Organophosphate pesticide exerts toxic effect on the optic nerve of glaucoma rats by promoting oxidative stress and inflammation

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Beyond intraocular pressure (IOP), environmental risk factors like pesticide exposure are increasingly implicated. Dimethyl phosphate (DMP), a key metabolite of organophosphorus pesticides, accumulates in the body and exhibits systemic toxicity. However, its direct role and mechanism in glaucoma pathogenesis remain entirely unexplored. Methods We investigated the impact of DMP on glaucoma progression using a rat glaucoma model. Animals were subjected to DMP exposure at varying concentrations. We assessed IOP, optic nerve thickness, and expression of neurotrophic factors (NGF, BDNF). Molecular mechanisms were elucidated via Western blotting for key signaling pathways and apoptosis/inflammation markers, complemented by ELISA for oxidative stress. Functional validation was performed using specific pathway agonists and inhibitors. Results DMP exposure exacerbated core glaucomatous pathology in a concentration-dependent manner, significantly elevating IOP, reducing optic nerve thickness, and downregulating NGF/BDNF. Mechanistically, DMP concurrently inhibited the pro-survival PI3K/Akt pathway while activating the pro-inflammatory JAK/STAT and NF-κB pathways, and the fibrotic Wnt/β-catenin pathway. This multi-pathway disruption synergistically amplified retinal oxidative stress and triggered RGC apoptosis. Rescue experiments confirmed that the modulation of these specific pathways directly influenced the observed oxidative injury and cellular damage. Conclusion This study provides the first evidence that dimethyl phosphate (DMP), a common organophosphorus metabolite, promotes glaucoma progression by regulating a multi-pathway signaling network that ultimately converges on oxidative stress and apoptosis. This work redefines DMP as a novel environmental risk factor for glaucoma, reveals its complex mechanism of action, and offers new perspectives for optic nerve protection strategies and environmental risk assessment. Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Drug discovery Biological sciences/Neuroscience Glaucoma Organophosphate exposure Retinal ganglion cell apoptosis Oxidative stress Neurodegeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction Glaucoma, a degenerative disease of the optic nerve, is characterized by optic disc depression, apoptosis of retinal ganglion cells, and loss of vision ( 1 ). The global prevalence of glaucoma is estimated to be approximately 3.5% among people aged 40 to 80 years. With the growing global elderly population, the number of glaucoma patients is expected to surge to 111.8 million by 2040, which will make it one of the leading causes of irreversible visual disability worldwide, posing a serious public health challenge ( 2 ). The main common types of glaucoma are primary open-angle glaucoma (POAG), primary closed-angle glaucoma (PCAG), and normal-tension glaucoma (NTG) ( 3 ). Although the pathophysiologic mechanisms of glaucoma are not fully understood, elevated intraocular pressure (IOP) is recognized as one of the major risk factors for the development of glaucoma. However, many glaucoma patients continue to progress even when treated with IOP-lowering therapy or when IOP itself is not high, prompting researchers to look for other possible interventions to slow or stop further damage in glaucoma ( 4 ). Organophosphorus compounds, which are extremely hazardous environmental chemicals, including pesticides used in agriculture and chemical warfare nerve agents (CWNAs), are often exposed to humans through occupational exposure or suicidal ingestion ( 5 ). As irreversible inhibitors of cholinesterase, poisoning by organophosphates leads to accumulation of acetylcholine, which in turn triggers hyperactivation of the cholinergic pathway, producing a variety of toxic effects. These acute symptoms include miosis, gastrointestinal and respiratory distress, muscle twitching, and prolonged seizures ( 5 , 6 ). However, because organophosphorus pesticides (OPPs) are inexpensive, effective, and easy to break down, they have been widely used in agriculture, horticulture, and the home in recent decades and have largely replaced organochlorine pesticides as the basic pesticide. Existing studies have not provided clear evidence of a link between organophosphate exposure and the development of glaucoma. Dimethyl phosphate (DMP) is one of the most important metabolites of organophosphorus pesticides in the human body. It is widely derived from the environmental degradation of common