Organophosphate pesticide DEDT promotes diabetic retinopathy progression via AMPK/Nrf2/HO-1 pathway

Organophosphate pesticide DEDT promotes diabetic retinopathy progression via AMPK/Nrf2/HO-1 pathway | 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 Organophosphate pesticide DEDT promotes diabetic retinopathy progression via AMPK/Nrf2/HO-1 pathway Biqing Ding, Siyu Gui, Xinchen Wang, Yumei Tao, Jianghui Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7849232/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Background The impact of environmental toxins, particularly organophosphate pesticides (OPs), on the progression of diabetic retinopathy (DR) remains insufficiently understood. Recent studies have highlighted the potential role of environmental pollutants in exacerbating diabetic complications, but the underlying mechanisms are still unclear. This study aims to explore the effect of diethyldithiophosphate (DEDT), an OP, on DR progression through modulation of the AMPK/Nrf2/HO-1 signaling pathway. Methods Human retinal microvascular endothelial cells (HRMECs) and retinal pigment epithelial cells (ARPE-19) were cultured under high-glucose conditions to simulate diabetic stress. Cells were exposed to various concentrations of DEDT, and their viability, oxidative stress, tight junction integrity, and inflammation were assessed. Western blot, quantitative PCR, and enzyme-linked immunosorbent assay (ELISA) techniques were employed to evaluate the expression of key proteins in the AMPK/Nrf2/HO-1 pathway and inflammatory cytokines. In vivo, diabetic rat models were treated with DEDT to assess retinal damage and oxidative stress. The effects of AMPK activation were also evaluated using AICAR, an AMPK activator, to further explore the mechanistic role of AMPK/Nrf2/HO-1 signaling. Results Our results demonstrated that DEDT exposure significantly reduces retinal cell viability and disrupts tight junction proteins (ZO-1, Occludin, Claudin-5) under high-glucose conditions. Mechanistically, DEDT inhibited the AMPK/Nrf2/HO-1 pathway, leading to increased oxidative stress, enhanced inflammation, and elevated levels of apoptotic markers (Bax and Bcl-2). In vivo, DEDT exposure exacerbated retinal damage and oxidative stress in diabetic rats. Activation of AMPK by AICAR reversed these effects, restoring Nrf2 and HO-1 expression, improving cell viability, and protecting the blood-retinal barrier. These findings indicated that DEDT promotes DR progression by disrupting the AMPK/Nrf2/HO-1 signaling pathway. Conclusion This study provided experimental evidence that DEDT accelerates diabetic retinopathy progression via inhibition of the AMPK/Nrf2/HO-1 pathway, contributing to increased oxidative stress and retinal barrier dysfunction. Our results emphasized the potential health risks associated with pesticide exposure, particularly in diabetic populations, and highlight the importance of regulating environmental toxins to prevent exacerbation of diabetic complications. Biological sciences/Biochemistry Biological sciences/Cell biology Health sciences/Diseases Health sciences/Endocrinology Biological sciences/Molecular biology Diabetic retinopathy AMPK/Nrf2/HO-1 organophosphate pesticides toxic effects Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Organophosphate pesticides (OPs) are widely used in agriculture, household pest control and public health, causing neurotoxicity by inhibiting the enzyme Acetylcholinesterase (AChE), leading to excessive accumulation of acetylcholine in the nervous system ( 1 ). However, recent epidemiological and experimental studies suggest that long-term low-dose exposure to OPs may cause metabolic disturbances and endocrine disruption, increasing the risk of chronic diseases. Additionally, it may exacerbate the development of microvascular complications in diabetic patients through mechanisms such as oxidative stress and inflammation ( 2 ). Eyes, as highly metabolically active and microvessel-rich organs, are susceptible to damage from environmental toxicants. Studies have shown that OPs can affect ocular health by inducing mitochondrial dysfunction and endothelial cell damage ( 3 ). Long-term exposure to OPs has been linked to an increased risk of retinal degeneration, lens opacity, and vascular abnormalities, which may further exacerbate diabetic microvascular complications ( 4 , 5 ). Diabetic retinopathy (DR) is one of the most common complications of diabetes and a leading cause of vision loss globally ( 6 ). With the rising prevalence of diabetes, the burden of DR is expected to increase, with over 100 million people projected to be affected by DR by 2030 ( 7 ). Studies show that the prevalence of DR in type 2 diabetes patients is approximately 35%, and even higher in type 1 diabetes patients ( 8 , 9 ). The pathophysiology of DR is complex, primarily involving retinal microvascular damage due to prolonged hyperglycemia, leading to microvascular changes and neovascularization ( 10 ). Early-stage DR typically presents with no noticeable symptoms, but as the condition progresses, patients may experience blurred vision, visual field defects, and even blindness ( 1 ). Moreover, the role of environmental factors in DR has gained increasing attention. Hua et al. pointed out that the environmental toxin 2-Ethylhexyl diphenyl phosphate (EHDPP) might impact retinal microcirculation through metabolic disruption and inflammatory pathways, leading to impaired retinal photoreceptor function in mice, which could offer new directions for DR prevention strategies ( 12 ). Previous studies have primarily focused on the role of heavy metals, air pollutants, and widely recognized endocrine-disrupting chemicals (EDCs), such as bisphenol A and phthalates, in the development of diabetic retinopathy ( 13 , 14 ). However, the potential contribution of organophosphate pesticide metabolites to DR risk remains unexplored ( 15 ). In recent years, the AMPK/Nrf2/HO-1 signaling pathway has attracted significant attention from researchers due to its crucial role in regulating cellular stress responses, antioxidant defense, and protecting vascular barrier function. 5'-AMP-activated protein kinase (AMPK) serves as a key regulator of cellular energy metabolism. It enhances cellular antioxidant responses by activating nuclear factor erythroid 2-related factor 2 (Nrf2), which in turn induces heme oxygenase-1 (HO-1) expression to further mitigate oxidative stress and inflammatory responses (Small-molecule agonist AdipoRon alleviates diabetic retinopathy through the AdipoR1/AMPK/EGR4 pathway). Previous studies indicate that activation of the AMPK/Nrf2/HO-1 pathway mitigates hyperglycemia-induced endothelial cell damage and plays a vital role in preserving vascular barrier integrity (Regulation of AMPK and GAPDH by Transglutaminase 2 Plays a Pivotal Role in Microvascular Leakage in Diabetic Retinas). However, systematic investigations into this pathway's role in diabetic retinopathy remain scarce. Therefore, this study aims to explore the regulatory effects of an organophosphorus pesticide DEDT on the AMPK/Nrf2/HO-1 pathway and elucidate its potential mechanisms in DR progression. By thoroughly examining alterations in this signaling pathway, we expect to provide novel insights and therapeutic targets for the prevention and treatment of diabetic retinopathy. 2. Methods Streptozotocin (STZ), Catalog: S0130, Sigma-Aldrich; Diethyl Dithiophosphate (DEDT), Catalog: 24617-47-0, ChemService; AICAR, Catalog: 99447-33-3, Tocris Bioscience; Cell Counting Kit-8 (CCK-8), Catalog: C0037, Beyotime Biotechnology; TRIzol Reagent, Catalog: 15596026, Thermo Fisher Scientific; SYBR Green Master Mix, Catalog: 4309155, Applied Biosystems; Cell Counting Kit-8 (CCK-8), Catalog: C0037, Beyotime Biotechnology; ZO-1 ELISA Kit, Catalog: PT518, Beyotime Biotechnology; Occludin ELISA Kit, Catalog: PT519, Beyotime Biotechnology; Claudin-5 ELISA Kit, Catalog: PT521, Beyotime Biotechnology; IL-6 ELISA Kit, Catalog: PI330, Beyotime Biotechnology; TNF-α ELISA Kit, Catalog: PI305, Beyotime Biotechnology; ; IL-8 ELISA Kit, Catalog: PI331, Beyotime Biotechnology; ; IL-1β ELISA Kit, Catalog: PI303, Beyotime Biotechnology; Bax ELISA Kit, Catalog: PA102, Beyotime Biotechnology; BCL-2 ELISA Kit, Catalog: PA101, Beyotime Biotechnology; Phospho-AMPK (Thr172) ELISA Kit, Catalog: PT428, Beyotime Biotechnology; Nrf2 ELISA Kit, Catalog: PT588, Beyotime Biotechnology; HO-1 ELISA Kit, Catalog: PT592, Beyotime Biotechnology; Glutathione (GSH) Assay Kit, Catalog: S0053, Beyotime Biotechnology; Malondialdehyde (MDA) test kit, Catalog: S0131S, Beyotime Biotechnology; Superoxide dismutase (SOD) test kit, Catalog: S0101, Beyotime Biotechnology; Catalase (CAT) test kit, Catalog: S0051, Beyotime Biotechnology. 