","firstName":"xiaofeng","middleName":"","lastName":"li","suffix":""}],"badges":[],"createdAt":"2025-05-09 06:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6625531/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6625531/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-18438-4","type":"published","date":"2025-09-23T15:57:33+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84325253,"identity":"2224bf61-75c0-4ea0-adcd-16a3d8f81621","added_by":"auto","created_at":"2025-06-10 15:00:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":72631,"visible":true,"origin":"","legend":"\u003cp\u003eBody weight and blood glucose changes in STZ-induced type 2 diabetic SD rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eBody weights of rats in different groups. \u003cstrong\u003e(B)\u003c/strong\u003e Blood glucose of rats in different groups. glucose levels in rats.Data are shown as mean±SEM,n = 3 per group. Compared with NC group: *p \u0026lt; 0.05, **p \u0026lt; 0.01,***p \u0026lt; 0.001;NC: normal control group. 0mM：T2DM group；0.01、0.1、1、10mM: levels in T2DM treated with MCC950 at different concentrations (0.01,0.1,1,10mM).\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6625531/v1/5fc8ac3960a821de870e14cd.jpg"},{"id":84325255,"identity":"2b35e544-1578-4d34-bed7-3556fa12c06a","added_by":"auto","created_at":"2025-06-10 15:00:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":398459,"visible":true,"origin":"","legend":"\u003cp\u003ePathologic structural changes in the retinal tissues of different groups of rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003ePhotographs of HE staining of rats in different groups.\u003cstrong\u003e(B)\u003c/strong\u003eAnalysis of the thickness of the subretinal layers.Data represents mean±SEM, n = 3 per group, Scale bar: 10 μm. Compared with NC group: *p \u0026lt; 0.05;Compared with T2DM group: #p \u0026lt; 0.05, ##p \u0026lt; 0.01. NC: normal control group.0mM：T2DM group；0.01、0.1、1、10mM: levels in T2DM treated with MCC950 at different concentrations ( 0.01,0.1,1,10mM).\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6625531/v1/b2a28853fd770e51b9317fce.jpg"},{"id":84325256,"identity":"090d0162-e9ce-414c-89ff-c66a5e49647c","added_by":"auto","created_at":"2025-06-10 15:00:50","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":647534,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of MCC950 on STZ-induced apoptosis in retinal tissues of type 2 diabetic SD rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A,C)\u003c/strong\u003eDetection of apoptotic cells in different groups of rat retina by TUNEL method .\u003cstrong\u003e (B-E)\u003c/strong\u003eDisplayed is the expression of Bax and Bcl-2 in rat retina in different groups. Tubulin served as an endogenous reference for normalization.Data represents mean±SEM, n = 3 per group, Scale bar: 50μm. Compared with NC group: *p \u0026lt; 0.05;Compared with T2DM group: #p \u0026lt; 0.05. NC: normal control group.0mM：T2DM group；0.01、0.1、1、10mM: levels in T2DM treated with MCC950 at different concentrations (0.01,0.1,1,10mM).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6625531/v1/671298be65f75dcad104df29.jpeg"},{"id":84325258,"identity":"f7b7a28a-0b3e-4da6-a362-3124ac1a159d","added_by":"auto","created_at":"2025-06-10 15:00:50","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":978710,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the fluorescence intensity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-F)\u003c/strong\u003eImmunofluorescence analysis of the expression of NEK7 and the levels of ROS in rat retina in different groups .\u003cstrong\u003e(G)\u003c/strong\u003eCorrelation analysis between ROS and NEK7 in retina of mice. n=3 per group; Data represents mean±SEM, n = 3 per group, Scale bar: 50μm. Compared with NC group: *p \u0026lt; 0.05;Compared with T2DM group: #p \u0026lt; 0.05. NC: normal control group.0mM：T2DM group；0.01、0.1、1、10mM: levels in T2DM treated with MCC950 at different concentrations (0.01,0.1,1,10mM).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6625531/v1/21e549d3df58b7827e7b29b5.jpeg"},{"id":84325261,"identity":"906f758f-de17-40df-93e0-66db50191e61","added_by":"auto","created_at":"2025-06-10 15:00:51","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":637246,"visible":true,"origin":"","legend":"\u003cp\u003eMCC950 inhibited the activation of NLRP3 inflammasome by western blot analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-D)\u003c/strong\u003eThe expression of NLRP3、NEK7 and ASC in rat retina in different groups. \u003cstrong\u003e(E-I)\u003c/strong\u003eThe expression of inflammatory factor Caspase1 and Cleaved-Caspase1、IL-18 and IL-1beta in rat retina in different groups.Tubulin served as an endogenous reference for normalization.Data represents mean±SEM, n = 3 per group. Compared with NC group: *p \u0026lt; 0.05;Compared with T2DM group: #p \u0026lt; 0.05. NC: normal control group.0.01、0.1、1、10mM: levels in T2DM treated with MCC950 at different concentrations (0.01,0.1,1,10mM).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6625531/v1/764fd38691c76701e5a82aca.jpeg"},{"id":92430468,"identity":"348cb404-7309-4492-b756-4eb1f0c3e2cf","added_by":"auto","created_at":"2025-09-29 16:05:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3253031,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6625531/v1/84cb5ebc-a9a4-4cf5-9443-16f165b2bb84.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"MCC950 targets the ROS-NEK7-NLRP3 axis to improve type 2 diabetic retinopathy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDiabetic retinopathy (DR), the most severe microvascular complication associated with diabetes mellitus, impacts approximately 34.6% of diabetic patients globally, potentially resulting in irreversible visual loss or blindness in its advanced phases\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e[4]\u003c/sup\u003e.