pesticides such as chlorpyrifos and dichlorvos, agricultural product residues, and occupational exposure. In recent years, numerous studies have revealed that DMP has independent toxic effects, the core mechanism of which is closely related to the inhibition of acetylcholinesterase activity and the induction of oxidative stress. In a neurological disease model, Wang et al. demonstrated that DMP exposure impaired children's neurodevelopment( 7 ). In addition, in a liver toxicity study, Li et al. found that DMP can destabilize the lipid bilayer of the cell membrane by peroxidizing polyunsaturated fatty acids, ultimately leading to liver cell damage( 8 ). Although there is currently no research directly exploring the relationship between DMP and eye diseases, oxidative stress has been proven to be the core pathogenic link of blinding eye diseases such as age-related macular degeneration( 9 ), diabetic retinopathy( 10 ) and glaucoma( 11 ). Therefore, based on the clear evidence that DMP promotes oxidative damage in nerve and liver tissues, we propose a scientific hypothesis: DMP may also play an important role in optic nerve degenerative diseases such as glaucoma by disrupting the redox balance in the retina and participating in the damage process of key structures such as retinal ganglion cells. This study aimed to investigate the association between DMP and glaucoma progression. We first examined intraocular pressure and oxidative stress levels after in vivo administration. We then used Western blot and ELISA to assess oxidative stress and apoptosis in animal models and validated the relevant signaling pathways. Furthermore, to verify the association between pathways and DMP treatment, we used pathway inhibitors. Our results demonstrated that DMP treatment promoted increased IOP, providing favorable evidence that DMP accelerates glaucoma progression. This study also offers new insights into clinical diagnosis and treatment. 2 Materials and Methods 2.1 Materials and Animals Hypertonic Saline (Sigma-Aldrich, S9888), RIPA Buffer (Thermo Fisher Scientific, 89900), BCA Protein Assay Kit (Thermo Fisher Scientific, 23225), Primary Antibodies: iNOS, ab15323 (Abcam); TNF-α, ab6671 (Abcam); IL-6, ab9324 (Abcam); IL-8, ab7747 (Abcam); IL-1β, ab9722 (Abcam); Bcl-2, ab32124 (Abcam); Caspase-3, ab13847 (Abcam); Caspase-8, ab25901 (Abcam); p-PI3K, ab182651 (Abcam); p-AKT, ab38449 (Abcam); p-mTOR, ab109268 (Abcam); Wnt3a, ab219412 (Abcam); Wnt7a, ab199252 (Abcam); β-catenin, ab32572 (Abcam); p-p65, ab86299 (Abcam); p-IκBα, ab133462 (Abcam); p-JAK1, ab138005 (Abcam); p-JAK2, ab32101 (Abcam); p-STAT1, ab109461 (Abcam); p-STAT3, ab76315 (Abcam); β-actin, ab8226 (Abcam). Secondary Antibodies (HRP-conjugated): Anti-Rabbit IgG, ab6721 (Abcam); Anti-Mouse IgG, ab6789 (Abcam). MDA (Malondialdehyde) ELISA Kit (Abcam, ab118970), Catalase (CAT) Activity Assay Kit (Abcam, ab83464), Glutathione Peroxidase (GPx) Assay Kit (Abcam, ab102530). Rat NGF (Nerve Growth Factor) ELISA Kit (Abcam, ab99986). Rat BDNF (Brain-Derived Neurotrophic Factor) ELISA Kit (Abcam, ab99978). Tonometer (Icare Finland, TonoLab), Optical Coherence Tomography (OCT) (Heidelberg Engineering). DMP (Sigma-Aldrich, D16659) is dissolved in saline to achieve the desired concentration (5mg/kg and 10mg/kg). Adult Sprague-Dawley (SD) rats were used in this study. The animals were housed under standard laboratory conditions (12-hour light/dark cycle, 22 ± 2°C, 50 ± 10% humidity) with free access to food and water. The animal experiments involved in this study were approved by the Ethics Committee of Anhui Medical University. All experiments were performed in accordance with ARRIVE guidelines and other relevant guidelines and regulations. 2.2 Construction of Animal Models All animal experiments were carried out in accordance with the relevant laws and guidelines of the Laboratory Animal Center of Anhui Medical University. Male Sprague–Dawley rats (8–10 weeks old, weighing 280–320 g) were obtained from the Experimental Animal Center of Anhui Medical University and maintained under standard conditions (22 ± 2°C, 12 h light/dark cycle, free access to food and water).Glaucoma was induced by injecting hypertonic saline into an extrascleral vein to elevate IOP. IOP was measured weekly using an IOP meter to confirm modeling success. Rats with a sustained IOP elevation of ≥ 25% compared with baseline were considered successful in modeling ( 12 ). Twenty-four hours later, low-dose (5 mg/kg) and high-dose (10 mg/kg) DMP were administered via tail vein injection once daily for 28 consecutive days. Grouping: Rats were randomly divided into four groups (n = 5 per group): Normal