2.1 Animal Model and Treatments Sprague-Dawley rats (8–10 weeks old, 250-300g) were obtained from the Animal Experiment Center of Anhui Medical University and were maintained under appropriate conditions. Animals were randomly divided into four experimental groups: normal control (NC, n = 5), NC with DEDT exposure (NC + DEDT, n = 5), DR model (DR, n = 5), and DR with DEDT exposure (DR + DEDT, n = 5). Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) (55 mg/kg), and animals with sustained blood glucose levels > 16.7 mmol/L for 3 consecutive days were included in the study. Based on previous toxicological studies, DEDT (10 mg/kg) was administered daily by oral gavage for 12 days ( 27 , 28 ). At the end of the experiment, all SD rats were deeply anesthetized with 3–5% isofluran and maintained at 1.5-2.0%. Once complete loss of pedal reflex was confirmed, euthanasia was performed by cervical dislocation. Death was confirmed by the absence of heartbeat and respiration before tissue collection. All animal procedures were approved by the Ethics Committee of Anhui Medical University and were conducted in accordance with ARRIVE guidelines and other relevant guidelines and regulations. 2.2 Cell Culture and Treatments Human retinal microvascular endothelial cells (HRMECs) and adult retinal pigment epithelial-19 (ARPE-19) cells were cultured under standard conditions. HRMEC cells was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China; Catalog No. CP-H130) and ARPE-19 cells was purchased from ATCC (Catalog No. CRL-2302). To mimic diabetic conditions, cells were maintained in medium containing 50 mM glucose (high glucose, HG), with 5.5 mM glucose serving as normal control (NC). Cells were treated with varying concentrations of DEDT (0–50 µM) for 0–48 hours. For rescue experiments, cells were co-treated with the AMPK activator AICAR (30 µM) for 2 h and then treated with HG for 24 h. 2.3 Cell Viability Assay (CCK-8) Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan) following the manufacturer’s protocol. Cells were seeded in 96-well plates and treated as indicated. After treatment, 10 µL of CCK-8 reagent was added to each well and incubated for 2 hours at 37°C. The absorbance was measured at 450 nm using a microplate reader. 2.4 Trans-Epithelial/Endothelial Electrical Resistance (TEER) Measurement TEER values were measured to assess barrier integrity of HRMECs monolayers cultured on Transwell inserts (0.4 µm pore size). Cells were treated with DEDT for different time points, and TEER was measured using an EVOM2 voltohmmeter (World Precision Instruments). Resistance values were normalized to the surface area and expressed in Ω·cm². 2.5 Dextran Permeability Assay HRMECs were cultured in high-glucose medium, then seeded into 24-well plates and cultured for 24 hours until reaching appropriate density. Subsequently, cells were exposed to high glucose and DEDT (40 µM) in different treatment groups, with experiments conducted after 48 hours of treatment. To assess blood-retinal barrier integrity, dextran solution (0.1 mg/mL) was added to the wells and incubated at 37°C for 1 hour. Supernatant was collected periodically throughout the experiment, and dextran absorbance was measured at 490 nm using a UV spectrophotometer to record transmittance at different time points. 2.6 Enzyme-Linked Immunosorbent Assay (ELISA) ELISA kits (R&D Systems, USA) were used to quantify protein levels of tight junction proteins (ZO-1, occludin, claudin-5) and inflammatory cytokines (IL-6, IL-8, IL-1β, TNF-α), AMPK, p-AMPK, Nrf2, HO-1, Bax, and BCL-2 in cell lysates or supernatants. Samples were processed according to the manufacturer's instructions, and absorbance was measured at 450 nm. 2.7 RNA Extraction and Quantitative Real-Time PCR (qPCR) Total RNA was extracted from treated cells using TRIzol reagent (Invitrogen, USA). cDNA synthesis was performed using a reverse transcription kit (Takara, Japan). qPCR was carried out with SYBR Green Master Mix (Applied Biosystems) on a StepOnePlus™ Real-Time PCR system. Gene expression was normalized to GAPDH and analyzed using the 2^–ΔΔCt method. Primer sequences are listed in Table S1 . 2.8 Measurement of Oxidative Stress Markers in Retinal Tissue Retinal tissues were homogenized in ice-cold lysis buffer, and oxidative stress markers including glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were quantified using corresponding colorimetric assay kits (Beyotime, China) according to the manufacturers’ protocols. 2.9 Quantification of Reactive Oxygen Species (ROS) in Tissues ROS levels were measured in systemic tissues (heart, liver, spleen, lung, kidney) and various ocular tissues (whole eye, cornea, vitreous body, retina, RPE/choroid). Tissues were homogenized, and ROS levels were assessed using a fluorescent ROS detection assay kit (DCFH-DA based, Nanjing Jiancheng Bioengineering Institute). Fluorescence intensity was detected at Ex/Em = 488/525 nm using a microplate reader and normalized to protein content. 2.10 Statistical analysis All experiments had at least six independent replicates. Data were expressed as mean ± SD. Statistical analyses were performed using GraphPad Prism software, and comparisons were analyzed by one-way or multifactor ANOVA followed by Tukey's post-hoc test, with p-values less than 0.05 considered statistically significant. 3. Results 3.1 DEDT Exacerbates High Glucose-Induced Decrease in Retinal Cell Activity and Barrier Function Impairment In HRMECs, cell viability decreased in a dose-dependent manner with increasing concentrations of DEDT (5–50 µM) (Fig. 1 A). A similar trend was observed in ARPE-19 cells, where DEDT exposure also led to a marked reduction in cell viability (Fig. 1 B). Furthermore, prolonged exposure to DEDT resulted in a time-dependent decline in cell viability in both HRMECs (Fig. 1 C) and ARPE-19 cells (Fig. 1 D). In addition, the expression levels of tight junction proteins including ZO-1, OCCLUDIN, and Claudin-5 were significantly reduced in the DR + DEDT group compared to the DR group, indicating that DEDT exacerbates the disruption of retinal barrier integrity (Fig. 1 E, 1 F). Moreover, TEER measurements demonstrated a progressive decline beginning at 3 hours after 40 µM DEDT exposure, indicating a time-dependent impairment of the cellular barrier function (Fig. 1 G). Simultaneously, results from the dextran permeability assay demonstrated a significant increase in the permeability of the cell monolayer to dextran (Fig. 1 H), further validating the disruptive effect of DEDT on the endothelial barrier. 3.2 DEDT Suppresses the AMPK/Nrf2/HO-1 Signaling Pathway and Exacerbates Oxidative Damage in Retinal Cells Under High-Sugar Conditions ROS detection results showed that DEDT significantly increased ROS levels in HRMECs under high glucose conditions, with the highest concentration exhibiting the most pronounced effect (Fig. 2 A). Western blot analysis revealed that in DR group, AMPK, Nrf2, and HO-1 protein expression decreased compared to the control group and was significantly suppressed in the DR + DEDT group (Fig. 2 B, 2 C), indicating that DEDT significantly impacts the AMPK/Nrf2/HO-1 signaling pathway, exacerbating oxidative stress. Following DEDT treatment under hyperglycemic conditions, the levels of pro-inflammatory cytokines (including IL-6, IL-1β, and TNF-α) in HRMECs were also significantly elevated, further confirming its pro-inflammatory effects (Fig. 2 D). As shown in Fig. 2 E, DEDT treatment not only enhanced the expression of the pro-apoptotic protein Bax but also suppressed the expression of the anti-apoptotic protein BCL-2, indicating that DEDT further promoted apoptosis following hyperglycemic stimulation. 3.3 DEDT Promotes Retinal Damage in Diabetic Rats and Inhibits the AMPK/Nrf2/HO-1 Signaling Pathway We observed that the retinal tissue structure in DEDT-treated diabetic rats exhibited more severe lesions compared to the DR group through HE staining, with disorganized cellular arrangement, suggesting that DEDT exacerbates diabetes-induced retinal damage (Fig. 3 A). Furthermore, DEDT significantly suppressed the expression of AMPK, Nrf2, and HO-1 proteins in the retinal tissue of diabetic rats (Figs. 3 B and 3 C). Similar results were observed for tight junction proteins ZO-1, Occludin, and Claudin-5 in Western blot analysis. As shown in Figs. 3 D and 3 E, tight junction protein expression was markedly reduced in the DEDT-treated group compared to the DR group, further confirming the destructive effect of DEDT on the retinal blood-retinal barrier. 