\u0026nbsp;Latest research indicates that the activation of NLRP3 inflammatory vesicles is a pivotal event in the pathogenesis of DR\u0026nbsp;\u0026ndash;\u0026nbsp;the mitochondrial dysfunction induced by high glucose levels leads to an excessive production of reactive oxygen species (ROS), which, in turn, activates NLRP3 inflammatory vesicles through a redox-sensitive signaling pathway. This activation then prompts IL-1\u0026beta;/IL-18-mediated chronic retinal inflammation and disrupts the blood-retinal barrier\u0026nbsp;\u003csup\u003e[5]\u003c/sup\u003e\u003csup\u003e[6]\u003c/sup\u003e. Of particular note, the interaction mechanism between NEK7, a crucial regulator of NLRP3 assembly, and ROS remains unexplored in the context of DR, offering a significant avenue for the investigation of novel therapeutic targets\u003csup\u003e[7]\u003c/sup\u003e\u003csup\u003e[8]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMCC950, as a specific NLRP3 inhibitor, has been shown to reduce plaque load by 83% in a mouse atherosclerosis model\u003csup\u003e[9]\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e but there are still key scientific issues that need to be resolved for its use in DR treatment: i) most of the existing studies have used a fixed dose (usually 10 mg/kg)\u0026nbsp;\u003csup\u003e[10]\u003c/sup\u003e, and there is a lack of dose-effect studies targeting localized administration to the retina; and ii) the mechanism of the regulation of NEK7-NLRP3 interactions and its interaction with the ROS pathway by MCC950 has not been clarified-NLRP3 and its interaction with the ROS pathway has not been clarified. In this study, we innovatively constructed a vitreous cavity drug delivery model to systematically evaluate the improvement effect of 0.01-10 mM MCC950 gradient treatment on retinal vascular injury in diabetic rats, and revealed for the first time that ROS activates NLRP3 inflammatory vesicles through up-regulating the molecular axis (ROS-NEK7-NLRP3 axis) of NEK7, which provides a theoretical basis for the precise anti-inflammatory treatment.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e2.1. Reagents and materials\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eContains information regarding the catalogue number of all reagents,and kits used.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"567\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ecategories\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003evendor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ecatalog number\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e\u003cstrong\u003edilution ratio\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003ePrimary antibodies\u003c/p\u003e\n \u003cp\u003eNLRP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eAffinity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eBF8029\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1:1000 (WB)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eNEK7\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eAffinity\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eDF4467\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e1:1000 (WB)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1:100 (IHC)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eASC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eAffinity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eDF6304\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e1:1000 (WB)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eCaspase-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eAffinity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eAF5418\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e1:1000 (WB)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eCleaved-caspase-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eAffinity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eAF4005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e1:1000 (WB)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eIL-1beta\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eAffinity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eAF4006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e1:1000 (WB)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eIL-18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eAbmart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eM027287\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e1:1000 (WB)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eBax\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eAffinity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eA19684\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e1:1000 (WB)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eBcl-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eAffinity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eA20777\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e1:1000 (WB)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eTubulin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eAffinity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eAF7011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e1:1000 (WB)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eSecondary antibodies\u0026nbsp;\u003c/p\u003e\n \u003cp\u003egoat anti-mouse IgG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eAbcolonal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eAS055\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e1:5000 (WB)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003egoat anti-rabbit IgG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eAbcolonal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eAS056\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e1:5000 (WB)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 276px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ereagents\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003evendor\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCatalog number\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 276px;\"\u003e\n \u003cp\u003eProteinase K\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eBeyotime\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003eST535\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 276px;\"\u003e\n \u003cp\u003eColorimetric TUNEL Apoptosis Assay Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eBeyotime\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003eC1091\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 276px;\"\u003e\n \u003cp\u003eBCA protein assay kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eBeyotime\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003eP0010\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 