Control (NC): Rats that received equal amounts of saline. Glaucoma model (Glu): rats with experimentally induced glaucoma. Glu + DMP (low): Glaucoma model rats receiving low dose of DMP (5mg/kg). Glu + DMP (high): Glaucoma model rats receiving high dose of DMP (10mg/kg). In addition, the dosing regimen of inhibitors for different signaling pathways including ZSTK474 (PI3K inhibitor, 25 mg/kg), ETC-159 (Wnt inhibitor, 10 mg/kg), BAY 11-7082 (p65 inhibitor, 5 mg/kg), Baricitinib (Jak inhibitor, 10 mg/kg). At the same time, the above inhibitors were administered by gavage. This study was approved by the Ethics Committee of Anhui Medical University. The experimental procedures were conducted following the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals: 2020 Edition to ensure humane endpoints and ethical compliance. After all experimental procedures, rats were humanely euthanized via intraperitoneal injection of sodium pentobarbital (≥ 150 mg/kg; Sigma-Aldrich, Cat. No. P3761), which corresponds to approximately three times the anesthetic dose recommended by the AVMA for laboratory rodents. This method induces rapid loss of consciousness followed by respiratory and cardiac arrest through central nervous system depression. Death was confirmed by the absence of heartbeat and respiratory movement, loss of corneal reflex, and the onset of rigor mortis. The average body weight of rats at the time of euthanasia was approximately 300 ± 20 g. All procedures were carried out by trained personnel under veterinary supervision. 2.3 Measurement of IOP and Retinal Nerve Fiber Layer (RNFL) Thickness IOP was measured weekly using a rebound tonometer. The average of three readings was recorded for each eye. RNFL thickness was assessed using optical coherence tomography (OCT) at the end of the experiment. 2.4 Western Blot Analysis Retinal and optic nerve tissues were collected and homogenized in RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using the BCA assay. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against iNOS, TNF-α, IL-6, IL-8, IL-1β, Bcl-2, Caspase-3, Caspase-8, p-PI3K, p-AKT, p-mTOR, Wnt3a, Wnt7a, β-catenin, p-p65, p-IκBα, p-JAK1, p-JAK2, p-STAT1, and p-STAT3. β-actin was used as a loading control. Band intensities were quantified using ImageJ software and normalized to β-actin. 2.5 ELISA Analysis The retinal tissue were homogenized and the supernatant were collected for ELISA. MDA, GSH and GPx levels were measured using the corresponding ELISA kits according to the instructions. The concentration of target proteins were measured at specific wavelength using the Microplate Reader. 2.6 Statistical Analysis Animal experimental data were expressed as mean ± SEM. Statistical significance was analyzed by one-way or two-way ANOVA followed by Tukey's post hoc test. We used GraphPad Prism 8.0 for statistical analysis and graphing. p values less than 0.05 were considered statistically significant. 3 Results 3.1 DMP Exacerbates Optic Nerve Injury and Reduces Neurotrophic Factors in rats with glaucoma Firstly, we validated the toxic effects of DMP in animal models. The results showed that IOP was significantly elevated in the glaucoma model group (Glu) compared to the normal control (NC) group (p < 0.0001). With the increase of DMP concentration, the IOP of glaucoma rats was further elevated in a dose-dependent manner (Fig. 1 A). In addition, the thickness of the optic nerve fiber layer was significantly reduced in the glaucoma group, with significant difference compared with NC group (p < 0.0001). DMP treatment further exacerbated the reduction of the optic nerve fiber layer thickness, especially in the high-dose group (p < 0.0001) (Fig. 1 B). We examined the expression of optic neurotrophic factors in rats by ELISA.The expression of both NGF and BNDF were significantly lower in the Glu group than in the NC group (p < 0.0001) (Fig. 1 C, 1 D). The DMP intervention further reduced the expression level of optic neurotrophic factors, which was relatively lowest in the high-dose group (p < 0.0001). 3.2 DMP Promotes Inflammation and Apoptosis of Optic Nerve in vivo We found that the expression of inflammation-related proteins (iNOS, TNF-α, IL-6, IL-8, IL-1β) in the optic nerve of glaucoma rats were significantly higher than that of the control group (p < 0.0001). Moreover, inflammatory proteins were increased about 1-fold in the Glu + DMP(high) group compared to the Glu group (Fig. 2 A, 2 B). DMP also promoted the expression of tissue apoptotic proteins Bax, Caspase-3, and Caspase-8 in glaucomatous rats, as well as significantly down-regulated the level of anti-apoptotic protein Bcl-2 (p < 0.0001) (Fig. 2 C, 2 D). 