3.4 DEDT Exacerbates Systemic and Ocular Oxidative Stress in Rats with Diabetic Retinopathy Compared to the NC and DR groups, the ROS levels in the heart, liver, spleen, lung, and kidney were significantly elevated in the DR + DEDT group (Fig. 4 A). When compared to the DR group, the ROS levels were notably higher in the DR + DEDT group, particularly in the retina and RPE/choroid. However, DEDT exposure did not result in significant changes in the ROS levels in the cornea of NC rats (Fig. 4 B). After DEDT treatment, levels of antioxidants (GSH, SOD, and CAT) were significantly reduced in the DR + DEDT group, while the lipid peroxidation product MDA was markedly increased, suggesting that DEDT further disrupts the redox balance in the diabetic retina (Fig. 4 C). 3.5 AICAR Reverses DEDT-Induced Retinal Damage by Activating AMPK Under high glucose conditions, the AMPK pathway is suppressed, and DEDT further inhibits the expression of key proteins in this pathway. However, the addition of the AMPK activator AICAR significantly restored cell viability in HRMECs under high glucose conditions (Fig. 5 A). As shown in Fig. 5 B, DEDT significantly reduced the mRNA expression levels of AMPK, Nrf2, and HO-1 under high glucose conditions, while AICAR treatment notably reversed these changes. Western blot analysis revealed that AICAR significantly restored the expression of AMPK, Nrf2, and HO-1. Furthermore, co-treatment with AICAR and DEDT reversed the inhibitory effect of DEDT on the AMPK/Nrf2/HO-1 signaling pathway (Fig. 5 C, 5 D). Moreover, AICAR treatment significantly reversed the inhibitory effect of DEDT on BCL-2 expression in the DR + DEDT group (Fig. 5 E) and also reversed the suppression of tight junction proteins (ZO-1, OCCLUDIN, and Claudin-5) induced by DEDT (Fig. 5 F, 5 G). Additionally, the levels of inflammatory cytokines IL-6 and IL-8 were significantly decreased in the AICAR + DR + DEDT group compared to the DR + DEDT group (Fig. 5 H). Discussion This study systematically elucidates the damaging effects of the organophosphorus pesticide DEDT on retinal cell function under diabetic hyperglycemic conditions and its underlying molecular mechanisms. Results demonstrate that DEDT significantly reduces the survival rates of HRMECs and ARPE-19 cells, exhibiting concentration-dependent and time-dependent effects under hyperglycemic conditions. Concurrently, DEDT treatment markedly downregulated the expression of tight junction proteins ZO-1, Occludin, and Claudin-5, increasing endothelial barrier permeability and indicating significant disruption of blood-retinal barrier integrity. At the molecular level, Western blot and qPCR results indicated that DEDT inhibits the activation of the AMPK/Nrf2/HO-1 signaling pathway. This weakens the cells' antioxidant defense capacity, increases ROS levels, and induces the release of inflammatory mediators (IL-6, TNF-α, IL-1β) along with the upregulation of the apoptosis-related protein Bax. Notably, the AMPK activator AICAR reversed these adverse effects by restoring Nrf2 and HO-1 expression while improving cell survival and barrier function, further validating the central role of the AMPK/Nrf2/HO-1 pathway in DEDT-mediated retinal damage. These findings indicate that DEDT promotes the onset and progression of diabetic retinopathy by inhibiting AMPK signaling activation, weakening cellular antioxidant responses, and disrupting the blood-retinal barrier. This provides new experimental evidence for understanding the mechanism by which environmental pollutants contribute to diabetic complications. Diethyl Phosphate (DEP) and Diethyl Dithiophosphate (DEDT) are the primary metabolites of organophosphate pesticides (OPs) in the human body, typically originating from environmental exposure, agricultural product residues, and occupational exposure. These metabolites serve as biomarkers for OPs exposure, reflecting the cumulative contact levels of individuals over time. Studies have shown that DEDT can affect the nervous system and metabolic processes ( 29 ). DEP, a hydrolysis product of OPs, has been found to interfere with neural conduction and immune system function, while DEDT, a characteristic metabolite of thiophosphate compounds, exhibits stronger toxicity ( 30 ). It may induce oxidative stress and inflammation, leading to damage to retinal vasculature. Although the effects of DEDT on ocular health are relatively understudied, existing experimental and epidemiological evidence suggests that OPs exposure may increase the risk of retinal and optic nerve diseases. Animal studies have shown that chronic low-dose DEDT exposure can damage retinal ganglion cells and disrupt retinal microcirculation ( 31 ). In contrast, DEDT, due to its thiophosphate structure, has higher lipophilicity, making it more readily absorbed by ocular tissues, where it induces reactive oxygen species (ROS) generation and causes microvascular damage to the retina ( 32 ). Despite the focus of ocular toxicology research primarily on neurotoxicity, studies on the effects of DEP and DEDT on retinal health remain limited. Previous epidemiological studies have mainly concentrated on their potential impacts on neurological disorders and diabetes. We clarified for the first time through a series of experimental methods that DEDT exposure promotes the development of DR, and discovered that DEDT exerts its toxic effects by inhibiting the AMPK/Nrf2/HO-1 pathway. This finding provides new epidemiological evidence for its potential ocular toxicity and offers critical data support for establishing stricter pesticide regulatory measures in the future. Given that diabetic patients already experience higher oxidative stress and inflammatory states, their susceptibility to environmental toxins may be higher than that of the general population. Therefore, our results suggest the need for enhanced environmental exposure monitoring in high-risk diabetic populations, particularly in agricultural communities with intensive pesticide use and in developing countries. Stricter pesticide control measures should be implemented to mitigate the adverse health effects of long-term low-dose exposure on diabetic individuals. This study has several significant advantages. First, we comprehensively evaluated the impact of DEDT on DR progression through a multi-angle experimental design. By examining multiple indicators including tight junction protein expression, oxidative stress, and inflammatory responses, this research deeply revealed the potential mechanisms of DEDT-induced retinal cell damage, thereby providing robust data support for the role of environmental pollutants in DR. Second, molecular biology experiments validated that DEDT exerts its retinal toxicity by modulating the AMPK/Nrf2/HO-1 signaling pathway. Furthermore, the combined use of cellular and animal models in the experimental design enhances the reliability and broad applicability of the findings. Although this study has made significant progress in exploring the mechanism of DEDT's effects on diabetic retinopathy, several limitations remain. First, while both diabetic rat models and human retinal microvascular endothelial cell models were employed, physiological differences between animal models and humans may impact the clinical translation of results. Diabetic rat models can simulate the pathological state of diabetes, but their immune responses, metabolic characteristics, and cellular functions may differ from those in humans. Therefore, additional animal models or preclinical studies are needed to validate the universality of the findings. Second, although the study employed a 40 µM DEDT concentration for treatment, it remains unclear whether this concentration accurately represents actual exposure levels in clinical or environmental settings. Future studies should further investigate the effects of DEDT on retinal damage across a range of concentrations to ensure the physiological relevance of experimental conditions. Furthermore, this study primarily focused on the short-term effects of DEDT on retinal cells, whereas diabetic retinopathy is a long-term chronic process. Consequently, chronic low-dose DEDT exposure may yield distinct biological effects. Future studies need to evaluate the sustained effects of DEDT on retinal damage under chronic exposure, particularly within a chronic diabetic environment. Finally, while this study examined the role of the AMPK/Nrf2/HO-1 signaling pathway, a comprehensive analysis of other potential mechanisms remains