276px;\"\u003e\n \u003cp\u003eROS assay kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eSigma\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003eD7008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 276px;\"\u003e\n \u003cp\u003eReactive oxygen species assay kit\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eBeyotime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003eS0033S\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 276px;\"\u003e\n \u003cp\u003eRIPA Lysis Buffer\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eBeyotime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003eP0013B\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 276px;\"\u003e\n \u003cp\u003eStreptozotocin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eSolarbio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003eS8050\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 276px;\"\u003e\n \u003cp\u003eHematoxylin-Eosin kit\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eSolarbio\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003eG1120\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 276px;\"\u003e\n \u003cp\u003eMCC950\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eMCE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003eHY-12815A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 276px;\"\u003e\n \u003cp\u003ePhenylmethanesulfonyl fluoride \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eBeyotime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003eST505\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 276px;\"\u003e\n \u003cp\u003ePVDF membrane \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eMillipore\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003eISEQ00010\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 276px;\"\u003e\n \u003cp\u003eFAS eye fixative solution\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eServicebio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003eG1109\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 276px;\"\u003e\n \u003cp\u003ePhosphatase inhibitor cocktail \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eBeyotime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003eP1082\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 276px;\"\u003e\n \u003cp\u003eAntifade mounting medium\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eBeyotime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003eP0131\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 276px;\"\u003e\n \u003cp\u003eFGSuper Sensitive ECL Luminescence Reagent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eMeilunbio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003eMA0186\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e2.2. \u0026nbsp;Diabetic rat modeling\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale SD rats, specific-pathogen-free and ranging from 6 to 8 weeks of age with a body weight between 230 and 260 grams, were procured from Liaoning Changsheng Biotechnology Co.Ltd.situated in Liaoning, China. Each rat involved in this study was guaranteed to be free of pathogens. The number of rats in each experimental group was decided based on our extensive past experience with comparable experimental protocols, as well as data available from other studies. During each experiment, the rats were allotted to various groups at random. All procedures and the care provided to the animals were conducted in strict adherence to the guidelines set forth by the Animal Care and Use Committee, and with the express approval of the Scientific Investigation Board of Xinhua Hospital, which is affiliated with Dalian University in Dalian, China. Every possible measure was taken to alleviate any distress experienced by the animals.\u003c/p\u003e\n\u003cp\u003eTo create a rat model of type 2 diabetes mellitus, the rats underwent a one-week period of acclimatization before being placed on a diet high in sugar and fat (compositionally, 45% of calories from fat) for a duration of 8 weeks. This dietary regimen was applied to all rats except for the normal control group. Subsequently, diabetes was induced through an intraperitoneal injection of streptozotocin (STZ, at a dose of 45mg/kg) following a 12-hour fasting period. A fasting blood glucose level of 16.7 mmol/L or higher, measured 72 hours post-injection, was used as the criterion for the successful induction of type 2 diabetes mellitus (T2DM). To expedite the progression of retinopathy, the high-fat diet was maintained for an additional 8 weeks. Meanwhile, the control group was given standard commercial rat chow and water. Blood glucose levels were periodically monitored using tail-vein blood samples and analyzed with yuwell test strips and a glucometer (manufactured by Yuwell Medical Equipment Co., Ltd., Jiangsu, China). Throughout the study, food and water were provided in standard conditions, with body weight and fasting blood glucose levels recorded every two weeks, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.3. Intravitreal injection\u003c/p\u003e\n\u003cp\u003eEight weeks after intraperitoneal administration of STZ, rats received intravitreal infusions of 2 \u0026mu;L of MCC950 at concentrations of 0, 0.01, 0.1, 1 and 10 mM into the vitreous chambers of both eyes, with each concentration forming its own group. Concurrently, the 0 mM concentration cohort received 2 \u0026mu;L of saline in both eyes as a comparative control. These injections were administered weekly and after four such infusions, all subjects were euthanised and their eyes and retinas were subsequently enucleated for analysis.