lacking. DEDT may contribute to DR progression through multiple signaling pathways. Consequently, future research should expand to investigate other relevant molecular pathways to achieve a more comprehensive understanding of DEDT's multifaceted role in DR. Conclusion This study firstly demonstrated that DEDT significantly accelerates diabetic retinopathy (DR) progression by inhibiting the AMPK/Nrf2/HO-1 signaling pathway. This showed as increased oxidative stress in retinal cells, decreased expression of tight junction proteins, impaired blood-retinal barrier function, heightened inflammatory responses, and increased apoptosis. In a high-sugar environment, DEDT exacerbates oxidative stress, inhibits AMPK activation, and consequently reduces Nrf2 upregulation, leading to decreased HO-1 expression. This impairs the effective clearance of harmful intracellular oxidants and inflammatory responses. This reflects abnormal changes in vascular endothelial function under hyperglycemic conditions, suggesting that prolonged exposure to environmental pollutants may exacerbate diabetic complications, carrying significant public health implications. This study provided novel theoretical support for early intervention in diabetic retinopathy and elucidated the impact of environmental pollutants on ocular health, offering potential targets for developing effective prevention and treatment strategies. Declarations Conflict of Interest Statement: None of the authors has any conflicts of interest to disclose. Funding: This study was supported by Anhui Medical University Youth Science Foundation (2022xkj026). Author Contribution BQD, SYG, XCW, YMT, JHW contributed equally in writing the manuscript and design the work. BQD analyzed and interpreted the patient data. SYG, XCW provided methodological support and software usage. YMT, JWS, YRL were responsible for writing the original draft. 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10:12:23","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":86004,"visible":true,"origin":"","legend":"","description":"","filename":"bdef570739f44697acc5677f320aaabd1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7849232/v1/9dcb5d224e09a3b97c80a5f2.xml"},{"id":96702659,"identity":"d3071066-9669-41f2-86b1-e458af9d8520","added_by":"auto","created_at":"2025-11-25 08:41:36","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":98348,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7849232/v1/841f613562e7446897ee9f26.html"},{"id":96702642,"identity":"5fd5be0e-d950-43df-a721-3db1b832d64a","added_by":"auto","created_at":"2025-11-25 08:41:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":582847,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDEDT impairs retinal cell viability and disrupts tight junction integrity.\u003c/strong\u003e (A-B) The effects of various concentrations of DEDT (0–50 μM) on cell viability under high glucose conditions were assessed in (A) HRMECs and (B) ARPE-19 cells. Data are presented as mean ± SD (n=8). (C-D) The time-dependent effects of 40 μM DEDT on cell viability under high glucose exposure were evaluated in (C) HRMECs and (D) ARPE-19 cells. (E-F) Protein expression levels of tight junction markers ZO-1, occludin, and claudin-5 were measured using WB analysis. (G) Transendothelial electrical resistance (TEER) was used to evaluate the barrier integrity of HRMECs treated with 40 μM DEDT under high glucose conditions at various time points. (H) Dextran permeability assay. TEER values were expressed in Ω·cm². Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, ns=not significant compared to the DR group.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7849232/v1/ab7eb3dfc54ede3a2048477e.png"},{"id":96702641,"identity":"0234d2e3-a004-4e0f-a98e-1b5e1e6d0247","added_by":"auto","created_at":"2025-11-25 08:41:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":338874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDEDT suppresses the AMPK/Nrf2/HO-1 signaling pathway and exacerbates inflammation and apoptosis in HRMECs.\u003c/strong\u003e (A) Effects of different doses of DEDT on ROS levels in HRMECs exposed to high glucose. (B-C) Western blot analysis and quantification of AMPK, Nrf2, and HO-1 expression. (D) ELISA measurement of inflammatory cytokines IL-6, TNF-α, and IL-1β in the culture supernatants. (E) Quantification of apoptotic-related proteins Bax and BCL-2 in HRMECs via ELISA analysis. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, ns=not significant compared to the DR group.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7849232/v1/db798f1180440c113c7e72a2.png"},{"id":96711253,"identity":"b6f1924b-0d35-48a1-aacf-8e5a88884835","added_by":"auto","created_at":"2025-11-25 10:11:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1338970,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDEDT exacerbates retinal damage and inhibits AMPK/Nrf2/HO-1 signaling pathway in diabetic rats. \u003c/strong\u003e(A) HE staining. (B-C) Western blot analysis of AMPK, Nrf2, and HO-1 protein expression. (D-E) Western blot analysis of tight junction proteins (ZO-1, Occludin, Claudin-5) in retinal tissue. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test (n=5, ****p\u0026lt;0.0001, ***p\u0026lt;0.001, **p\u0026lt;0.01, *p\u0026lt;0.05, ns=not significant).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7849232/v1/249a7d9aebf0495decb3a905.png"},{"id":96702643,"identity":"18906f54-4455-4df2-9082-4cd7d2729efc","added_by":"auto","created_at":"2025-11-25 08:41:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":268063,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDEDT exacerbates systemic and ocular oxidative stress in diabetic retinopathy rats. \u003c/strong\u003e(A) Quantitative analysis of reactive oxygen species (ROS) levels was performed in major systemic organs, including the heart, liver, spleen, lung, and kidney. (B) Tissue-specific ROS quantification was also conducted in ocular structures, including the whole eye, cornea, vitreous body, retina, and RPE/choroid layer. (C) Redox balance in the retina was evaluated by measuring oxidative stress markers and antioxidant defense components. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test (n=5, ****p\u0026lt;0.0001, ***p\u0026lt;0.001, **p\u0026lt;0.01, *p\u0026lt;0.05, ns=not significant).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7849232/v1/0d4329c0fb0ff25b871abee1.png"},{"id":96702644,"identity":"f76bd8f2-83ba-4c79-9dfa-c9701e2c2912","added_by":"auto","created_at":"2025-11-25 08:41:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":950382,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAICAR-mediated AMPK activation rescues DEDT-induced retinal damage. \u003c/strong\u003e(A) The effect of the AMPK activator AICAR on retinal cell viability under high glucose conditions with DEDT exposure. (B) Quantitative PCR was performed to analyze the mRNA expression of key components in the AMPK/Nrf2/HO-1 signaling pathway. (C-D) Protein levels of AMPK, Nrf2, and HO-1 were quantified by WB and quantitative analysis. (E) The expression of the anti-apoptotic protein BCL-2 was evaluated using ELISA. (F-G) Levels of tight junction proteins. (H) Inflammatory cytokines, including IL-6 and IL-8, were quantified by ELISA. Data are presented as mean ± SD (n=6). Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test, ****p\u0026lt;0.0001, ***p\u0026lt;0.001, **p\u0026lt;0.01, *p\u0026lt;0.05, ns=not significant. AICAR: AMPK activator.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7849232/v1/0aa4d52bf055f737b1427df3.png"},{"id":105223412,"identity":"4ec7b45c-2419-46dd-a04e-4994cd1d532e","added_by":"auto","created_at":"2026-03-23 16:05:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4463460,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7849232/v1/a09f3924-9c1e-421f-ac04-2ea36a2cde03.pdf"},{"id":96702663,"identity":"3a847c9d-0128-4292-ab65-932fe9e03be8","added_by":"auto","created_at":"2025-11-25 08:41:38","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":43070035,"visible":true,"origin":"","legend":"","description":"","filename":"10.19Supplemtarymaterial.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7849232/v1/b84a5f7609560bbbdeabcd83.