\u003c/p\u003e\n\u003cp\u003e2.4. Hematoxylin-eosin (HE) staining\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFollowing the induction of anesthesia in the rats with pentobarbital sodium (administered at a dose of 50 mg/kg body weight), immediately after removal of the rat eyeballs, they were immersed in FAS eyeball fixative for at least 24 hours.The specimens were embedded in wax, cooled, and sectioned into slices of 4 micrometers thickness using a microtome ,and the sections were baked in a constant temperature oven at 37\u0026deg;C for 30 minutes. After removal, the sections were immersed in xylene I for 10 minutes, xylene II for 10 minutes and then in different concentrations of alcohol for 5 minutes (75%, 85%, 95% and anhydrous ethanol) for gradient dehydration.These sections were subjected to a pretreatment with HD constant staining solution for one minute, followed by hematoxylin dyeing for ten minutes and a water rinse for one minute. Further processing included brief dips in the constant dye solution for ten seconds with subsequent rinsing for twenty seconds, immersion in the constant dye back blue solution for two minutes followed by a water rinse of twenty seconds. The sections were then treated with 95% ethanol for thirty seconds and eosin Y dye solution for five minutes. Finally, the sections were transferred through anhydrous ethanol (three stages of one minute each), xylenel sulfoxide for ten minutes, and xyleneⅡ\u0026nbsp;for an additional ten minutes to achieve transparency, before being mounted with neutral gum.\u003c/p\u003e\n\u003cp\u003eThe specimens were subsequently secured using a neutral adhesive, and subjected to examination under a microscope . Retinal thickness was measured using ImageJ software, including the inner limiting membrane (ILM), nerve fibre layer (NFL), ganglion cell layer (GCL) (quantification of the GCL includes the ILM and NFL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL) and outer nuclear layer (ONL).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.5. Determination of ROS\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFollowing humane euthanasia, the rats\u0026apos; eyes were promptly enucleated. The excised tissues were meticulously embedded in OCT compound, subsequently frozen and sectioned utilizing a cryostat . To assess the retinal ROS levels and oxidative stress,freshly cut retinal cryosections underwent dihydroethidium staining and adhering to the protocol delineated by the manufacturer. Intracellular ROS concentrations were quantified with a ROS assay kit, following the producer\u0026apos;s guidelines. After staining, the sections were sealed with antifade mounting medium and immediately examined under a fluorescence microscope.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.6. TUNEL staining.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCell death was measured using TUNEL, according to the manufacturer\u0026rsquo;s instructions. Retinal sections from different groups were permeabilized by immersion in permeabilizing solution (dissolved in sodium citrate, freshly prepared) and rinsed twice (rinsing times in 5 minutes). The retinal sections were then stained according to the instructions of the In Situ Apoptosis Detection Kit.The sections were sealed with an antifade mounting medium and immediately observed under a fluorescence microscope.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.7. Immunofluorescence\u003c/p\u003e\n\u003cp\u003eParaffin sections of rat retina were dewaxed with xylene, rehydrated in graded alcohol solutions including anhydrous ethanol, 95%, 85% and 75% alcohol, blocked with immunostaining sequestering solution. The antigen was retrieved by incubation at 95 ℃ in antigen retrieval solution (0.01M citrate, pH=6)for 30 mins, after which the slices incubated with primary antibodies (anti-NEK7) at 4℃ overnight, and subsequently incubated with fluorescent-dye conjugated secondary antibodies for 2 h. The sections were sealed with an antifade mounting medium and immediately observed under a fluorescence microscope.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.8. Western blotting\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe rats were euthanised at the conclusion of the treatment period, and the eyes were extracted expeditiously. The cornea was then meticulously divided along the corneoscleral rim under the microscope. The lens and vitreous were completely expelled, and ophthalmic forceps were inserted into the subretinal cavity to bluntly separate the sclera between the periphery of the retina and the optic papilla.Total protein was extracted from tissues using RIPA lysis buffer containing protease and phosphatase inhibitors. The retina was de-abraded using a tissue grinding pestle and mortar to obtain supernatant proteins, which were quantified using a BCA protein assay kit. Proteins were separated on a 10-15% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes. After being blocked with 5% skim milk, the membranes were incubated with primary antibodies (anti-NLRP3, anti-NEK7, anti-Caspase1, anti-Cleaved-Caspase1, anti-ASC, anti-IL-18, anti-IL-1beta, anti-BAX, anti-BCL-2, anti-Tubulin) at 4℃\u0026nbsp;overnight, then incubated with secondary HRP-conjugated antibody for 2 h. Blots were then developed using a chemiluminescent kit according to the manufacturer\u0026apos;s instructions.The results were quantified using Image J software (U.S. National Institutes of Health,Bethesda, MD, USA) with Tubulin as an internal control.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2.9. Statistical analysis\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using IBM SPSS Statistics 30.0(IBM, Armonk, NY, USA). All results are presented as mean\u0026plusmn;SEM. One-way ANOVA was applied to compare the data among multiple groups, TUKEY was used for multiple comparisons among sample data, and Spearman was used for correlation analysis. Statistical significance was defined as p \u0026lt; 0.05. Graphs were prepared using GraphPad Prism 10(Graphpad, San Diego, CA, USA).\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1 Changes in body weight and blood glucose in rats\u003c/p\u003e\n\u003cp\u003eThroughout the study period, the body weights and blood glucose levels of the rats were meticulously monitored biweekly. Figure 1A illustrates that the rats in the NC group experienced a steady increase in body weight. Following a 2-week period of acclimatization to the diet, the rats' blood glucose levels remained within the range of 3.6-4.3 mmol/L. As the rats continued on a high-sugar and high-fat diet, the STZ-induced diabetic rats exhibited a more rapid increase in both body weight and blood glucose levels compared to the NC group, with statistically significant differences emerging from the 8th week onwards (p \u0026lt; 0.05). Conversely, immediately after the induction of diabetes via intraperitoneal STZ injection, the diabetic rats' body weight decreased sharply, reaching a level significantly below that of the NC group (p \u0026lt; 0.01, Fig.1A). By the 10th week, a marked rise in the blood glucose levels of the diabetic rats was observed, with all differences being statistically significant (p\u0026lt;0.05). Meanwhile, the blood glucose levels in the diabetic rats treated with the MCC950 gradient did not show a statistically significant change.(p\u0026gt;0.05,Fig.1B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.2 Effects of different concentrations of MCC950 on histopathological damage of rat retina\u003c/p\u003e\n\u003cp\u003eWith the progression of T2DM, pathological damage occurred in the rat retina.HE staining showed that the retinal thickness in the NC group was significantly higher than that in the other groups (Fig.2 B).STZ-induced diabetic rat retinas underwent pathological damage:structural disorganization of retinal layers, pre-retinal neovascularization and arteriolar thrombosis (Fig.2A), and a decrease in the retinal ganglion cells.However, the thickness of the GCL did not differ significantly among the groups. There was no significant difference between the groups. Cell nuclei (black arrows) were also seen in the T2DM group. The inner plexiform layer (IPL), inner nuclear layer (INL) and outer nuclear layer (ONL) were thinner than in the NC group, and the MCC950 gradient group dose-dependently improved their thickness. The outer plexiform layer (OPL) was thinner than in the NC group, with no significant difference between the groups. Compared with the T2DM group, the structure of the inner plexiform layer (IPL), inner nuclear layer (INL) and outer nuclear layer (ONL) was significantly improved under the treatment of 1 mM concentration of MCC950, and the cells of the IPL and ONL tended to be neatly aligned, and the thickening of the vascular basement membrane and exudative lesions were significantly reduced.\u003c/p\u003e\n\u003cp\u003e3.3 Effects of different concentrations of MCC950 on apoptosis in rat retinal tissues\u003c/p\u003e\n\u003cp\u003eIn order to evaluate the effects of different concentrations of MCC950 on the apoptosis of retinal tissue cells, we detected the apoptosis of retinal tissue cells in rats by TUNEL method. The results showed that the retinal apoptosis in STZ-induced diabetic rats was significantly increased, and the apoptosis of retinal tissue cells caused by long-term hyperglycemia decreased dose-dependently with the increase of MCC950 concentration in the concentration range of 0.01 mM to 1 mM, and the difference of retinal apoptosis under the treatment of MCC950 at the concentration of 10 mM was not statistically significant comparing with that of \u0026nbsp;T2DM group ( Fig.3 A,C).Western blot results showed that Bcl-2 protein expression was up-regulated and Bax protein expression was down-regulated in a gradient of MCC950 concentration, and the Bcl-2/Bax ratio was increased, which further confirmed its anti-apoptotic effect (Fig.3 D-E).\u003c/p\u003e\n\u003cp\u003e3.4 Effects of Different Concentrations of MCC950 on ROS Expression in Rat Retinal Tissues and Correlation Analysis with NEK7 Expression\u003c/p\u003e\n\u003cp\u003eIn order to verify whether MCC950 can block the NEK7/NLRP3 pathway in diabetic retinopathy by binding specifically with NLRP3 and thus achieve the therapeutic effect, we used immunofluorescence localization (Fig.4 A) to analyze the NEK7 expression level in the retina. The results showed that in the NC group, the NEK7 immunofluorescence signal (green) was mainly localized in the ganglion cell layer (GCL) and the inner nuclear layer (INL) of the retina, and was weakly expressed, i.e., the basal expression level of NEK7 was low in the physiological state. In the T2DM group, the fluorescence signal of NEK7 was significantly enhanced and widely distributed in the ganglion cell layer, inner nuclear layer and perivascular area. Quantitative analysis showed that the fluorescence intensity was significantly higher in the T2DM group than in the NC group (p \u0026lt; 0.001, Fig. 4 B), indicating that diabetic pathology induced the up-regulation of NEK7 expression.In the MCC950 gradient treatment group, the expression level of NEK7 was significantly reduced in the 10 mM concentration group (p \u0026lt; 0.05), and the fluorescence intensity of NEK7 decreased dose-dependently and signal distribution gradually in a range of concentrations from 0.01 mM to 1 mM. dependent decrease, and the signal distribution gradually weakened (Fig.4 B,D,E). This indicates that MCC950 reverses the T2DM-induced abnormal activation of NEK7 by dose-dependently inhibiting NEK7expression.\u003c/p\u003e\n\u003cp\u003eIn addition, to verify the protective effect of concentration gradient MCC950 against apoptosis and oxidative damage in the retina of diabetic rats in the T2DM group, we detected the expression of ROS in the retinal tissues of rats in each group and further analyzed the correlation between ROS and NEK7 expression. The results suggested that compared with diabetic rats in the T2DM group, ROS accumulation was reduced under treatment in the 10 mM concentration group, but there was no statistically significant difference; in the concentration range of 0.01 mM to 1 mM, the expression of ROS gradually decreased with the increase of the concentration of MCC950, and the expression was the lowest at the 1 mM concentration (p\u0026lt;0.001), and the difference was statistically significant (Fig. 4 C,D,F) Spearman correlation analysis showed a significant positive correlation between NEK7 fluorescence intensity and retinal ROS levels in rat retinal tissues (p\u0026lt;0.05,r=0.8857, Fig.4 G), suggesting that NEK7 plays an important role in regulating oxidative stress in the retina and verifying the activation of NLRP3 inflammatory vesicles by ROS through upregulation of NEK7.\u003c/p\u003e\n\u003cp\u003e3.5 Gradient MCC950 Inhibits NEK7/NLRP3 Pathway-Related Protein Expression\u003c/p\u003e\n\u003cp\u003eTo verify the effects of different concentrations of MCC950 on the activation of the NEK7/NLRP3 pathway in rat retinal tissues, we examined the expression of NLRP3, NEK7, ASC and other proteins in the retinal tissues of rats in each group by Western-blot. The results suggested that the expression of each protein in diabetic rats in the T2DM group was significantly higher compared with that in the NC group (p\u0026lt;0.05).The concentration gradient of MCC950 significantly inhibited the expression of proteins related to the NLRP3-NEK7 pathway, which was dose-dependently reduced in the concentration range of 0.01 mM to 1 mM, and the differences were all statistically significant (p\u0026lt;0.05,Fig.5 A-D).