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Organophosphate pesticide DEDT promotes diabetic retinopathy progression via AMPK/Nrf2/HO-1 pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOrganophosphate pesticides (OPs) are widely used in agriculture, household pest control and public health, causing neurotoxicity by inhibiting the enzyme Acetylcholinesterase (AChE), leading to excessive accumulation of acetylcholine in the nervous system (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). However, recent epidemiological and experimental studies suggest that long-term low-dose exposure to OPs may cause metabolic disturbances and endocrine disruption, increasing the risk of chronic diseases. Additionally, it may exacerbate the development of microvascular complications in diabetic patients through mechanisms such as oxidative stress and inflammation (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Eyes, as highly metabolically active and microvessel-rich organs, are susceptible to damage from environmental toxicants. Studies have shown that OPs can affect ocular health by inducing mitochondrial dysfunction and endothelial cell damage (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Long-term exposure to OPs has been linked to an increased risk of retinal degeneration, lens opacity, and vascular abnormalities, which may further exacerbate diabetic microvascular complications (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDiabetic retinopathy (DR) is one of the most common complications of diabetes and a leading cause of vision loss globally (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). With the rising prevalence of diabetes, the burden of DR is expected to increase, with over 100\u0026nbsp;million people projected to be affected by DR by 2030 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Studies show that the prevalence of DR in type 2 diabetes patients is approximately 35%, and even higher in type 1 diabetes patients (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The pathophysiology of DR is complex, primarily involving retinal microvascular damage due to prolonged hyperglycemia, leading to microvascular changes and neovascularization (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Early-stage DR typically presents with no noticeable symptoms, but as the condition progresses, patients may experience blurred vision, visual field defects, and even blindness (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Moreover, the role of environmental factors in DR has gained increasing attention. Hua et al. pointed out that the environmental toxin 2-Ethylhexyl diphenyl phosphate (EHDPP) might impact retinal microcirculation through metabolic disruption and inflammatory pathways, leading to impaired retinal photoreceptor function in mice, which could offer new directions for DR prevention strategies (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Previous studies have primarily focused on the role of heavy metals, air pollutants, and widely recognized endocrine-disrupting chemicals (EDCs), such as bisphenol A and phthalates, in the development of diabetic retinopathy (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). However, the potential contribution of organophosphate pesticide metabolites to DR risk remains unexplored (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn recent years, the AMPK/Nrf2/HO-1 signaling pathway has attracted significant attention from researchers due to its crucial role in regulating cellular stress responses, antioxidant defense, and protecting vascular barrier function. 5'-AMP-activated protein kinase (AMPK) serves as a key regulator of cellular energy metabolism. It enhances cellular antioxidant responses by activating nuclear factor erythroid 2-related factor 2 (Nrf2), which in turn induces heme oxygenase-1 (HO-1) expression to further mitigate oxidative stress and inflammatory responses (Small-molecule agonist AdipoRon alleviates diabetic retinopathy through the AdipoR1/AMPK/EGR4 pathway). Previous studies indicate that activation of the AMPK/Nrf2/HO-1 pathway mitigates hyperglycemia-induced endothelial cell damage and plays a vital role in preserving vascular barrier integrity (Regulation of AMPK and GAPDH by Transglutaminase 2 Plays a Pivotal Role in Microvascular Leakage in Diabetic Retinas). However, systematic investigations into this pathway's role in diabetic retinopathy remain scarce. Therefore, this study aims to explore the regulatory effects of an organophosphorus pesticide DEDT on the AMPK/Nrf2/HO-1 pathway and elucidate its potential mechanisms in DR progression. By thoroughly examining alterations in this signaling pathway, we expect to provide novel insights and therapeutic targets for the prevention and treatment of diabetic retinopathy.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cp\u003eStreptozotocin (STZ), Catalog: S0130, Sigma-Aldrich; Diethyl Dithiophosphate (DEDT), Catalog: 24617-47-0, ChemService; AICAR, Catalog: 99447-33-3, Tocris Bioscience; Cell Counting Kit-8 (CCK-8), Catalog: C0037, Beyotime Biotechnology; TRIzol Reagent, Catalog: 15596026, Thermo Fisher Scientific; SYBR Green Master Mix, Catalog: 4309155, Applied Biosystems; Cell Counting Kit-8 (CCK-8), Catalog: C0037, Beyotime Biotechnology; ZO-1 ELISA Kit, Catalog: PT518, Beyotime Biotechnology; Occludin ELISA Kit, Catalog: PT519, Beyotime Biotechnology; Claudin-5 ELISA Kit, Catalog: PT521, Beyotime Biotechnology; IL-6 ELISA Kit, Catalog: PI330, Beyotime Biotechnology; TNF-α ELISA Kit, Catalog: PI305, Beyotime Biotechnology; ; IL-8 ELISA Kit, Catalog: PI331, Beyotime Biotechnology; ; IL-1β ELISA Kit, Catalog: PI303, Beyotime Biotechnology; Bax ELISA Kit, Catalog: PA102, Beyotime Biotechnology; BCL-2 ELISA Kit, Catalog: PA101, Beyotime Biotechnology; Phospho-AMPK (Thr172) ELISA Kit, Catalog: PT428, Beyotime Biotechnology; Nrf2 ELISA Kit, Catalog: PT588, Beyotime Biotechnology; HO-1 ELISA Kit, Catalog: PT592, Beyotime Biotechnology; Glutathione (GSH) Assay Kit, Catalog: S0053, Beyotime Biotechnology; Malondialdehyde (MDA) test kit, Catalog: S0131S, Beyotime Biotechnology; Superoxide dismutase (SOD) test kit, Catalog: S0101, Beyotime Biotechnology; Catalase (CAT) test kit, Catalog: S0051, Beyotime Biotechnology.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Animal Model and Treatments\u003c/h2\u003e\u003cp\u003eSprague-Dawley rats (8\u0026ndash;10 weeks old, 250-300g) were obtained from the Animal Experiment Center of Anhui Medical University and were maintained under appropriate conditions. Animals were randomly divided into four experimental groups: normal control (NC, n\u0026thinsp;=\u0026thinsp;5), NC with DEDT exposure (NC\u0026thinsp;+\u0026thinsp;DEDT, n\u0026thinsp;=\u0026thinsp;5), DR model (DR, n\u0026thinsp;=\u0026thinsp;5), and DR with DEDT exposure (DR\u0026thinsp;+\u0026thinsp;DEDT, n\u0026thinsp;=\u0026thinsp;5). Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) (55 mg/kg), and animals with sustained blood glucose levels\u0026thinsp;\u0026gt;\u0026thinsp;16.7 mmol/L for 3 consecutive days were included in the study. Based on previous toxicological studies, DEDT (10 mg/kg) was administered daily by oral gavage for 12 days (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). At the end of the experiment, all SD rats were deeply anesthetized with 3\u0026ndash;5% isofluran and maintained at 1.5-2.0%. Once complete loss of pedal reflex was confirmed, euthanasia was performed by cervical dislocation. Death was confirmed by the absence of heartbeat and respiration before tissue collection. All animal procedures were approved by the Ethics Committee of Anhui Medical University and were conducted in accordance with ARRIVE guidelines and other relevant guidelines and regulations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Cell Culture and Treatments\u003c/h2\u003e\u003cp\u003eHuman retinal microvascular endothelial cells (HRMECs) and adult retinal pigment epithelial-19 (ARPE-19) cells were cultured under standard conditions. HRMEC cells was purchased from Procell Life Science \u0026amp; Technology Co., Ltd. (Wuhan, China; Catalog No. CP-H130) and ARPE-19 cells was purchased from ATCC (Catalog No. CRL-2302). To mimic diabetic conditions, cells were maintained in medium containing 50 mM glucose (high glucose, HG), with 5.5 mM glucose serving as normal control (NC). Cells were treated with varying concentrations of DEDT (0\u0026ndash;50 \u0026micro;M) for 0\u0026ndash;48 hours. For rescue experiments, cells were co-treated with the AMPK activator AICAR (30 \u0026micro;M) for 2 h and then treated with HG for 24 h.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Cell Viability Assay (CCK-8)\u003c/h2\u003e\u003cp\u003eCell viability was evaluated using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan) following the manufacturer\u0026rsquo;s protocol. Cells were seeded in 96-well plates and treated as indicated. After treatment, 10 \u0026micro;L of CCK-8 reagent was added to each well and incubated for 2 hours at 37\u0026deg;C. The absorbance was measured at 450 nm using a microplate reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Trans-Epithelial/Endothelial Electrical Resistance (TEER) Measurement\u003c/h2\u003e\u003cp\u003eTEER values were measured to assess barrier integrity of HRMECs monolayers cultured on Transwell inserts (0.4 \u0026micro;m pore size). Cells were treated with DEDT for different time points, and TEER was measured using an EVOM2 voltohmmeter (World Precision Instruments). Resistance values were normalized to the surface area and expressed in Ω\u0026middot;cm\u0026sup2;.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Dextran Permeability Assay\u003c/h2\u003e\u003cp\u003eHRMECs were cultured in high-glucose medium, then seeded into 24-well plates and cultured for 24 hours until reaching appropriate density. Subsequently, cells were exposed to high glucose and DEDT (40 \u0026micro;M) in different treatment groups, with experiments conducted after 48 hours of treatment. To assess blood-retinal barrier integrity, dextran solution (0.1 mg/mL) was added to the wells and incubated at 37\u0026deg;C for 1 hour. Supernatant was collected periodically throughout the experiment, and dextran absorbance was measured at 490 nm using a UV spectrophotometer to record transmittance at different time points.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Enzyme-Linked Immunosorbent Assay (ELISA)\u003c/h2\u003e\u003cp\u003eELISA kits (R\u0026amp;D Systems, USA) were used to quantify protein levels of tight junction proteins (ZO-1, occludin, claudin-5) and inflammatory cytokines (IL-6, IL-8, IL-1β, TNF-α), AMPK, p-AMPK, Nrf2, HO-1, Bax, and BCL-2 in cell lysates or supernatants. Samples were processed according to the manufacturer's instructions, and absorbance was measured at 450 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 RNA Extraction and Quantitative Real-Time PCR (qPCR)\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from treated cells using TRIzol reagent (Invitrogen, USA). cDNA synthesis was performed using a reverse transcription kit (Takara, Japan). qPCR was carried out with SYBR Green Master Mix (Applied Biosystems) on a StepOnePlus\u0026trade; Real-Time PCR system. Gene expression was normalized to GAPDH and analyzed using the 2^\u0026ndash;ΔΔCt method. Primer sequences are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Measurement of Oxidative Stress Markers in Retinal Tissue\u003c/h2\u003e\u003cp\u003eRetinal tissues were homogenized in ice-cold lysis buffer, and oxidative stress markers including glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were quantified using corresponding colorimetric assay kits (Beyotime, China) according to the manufacturers\u0026rsquo; protocols.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Quantification of Reactive Oxygen Species (ROS) in Tissues\u003c/h2\u003e\u003cp\u003eROS levels were measured in systemic tissues (heart, liver, spleen, lung, kidney) and various ocular tissues (whole eye, cornea, vitreous body, retina, RPE/choroid). Tissues were homogenized, and ROS levels were assessed using a fluorescent ROS detection assay kit (DCFH-DA based, Nanjing Jiancheng Bioengineering Institute). Fluorescence intensity was detected at Ex/Em\u0026thinsp;=\u0026thinsp;488/525 nm using a microplate reader and normalized to protein content.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e\u003cp\u003eAll experiments had at least six independent replicates. Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical analyses were performed using GraphPad Prism software, and comparisons were analyzed by one-way or multifactor ANOVA followed by Tukey's post-hoc test, with p-values less than 0.05 considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 DEDT Exacerbates High Glucose-Induced Decrease in Retinal Cell Activity and Barrier Function Impairment\u003c/h2\u003e\u003cp\u003eIn HRMECs, cell viability decreased in a dose-dependent manner with increasing concentrations of DEDT (5\u0026ndash;50 \u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). A similar trend was observed in ARPE-19 cells, where DEDT exposure also led to a marked reduction in cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Furthermore, prolonged exposure to DEDT resulted in a time-dependent decline in cell viability in both HRMECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and ARPE-19 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In addition, the expression levels of tight junction proteins including ZO-1, OCCLUDIN, and Claudin-5 were significantly reduced in the DR\u0026thinsp;+\u0026thinsp;DEDT group compared to the DR group, indicating that DEDT exacerbates the disruption of retinal barrier integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Moreover, TEER measurements demonstrated a progressive decline beginning at 3 hours after 40 \u0026micro;M DEDT exposure, indicating a time-dependent impairment of the cellular barrier function (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Simultaneously, results from the dextran permeability assay demonstrated a significant increase in the permeability of the cell monolayer to dextran (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), further validating the disruptive effect of DEDT on the endothelial barrier.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2 DEDT Suppresses the AMPK/Nrf2/HO-1 Signaling Pathway and Exacerbates Oxidative Damage in Retinal Cells Under High-Sugar Conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eROS detection results showed that DEDT significantly increased ROS levels in HRMECs under high glucose conditions, with the highest concentration exhibiting the most pronounced effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Western blot analysis revealed that in DR group, AMPK, Nrf2, and HO-1 protein expression decreased compared to the control group and was significantly suppressed in the DR\u0026thinsp;+\u0026thinsp;DEDT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), indicating that DEDT significantly impacts the AMPK/Nrf2/HO-1 signaling pathway, exacerbating oxidative stress. Following DEDT treatment under hyperglycemic conditions, the levels of pro-inflammatory cytokines (including IL-6, IL-1β, and TNF-α) in HRMECs were also significantly elevated, further confirming its pro-inflammatory effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, DEDT treatment not only enhanced the expression of the pro-apoptotic protein Bax but also suppressed the expression of the anti-apoptotic protein BCL-2, indicating that DEDT further promoted apoptosis following hyperglycemic stimulation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3 DEDT Promotes Retinal Damage in Diabetic Rats and Inhibits the AMPK/Nrf2/HO-1 Signaling Pathway\u003c/h2\u003e\u003cp\u003eWe observed that the retinal tissue structure in DEDT-treated diabetic rats exhibited more severe lesions compared to the DR group through HE staining, with disorganized cellular arrangement, suggesting that DEDT exacerbates diabetes-induced retinal damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Furthermore, DEDT significantly suppressed the expression of AMPK, Nrf2, and HO-1 proteins in the retinal tissue of diabetic rats (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Similar results were observed for tight junction proteins ZO-1, Occludin, and Claudin-5 in Western blot analysis. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, tight junction protein expression was markedly reduced in the DEDT-treated group compared to the DR group, further confirming the destructive effect of DEDT on the retinal blood-retinal barrier.