\u003c/p\u003e\n\u003cp\u003e3.6 Effect of different concentrations of MCC950 on the expression of inflammatory factors in rat retina\u003c/p\u003e\n\u003cp\u003eTo verify the effect of different concentrations on each inflammatory factor in rat retina, the expression of inflammatory factors Caspase-1, Cleaved-Caspase-1, IL-1β and IL-18 in all rat retinal tissues were detected by WB. The results suggested that the expression of inflammatory factors was significantly increased in diabetic rats in the T2DM group compared to NC, and the differences were all statistically significant (p\u0026lt;0.05).The expression of Caspase-1, Cleaved-Caspase-1, IL-1β, and IL-18 was decreased under MCC950 treatment at a concentration of 10 mM (p\u0026lt;0.05); and in the 0.01 to 1 mM concentration range, the release of these inflammatory factors was dose-dependently reduced (all p\u0026lt;0.01, Fig. 5 E-I).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eStudies have confirmed that elevated glucose levels can trigger an excessive accumulation of mitochondrial ROS (reactive oxygen species), which in turn promotes the assembly of the NLRP3 inflammasome complex by augmenting the structural compatibility between NEK7 and NLRP3\u003csup\u003e[14]\u003c/sup\u003e. This activation of NLRP3-containing inflammatory vesicles not only leads to the subsequent proteolysis of pro-IL-1\u0026beta;\u0026nbsp;and pro-IL-18 but also facilitates the secretion of their respective mature forms. Consequently, this mechanism elicits an upsurge in the expression of IL-1\u0026beta;\u0026nbsp;and IL-18, initiating a chronic inflammatory response that is implicated in the exacerbation of diabetic retinopathy\u003csup\u003e[15]\u003c/sup\u003e\u003csup\u003e[16]\u003c/sup\u003e.The experimental evidence clearly demonstrates that MCC950 effectively halts the mentioned pathological processes through a dual-mode mechanism. This mechanism involves (i) directly binding to the NACHT domain of NLRP3, thereby preventing the interaction between NEK7 and NLRP3, and (ii) suppressing mitochondrial ROS production to counteract oxidative modification of NEK7\u003csup\u003e[17]\u003c/sup\u003e\u003csup\u003e[25]\u003c/sup\u003e.As a targeted inhibitor of the NLRP3 pathway, MCC950 has proven to markedly alleviate inflammatory cytokine storms and vascular injury by focusing on the ROS-NEK7-NLRP3 axis, thus offering a precise intervention approach for the treatment of diabetic retinopathy\u003csup\u003e[18]\u003c/sup\u003e\u003csup\u003e.\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eTo begin with, it is confirmed that mitochondrial ROS (reactive oxygen species) serve as activators for NEK7-NLRP3 inflammatory vesicles\u003csup\u003e[19]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWestern blot findings revealed a marked elevation in the expression of NEK7 within the retinal tissues of diabetic rodents, with a notable positive association between NEK7 expression levels and the fluorescence intensity indicative of mitochondrial ROS\u003csup\u003e[20]\u003c/sup\u003e. Furthermore, the formation of NLRP3 inflammatory vesicles was detected, and immunofluorescence staining localized NEK7 to aggregated clusters within the retinal vascular endothelial cells, concurrent with an increase in ASC oligomerization.\u003c/p\u003e\n\u003cp\u003eSecondly, evidence has clearly shown that the activation of NRLP3 triggers an inflammatory response, ultimately resulting in retinal injury. Notably, the levels of cleaved Caspase-1, IL-1\u0026beta;, and IL-18 were heightened in the retinas of the diabetic cohort, yet these were effectively mitigated by the application of MCC950 at diverse concentrations. HE staining outcomes validated the rejuvenation of retinal layer architecture, a restoration that appeared to beboth dose-dependent and a consequence of MCC950 treatment.Furthermore, this study delves into the twin inhibitory actions of MCC950.\u003c/p\u003e\n\u003cp\u003eIt directly impedes the NEK7-NLRP3 interaction: The western blot findings revealed that MCC950 potently inhibits NEK7 from binding to NLRP3 in a manner proportional to the drug concentration. This interference likely prevents the structural activation of NLRP3 by blocking the ATP-binding site within its NACHT domain. The elimination of mitochondrial ROS (reactive oxygen species) is a topic of great significance. The ROS levels in retinal mitochondria were notably diminished following treatment with MCC950.Subsequently, a noteworthy occurrence of synergistic inhibition between apoptosis and inflammation has been identified.\u003c/p\u003e\n\u003cp\u003eTUNEL and Apoptotic Pathway: An elevated rate of apoptosis in retinal tissue cells was documented within the T2DM cohort, whereas a marked decrease in apoptosis was evident in the MCC950-treated group. Through Western blotting, it was determined that MCC950 enhanced the expression of the anti-apoptotic protein Bcl-2 and diminished the levels of the pro-apoptotic protein Bax, effectively restoring the Bcl-2/Bax ratio to its normal state.\u003c/p\u003e\n\u003cp\u003eOur investigation has elucidated that MCC950 exhibits a maximal efficacy threshold within the concentration bracket of 0.01-10 mM, indicating a saturation effect. This discovery lays a solid foundation for fine-tuning the dosage of intravitreal injections. The compound\u0026apos;s dual mode of action\u0026mdash;blocking NLRP3 complex assembly and neutralizing reactive oxygen species (ROS)\u0026mdash;\u0026nbsp;orchestrates a potent interruption of the inflammation-oxidative stress feedback loop, surpassing the efficacy of single-site inhibitors such as the NLRP3-specific monoclonal antibody, which has been documented to decrease vascular permeability by merely 40%\u003csup\u003e[21]\u003c/sup\u003e\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn the course of this investigation, we discovered that the intravitreal administration of MCC950 markedly mitigated the retinal pathological injury in diabetic rats by suppressing the NEK7/NLRP3 signaling pathway.Additionally, it exerted inhibitory effects on oxidative stress, inflammation, and cellular apoptosis. Moreover, a distinct dose-response relationship was observed for the therapeutic efficacy of MCC950 on diabetic retinopathy (DR) within a specific concentration spectrum. Our findings also underscore the integral role of the ROS-NEK7-NLRP3 axis in the pathogenesis of DR, as evidenced by the significant positive correlation between NEK7 and reactive oxygen species (ROS).