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4 DEDT Exacerbates Systemic and Ocular Oxidative Stress in Rats with Diabetic Retinopathy\u003c/h2\u003e\u003cp\u003eCompared to the NC and DR groups, the ROS levels in the heart, liver, spleen, lung, and kidney were significantly elevated in the DR\u0026thinsp;+\u0026thinsp;DEDT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). When compared to the DR group, the ROS levels were notably higher in the DR\u0026thinsp;+\u0026thinsp;DEDT group, particularly in the retina and RPE/choroid. However, DEDT exposure did not result in significant changes in the ROS levels in the cornea of NC rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). After DEDT treatment, levels of antioxidants (GSH, SOD, and CAT) were significantly reduced in the DR\u0026thinsp;+\u0026thinsp;DEDT group, while the lipid peroxidation product MDA was markedly increased, suggesting that DEDT further disrupts the redox balance in the diabetic retina (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.5 AICAR Reverses DEDT-Induced Retinal Damage by Activating AMPK\u003c/h2\u003e\u003cp\u003eUnder high glucose conditions, the AMPK pathway is suppressed, and DEDT further inhibits the expression of key proteins in this pathway. However, the addition of the AMPK activator AICAR significantly restored cell viability in HRMECs under high glucose conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, DEDT significantly reduced the mRNA expression levels of AMPK, Nrf2, and HO-1 under high glucose conditions, while AICAR treatment notably reversed these changes. Western blot analysis revealed that AICAR significantly restored the expression of AMPK, Nrf2, and HO-1. Furthermore, co-treatment with AICAR and DEDT reversed the inhibitory effect of DEDT on the AMPK/Nrf2/HO-1 signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Moreover, AICAR treatment significantly reversed the inhibitory effect of DEDT on BCL-2 expression in the DR\u0026thinsp;+\u0026thinsp;DEDT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) and also reversed the suppression of tight junction proteins (ZO-1, OCCLUDIN, and Claudin-5) induced by DEDT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Additionally, the levels of inflammatory cytokines IL-6 and IL-8 were significantly decreased in the AICAR\u0026thinsp;+\u0026thinsp;DR\u0026thinsp;+\u0026thinsp;DEDT group compared to the DR\u0026thinsp;+\u0026thinsp;DEDT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study systematically elucidates the damaging effects of the organophosphorus pesticide DEDT on retinal cell function under diabetic hyperglycemic conditions and its underlying molecular mechanisms. Results demonstrate that DEDT significantly reduces the survival rates of HRMECs and ARPE-19 cells, exhibiting concentration-dependent and time-dependent effects under hyperglycemic conditions. Concurrently, DEDT treatment markedly downregulated the expression of tight junction proteins ZO-1, Occludin, and Claudin-5, increasing endothelial barrier permeability and indicating significant disruption of blood-retinal barrier integrity. At the molecular level, Western blot and qPCR results indicated that DEDT inhibits the activation of the AMPK/Nrf2/HO-1 signaling pathway. This weakens the cells' antioxidant defense capacity, increases ROS levels, and induces the release of inflammatory mediators (IL-6, TNF-α, IL-1β) along with the upregulation of the apoptosis-related protein Bax. Notably, the AMPK activator AICAR reversed these adverse effects by restoring Nrf2 and HO-1 expression while improving cell survival and barrier function, further validating the central role of the AMPK/Nrf2/HO-1 pathway in DEDT-mediated retinal damage. These findings indicate that DEDT promotes the onset and progression of diabetic retinopathy by inhibiting AMPK signaling activation, weakening cellular antioxidant responses, and disrupting the blood-retinal barrier. This provides new experimental evidence for understanding the mechanism by which environmental pollutants contribute to diabetic complications.\u003c/p\u003e\u003cp\u003eDiethyl Phosphate (DEP) and Diethyl Dithiophosphate (DEDT) are the primary metabolites of organophosphate pesticides (OPs) in the human body, typically originating from environmental exposure, agricultural product residues, and occupational exposure. These metabolites serve as biomarkers for OPs exposure, reflecting the cumulative contact levels of individuals over time. Studies have shown that DEDT can affect the nervous system and metabolic processes (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). DEP, a hydrolysis product of OPs, has been found to interfere with neural conduction and immune system function, while DEDT, a characteristic metabolite of thiophosphate compounds, exhibits stronger toxicity (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). It may induce oxidative stress and inflammation, leading to damage to retinal vasculature. Although the effects of DEDT on ocular health are relatively understudied, existing experimental and epidemiological evidence suggests that OPs exposure may increase the risk of retinal and optic nerve diseases. Animal studies have shown that chronic low-dose DEDT exposure can damage retinal ganglion cells and disrupt retinal microcirculation (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). In contrast, DEDT, due to its thiophosphate structure, has higher lipophilicity, making it more readily absorbed by ocular tissues, where it induces reactive oxygen species (ROS) generation and causes microvascular damage to the retina (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Despite the focus of ocular toxicology research primarily on neurotoxicity, studies on the effects of DEP and DEDT on retinal health remain limited. Previous epidemiological studies have mainly concentrated on their potential impacts on neurological disorders and diabetes. We clarified for the first time through a series of experimental methods that DEDT exposure promotes the development of DR, and discovered that DEDT exerts its toxic effects by inhibiting the AMPK/Nrf2/HO-1 pathway. This finding provides new epidemiological evidence for its potential ocular toxicity and offers critical data support for establishing stricter pesticide regulatory measures in the future. Given that diabetic patients already experience higher oxidative stress and inflammatory states, their susceptibility to environmental toxins may be higher than that of the general population. Therefore, our results suggest the need for enhanced environmental exposure monitoring in high-risk diabetic populations, particularly in agricultural communities with intensive pesticide use and in developing countries. Stricter pesticide control measures should be implemented to mitigate the adverse health effects of long-term low-dose exposure on diabetic individuals.\u003c/p\u003e\u003cp\u003eThis study has several significant advantages. First, we comprehensively evaluated the impact of DEDT on DR progression through a multi-angle experimental design. By examining multiple indicators including tight junction protein expression, oxidative stress, and inflammatory responses, this research deeply revealed the potential mechanisms of DEDT-induced retinal cell damage, thereby providing robust data support for the role of environmental pollutants in DR. Second, molecular biology experiments validated that DEDT exerts its retinal toxicity by modulating the AMPK/Nrf2/HO-1 signaling pathway. Furthermore, the combined use of cellular and animal models in the experimental design enhances the reliability and broad applicability of the findings.\u003c/p\u003e\u003cp\u003eAlthough this study has made significant progress in exploring the mechanism of DEDT's effects on diabetic retinopathy, several limitations remain. First, while both diabetic rat models and human retinal microvascular endothelial cell models were employed, physiological differences between animal models and humans may impact the clinical translation of results. Diabetic rat models can simulate the pathological state of diabetes, but their immune responses, metabolic characteristics, and cellular functions may differ from those in humans. Therefore, additional animal models or preclinical studies are needed to validate the universality of the findings. Second, although the study employed a 40 \u0026micro;M DEDT concentration for treatment, it remains unclear whether this concentration accurately represents actual exposure levels in clinical or environmental settings. Future studies should further investigate the effects of DEDT on retinal damage across a range of concentrations to ensure the physiological relevance of experimental conditions. Furthermore, this study primarily focused on the short-term effects of DEDT on retinal cells, whereas diabetic retinopathy is a long-term chronic process. Consequently, chronic low-dose DEDT exposure may yield distinct biological effects. Future studies need to evaluate the sustained effects of DEDT on retinal damage under chronic exposure, particularly within a chronic diabetic environment. Finally, while this study examined the role of the AMPK/Nrf2/HO-1 signaling pathway, a comprehensive analysis of other potential mechanisms remains lacking. DEDT may contribute to DR progression through multiple signaling pathways. Consequently, future research should expand to investigate other relevant molecular pathways to achieve a more comprehensive understanding of DEDT's multifaceted role in DR.