\u003c/p\u003e\n\u003cp\u003eThis study reveals the pivotal role of the ROS-NEK7-NLRP3 axis in diabetic retinopathy (DR). Hyperglycemia-induced mitochondrial oxidative stress triggers NEK7 upregulation, which in turn promotes NLRP3 inflammasome assembly\u003csup\u003e[22]\u003c/sup\u003e\u003csup\u003e[23]\u003c/sup\u003e.This process ultimately leads to IL-1\u0026beta;/IL-18-mediated retinal vascular injury\u003csup\u003e[24]\u003c/sup\u003e\u003csup\u003e[25]\u003c/sup\u003e.The present study has demonstrated the dual inhibitory effects of intravitreal delivery of MCC950. Within a certain concentration range, MCC950 has been shown to disrupt the NLRP3-ASC interaction, as well as to suppress NEK7 expression and ROS accumulation. Furthermore, a robust positive correlation has been observed between NEK7 and ROS. These findings establish the first dose-optimised therapeutic regimen for DR and identify targetable nodes of the redox-inflammatory crosstalk, paving the way for developing NEK7/NLRP3 dual-pathway inhibitors.\u003c/p\u003e\n\u003cp\u003eThis comprehensive study has uncovered the critical importance of the ROS-NEK7-NLRP3 signaling axis in the development and progression of diabetic retinopathy (DR). The research delves into the molecular mechanisms by which hyperglycemia, a hallmark of diabetes, induces mitochondrial oxidative stress. This stress, in turn, activates the expression of NEK7, a kinase that plays a crucial role in cell division and the cell cycle. The upregulation of NEK7 facilitates the assembly of the NLRP3 inflammasome, a multiprotein complex that is central to the innate immune response. The activation of this inflammasome leads to the maturation and release of pro-inflammatory cytokines such as interleukin-1 beta (IL-1\u0026beta;) and interleukin-18 (IL-18), which are known to cause significant damage to the retinal vasculature\u003csup\u003e[27]\u003c/sup\u003e\u003csup\u003e[32]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe study has meticulously examined the effects of MCC950, a potent and selective inhibitor of the NLRP3 inflammasome, when administered intravitreally. It has been observed that within a specific concentration range, MCC950 effectively disrupts the interaction between NLRP3 and ASC (apoptosis-associated speck-like protein containing a CARD), a critical step in the activation of the inflammasome\u003csup\u003e[31]\u003c/sup\u003e\u003csup\u003e[33]\u003c/sup\u003e. Additionally, the compound has been shown to suppress the expression of NEK7 and reduce the accumulation of reactive oxygen species (ROS), which are byproducts of oxidative stress and contribute to cellular damage.\u003c/p\u003e\n\u003cp\u003eWe have also highlighted a strong positive correlation between the levels of NEK7 and ROS, suggesting that the two factors may mutually reinforce each other\u0026apos;s effects, thereby exacerbating the inflammatory response and tissue damage in diabetic retinopathy. This correlation underscores the importance of targeting both NEK7 and ROS in the treatment of DR.\u003csup\u003e[18]\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThe findings of this study are groundbreaking as they establish the first dose-optimized therapeutic regimen for diabetic retinopathy. By identifying the key nodes of interaction between oxidative stress and inflammation, the research paves the way for the development of novel dual-pathway inhibitors that target both NEK7 and NLRP3\u003csup\u003e[28]\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e.Such inhibitors could potentially offer a more effective treatment strategy for DR, addressing both the inflammatory and oxidative components of the disease. This could lead to significant improvements in patient outcomes by reducing the incidence and severity of vision-threatening complications associated with diabetic retinopathy.\u003csup\u003e[29]\u003c/sup\u003e\u003csup\u003e[30]\u003c/sup\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Scientific Investigation Board of Xinhua Hospital Affiliated to Dalian University, and was performed in accordance with relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Liaoning Provincial Department of Education Science and Technology Research Program (No. LJKZ1193).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiaofeng Li and Kexuan Ren designed the study, \u0026nbsp; revised the manuscript accordingly. Kexuan Ren performed all experiments,analyzed the data.All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical trial number: not applicable.\u003c/p\u003e\n\u003cp\u003eLaboratory Animal Ethical Review Number:2025-018-01\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available from the first author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Xinhua Hospital Affiliated to Dalian University for providing the scientific research platform.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePan, C. W., Wang, S., Xu, C. L., \u0026amp; Song, E. (2018). Combined effect of glycemic and blood pressure control on diabetic retinopathy among Chinese with type-2 diabetes mellitus. Diabetology \u0026amp; metabolic syndrome, 10, 73. https://doi.org/10.1186/s13098-018-0377-7\u003c/li\u003e\n\u003cli\u003eAccili, D., Deng, Z. \u0026amp; Liu, Q. Insulin resistance in type 2 diabetes mellitus. Nat Rev Endocrinol (2025). https://doi.org/10.1038/s41574-025-01114-y\u003c/li\u003e\n\u003cli\u003eZheng, X., Wan, J., \u0026amp; Tan, G. (2023). The mechanisms of NLRP3 inflammasome/pyroptosis activation and their role in diabetic retinopathy. Frontiers in immunology, 14, 1151185. https://doi.org/10.3389/fimmu.2023.1151185\u003c/li\u003e\n\u003cli\u003eLi, H., Liu, X., Zhong, H., Fang, J., Li, X., Shi, R., \u0026amp; Yu, Q. (2023). Research progress on the pathogenesis of diabetic retinopathy. BMC ophthalmology, 23(1), 372. https://doi.org/10.1186/s12886-023-03118-6\u003c/li\u003e\n\u003cli\u003eTao Ye, Wei-yan Tao, Xiao-yi Chen, Cheng Jiang, Bin Di, Li-li Xu,Mechanisms of NLRP3 inflammasome activation and the development of peptide inhibitors,Cytokine \u0026amp; Growth Factor Reviews,Volume 74,2023,Pages 1-13,ISSN 1359-6101,https://doi.org/10.1016/j.cytogfr.2023.09.007.\u003c/li\u003e\n\u003cli\u003eZheng, D., Liwinski, T. \u0026amp; Elinav, E. Inflammasome activation and regulation: toward a better understanding of complex mechanisms. Cell Discov 6, 36 (2020). https://doi.org/10.1038/s41421-020-0167-x.\u003c/li\u003e\n\u003cli\u003eHe, Y., Zeng, M., Yang, D. et al. NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux. Nature 530, 354\u0026ndash;357 (2016). https://doi.org/10.1038/nature16959.\u003c/li\u003e\n\u003cli\u003eSharif, H., Wang, L., Wang, W.L. et al. Structural mechanism for NEK7-licensed activation of NLRP3 inflammasome. Nature 570, 338\u0026ndash;343 (2019). https://doi.org/10.1038/s41586-019-1295-z.\u003c/li\u003e\n\u003cli\u003eJiao J. Zhao G. Wang Y. Ren P. \u0026amp; Wu M. (2020). MCC950 a Selective Inhibitor of NLRP3 Inflammasome Reduces the Inflammatory Response and Improves Neurological Outcomes in Mice Model of Spinal Cord Injury. Frontiers in molecular biosciences 7 37. https://doi.org/10.3389/fmolb.2020.00037\u003c/li\u003e\n\u003cli\u003eYadong Z. Xiangbao M.Tianyuan Y. Weijie X. Guibo S. \u0026amp; Xiaobo S. . (2018). Inhibiting the nlrp3 inflammasome activation with mcc950 ameliorates diabetic encephalopathy in db/db mice. 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Cell Death Dis 8, e2941 (2017). https://doi.org/10.1038/cddis.2017.308\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"MCC950, concentration gradient, diabetic retinopathy, ROS-NEK7-NLRP3 axis, Type 2 diabetes mellitus, vitreous injection","lastPublishedDoi":"10.21203/rs.3.rs-6625531/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6625531/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlobally, type 2 diabetes mellitus (T2DM) constitutes more than 90% of all diabetes cases, with diabetic retinopathy (DR) emerging as the predominant microvascular complication, resulting in vision loss in roughly 30% of affected individuals \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. In contrast to type 1 diabetes, DR associated with T2DM is marked by insulin resistance and persistent low-grade inflammation, which may intensify the activation of the NLRP3 inflammasome \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The pathogenesis of diabetic retinopathy is fundamentally driven by the activation of the NLRP3 inflammasome due to redox disequilibrium\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.In this study, we elucidate a novel mechanism through which MCC950, a selective inhibitor of NLRP3, mitigates DR via the ROS-NEK7-NLRP3 pathway. Diabetic rats induced by streptozotocin were administered intravitreal injections of MCC950 at varying concentrations (0.01, 0.1, 1, 10 mM). Quantitative assessments revealed that a concentration of 1 mM MCC950 markedly improved retinal histopathological alterations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and modulated retinal apoptosis and oxidative stress to a considerable degree (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On a mechanistic level, MCC950 suppressed NLRP3 inflammasome activation (manifested by reduced levels of Cleaved Caspase-1, IL-1β, and IL-18, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in a dose-dependent manner by disrupting the interaction between NEK7 and NLRP3. Notably, there was a strong positive correlation between the intensity of ROS fluorescence and the fluorescence expression of NEK7 (r\u0026thinsp;=\u0026thinsp;0.8857, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with MCC950 treatment significantly lowering retinal ROS levels at the 1 mM concentration. Moreover, pharmacological inhibition of NEK7 potentiated the therapeutic efficacy of MCC950. This research establishes the upregulation of NEK7 mediated by ROS as a critical factor in NLRP3 activation in DR, and provides pioneering evidence for the efficacy of dose-optimized MCC950 therapy targeting this axis.\u003c/p\u003e","manuscriptTitle":"MCC950 targets the ROS-NEK7-NLRP3 axis to improve type 2 diabetic retinopathy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-10 15:00:46","doi":"10.21203/rs.3.rs-6625531/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-27T07:15:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-27T06:14:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"183741272004520875687909083900312593818","date":"2025-06-26T16:23:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-17T00:38:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"228325113972439388036161857658355413025","date":"2025-06-15T22:45:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-14T05:15:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"26184608313063791793551338656189542446","date":"2025-06-06T06:04:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308329838541101393470266387136102902252","date":"2025-06-05T12:14:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87700392771567191218046016065640238123","date":"2025-06-05T07:13:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-05T01:13:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-05T01:10:29+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-26T13:25:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-23T10:09:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-09T06:13:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6ce47cea-f38f-42b8-8c24-9e243fc5c90d","owner":[],"postedDate":"June 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":49620441,"name":"Health sciences/Diseases/Endocrine system and metabolic diseases"},{"id":49620442,"name":"Health sciences/Diseases/Eye diseases"}],"tags":[],"updatedAt":"2025-09-29T16:00:30+00:00","versionOfRecord":{"articleIdentity":"rs-6625531","link":"https://doi.org/10.1038/s41598-025-18438-4","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-09-23 15:57:33","publishedOnDateReadable":"September 23rd, 2025"},"versionCreatedAt":"2025-06-10 15:00:46","video":"","vorDoi":"10.1038/s41598-025-18438-4","vorDoiUrl":"https://doi.org/10.1038/s41598-025-18438-4","workflowStages":[]},"version":"v1","identity":"rs-6625531","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6625531","identity":"rs-6625531","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}