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study firstly demonstrated that DEDT significantly accelerates diabetic retinopathy (DR) progression by inhibiting the AMPK/Nrf2/HO-1 signaling pathway. This showed as increased oxidative stress in retinal cells, decreased expression of tight junction proteins, impaired blood-retinal barrier function, heightened inflammatory responses, and increased apoptosis. In a high-sugar environment, DEDT exacerbates oxidative stress, inhibits AMPK activation, and consequently reduces Nrf2 upregulation, leading to decreased HO-1 expression. This impairs the effective clearance of harmful intracellular oxidants and inflammatory responses. This reflects abnormal changes in vascular endothelial function under hyperglycemic conditions, suggesting that prolonged exposure to environmental pollutants may exacerbate diabetic complications, carrying significant public health implications. This study provided novel theoretical support for early intervention in diabetic retinopathy and elucidated the impact of environmental pollutants on ocular health, offering potential targets for developing effective prevention and treatment strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of Interest Statement:\u003c/h2\u003e\u003cp\u003eNone of the authors has any conflicts of interest to disclose.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis study was supported by Anhui Medical University Youth Science Foundation (2022xkj026).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eBQD, SYG, XCW, YMT, JHW contributed equally in writing the manuscript and design the work. BQD analyzed and interpreted the patient data. SYG, XCW provided methodological support and software usage. YMT, JWS, YRL were responsible for writing the original draft. ZXJ, LMT reviewed and substantively revised the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data included in this study can be obtained upon request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMatsushita T, Fujita Y, Omori K, Huang Y, Matsui Y, Shirasaki N. Effect of chlorination on anti-acetylcholinesterase activity of organophosphorus insecticide solutions and contributions of the parent insecticides and their oxons to the activity. Chemosphere. 2020;261:127743.\u003c/li\u003e\n\u003cli\u003eLang Q, Qin X, Yu X, Wei S, Wei J, Zhang M, et al. Association of joint exposure to organophosphorus flame retardants and phthalate acid esters with gestational diabetes mellitus: a nested case-control study. 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Organophosphate esters exposure in relation to glucose homeostasis and type 2 diabetes in adults: A national cross-sectional study from the national health and nutrition survey. Chemosphere. 2022;301:134669.\u003c/li\u003e\n\u003cli\u003eJayatilaka NK, Restrepo P, Davis Z, Vidal M, Calafat AM, Ospina M. Quantification of 16 urinary biomarkers of exposure to flame retardants, plasticizers, and organophosphate insecticides for biomonitoring studies. Chemosphere. 2019;235:481-91.\u003c/li\u003e\n\u003cli\u003eSchaap-Fogler M, Bahar I, Rephaeli A, Dahbash M, Nudelman A, Livny E, et al. Effect of Histone Deacetylase Inhibitor, Butyroyloxymethyl-Diethyl Phosphate (AN-7), on Corneal Neovascularization in a Mouse Model. J Ocul Pharmacol Ther. 2017;33(6):480-6.\u003c/li\u003e\n\u003cli\u003eOlivares-Banuelos TN, Martinez-Hernandez I, Hernandez-Kelly LC, Chi-Castaneda D, Vega L, Ortega A. The neurotoxin diethyl dithiophosphate impairs glutamate transport in cultured Bergmann glia cells. 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JAMA Ophthalmol. 2024;142(3):171-7.\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":"Diabetic retinopathy, AMPK/Nrf2/HO-1, organophosphate pesticides, toxic effects","lastPublishedDoi":"10.21203/rs.3.rs-7849232/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7849232/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe impact of environmental toxins, particularly organophosphate pesticides (OPs), on the progression of diabetic retinopathy (DR) remains insufficiently understood. Recent studies have highlighted the potential role of environmental pollutants in exacerbating diabetic complications, but the underlying mechanisms are still unclear. This study aims to explore the effect of diethyldithiophosphate (DEDT), an OP, on DR progression through modulation of the AMPK/Nrf2/HO-1 signaling pathway.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eHuman retinal microvascular endothelial cells (HRMECs) and retinal pigment epithelial cells (ARPE-19) were cultured under high-glucose conditions to simulate diabetic stress. Cells were exposed to various concentrations of DEDT, and their viability, oxidative stress, tight junction integrity, and inflammation were assessed. Western blot, quantitative PCR, and enzyme-linked immunosorbent assay (ELISA) techniques were employed to evaluate the expression of key proteins in the AMPK/Nrf2/HO-1 pathway and inflammatory cytokines. In vivo, diabetic rat models were treated with DEDT to assess retinal damage and oxidative stress. The effects of AMPK activation were also evaluated using AICAR, an AMPK activator, to further explore the mechanistic role of AMPK/Nrf2/HO-1 signaling.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eOur results demonstrated that DEDT exposure significantly reduces retinal cell viability and disrupts tight junction proteins (ZO-1, Occludin, Claudin-5) under high-glucose conditions. Mechanistically, DEDT inhibited the AMPK/Nrf2/HO-1 pathway, leading to increased oxidative stress, enhanced inflammation, and elevated levels of apoptotic markers (Bax and Bcl-2). In vivo, DEDT exposure exacerbated retinal damage and oxidative stress in diabetic rats. Activation of AMPK by AICAR reversed these effects, restoring Nrf2 and HO-1 expression, improving cell viability, and protecting the blood-retinal barrier. These findings indicated that DEDT promotes DR progression by disrupting the AMPK/Nrf2/HO-1 signaling pathway.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThis study provided experimental evidence that DEDT accelerates diabetic retinopathy progression via inhibition of the AMPK/Nrf2/HO-1 pathway, contributing to increased oxidative stress and retinal barrier dysfunction. Our results emphasized the potential health risks associated with pesticide exposure, particularly in diabetic populations, and highlight the importance of regulating environmental toxins to prevent exacerbation of diabetic complications.\u003c/p\u003e","manuscriptTitle":"Organophosphate pesticide DEDT promotes diabetic retinopathy progression via AMPK/Nrf2/HO-1 pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-25 08:41:31","doi":"10.21203/rs.3.rs-7849232/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-04T11:23:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-04T05:32:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-28T07:50:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"249786026125176678918605493564643828184","date":"2025-11-18T06:32:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"57862325043686510394421400369656447477","date":"2025-11-14T10:31:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-14T07:27:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-14T07:23:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-10T12:35:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-22T12:54:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-22T08:15:25+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":"00b8094a-8316-43ca-a415-a2f6bb8ad1f5","owner":[],"postedDate":"November 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58484933,"name":"Biological sciences/Biochemistry"},{"id":58484934,"name":"Biological sciences/Cell biology"},{"id":58484935,"name":"Health sciences/Diseases"},{"id":58484936,"name":"Health sciences/Endocrinology"},{"id":58484937,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-03-23T16:02:02+00:00","versionOfRecord":{"articleIdentity":"rs-7849232","link":"https://doi.org/10.1038/s41598-026-37183-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-16 15:57:43","publishedOnDateReadable":"March 16th, 2026"},"versionCreatedAt":"2025-11-25 08:41:31","video":"","vorDoi":"10.1038/s41598-026-37183-w","vorDoiUrl":"https://doi.org/10.1038/s41598-026-37183-w","workflowStages":[]},"version":"v1","identity":"rs-7849232","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7849232","identity":"rs-7849232","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}