3.3 DMP affects multiple signaling pathways in optic nerve damage in glaucomatous rats WB analysis showed that DMP affected the expression of key proteins related to PI3K/AKT/mTOR, Wnt/β-catenin, NF-κB, and JAK/STAT pathways in rat optic nerve tissues. In the glaucoma group, the phosphorylation levels of PI3K, AKT, and mTOR were significantly reduced compared with NC, and DMP intervention further inhibited the expression of these phosphorylated proteins (p < 0.0001) (Fig. 3 A, 3 B). Similarly, the expression of Wnt3a, Wnt7a, and β-catenin in the Wnt/β-catenin pathway was significantly downregulated in the Glu group, and their low expression was exacerbated by DMP exposure (p < 0.0001), suggesting that optic nerve regeneration and repair may be impaired (Fig. 3 C, 3 D). In addition, the NF-κB pathway was overactivated in the Glu group, as evidenced by increased phosphorylation levels of p65 and IκBα (p < 0.0001) (Fig. 3 E, 3 F). In the JAK/STAT pathway, the phosphorylation levels of JAK1, JAK2, STAT1, and STAT3 were elevated in the Glu group (p < 0.0001), but the phosphorylated proteins were higher in the DMP group (Fig. 3 G, 3 H). In addition, we verified the corresponding inhibitors for the mechanism pathway verified above. The experimental results showed that after the action of ZSTK474, the phosphorylation levels of PI3K, AKT, and mTOR were significantly increased compared with Glu and Glu + DMP groups (Fig. 4 A, 4 B). Moreover, We found that after applying ETC-159, the protein levels of Wnt3a, Wnt7a and β-catenin were significantly recreased compared with Glu and Glu + DMP groups (Fig. 4 C, 4 D). Meanwhile, The experimental results of BAY 11-7082 and Baricitinib were similar to those of inhibitor 2 (Fig. 4 E-H), which further indicated that organophosphate exposure accelerated the progression of glaucoma through the above pathway. 3.4 Pathway-Dependent Modulation of Oxidative Stress and Intraocular Pressure by DMP Based on experimental validation of the aforementioned pathways, we further investigated the effects of DMP on oxidative stress levels and IOP in the presence of different pathway inhibitors. After application of ZSTK474, MDA, SOD and IOP levels were significantly reduced, and Gpx expression was significantly increased, compared to the Glu group and the Clu + DMP group (Fig. 5 A, B). Similar results were observed with BAY 11-7082 (Fig. 5 C, D) and Baricitinib (Fig. 5 E, F), indicating that organophosphate exposure significantly affects oxidative stress levels through the aforementioned pathways. 4 Discussion This study revealed the relationship between the organophosphorus pesticide metabolite DMP and glaucoma progression. Experimental results showed that in a glaucoma rat model, DMP increased intraocular pressure, reduced optic nerve thickness, and decreased NGF and BNDF levels in a concentration-dependent manner. Western blot analysis revealed that DMP significantly increased the levels of inflammation- and apoptosis-related proteins in the glaucoma rat model, inhibited the PI3K/Akt pathway, and promoted the JAK/STAT, NF-κB, and Wnt/β-catenin pathways. Functional validation experiments, including ELISA, demonstrated that DMP significantly increased oxidative stress levels in the retina of glaucomatous rats through these pathways. These findings suggest that DMP promotes oxidative stress and apoptosis by regulating multiple signaling pathways, thereby driving the development and progression of glaucoma. This discovery provides new experimental evidence for a deeper understanding of the effects and mechanisms of organophosphorus pesticide exposure on the body. Glaucoma is a progressive optic neuropathy characterized by retinal ganglion cell degeneration and resulting optic nerve head changes ( 13 ). In addition to oxidative stress ( 11 ), retinal ischemia-reperfusion injury ( 14 ) and hypertension ( 15 ), organophosphorus pesticide exposure is increasingly being considered as a risk factor for glaucoma. Organophosphorus pesticides inhibit the enzyme acetylcholinesterase, causing hyperexcitability of the nervous system, which may impede the regulation of intraocular pressure, which in turn affects glaucoma ( 16 ). Organophosphorus pesticide exposure inhibits acetylcholinesterase (AChE) at synapses and neuromuscular junctions in the cholinergic pathway, and some organophosphorus pesticides even cause permanent inactivation of phosphorylated AchE, leading to accumulation of acetylcholine and post-synaptic hyperexcitability of muscarinic and nicotinic receptors ( 16 ). Low concentrations of acetylcholine can lower intraocular pressure by shrinking the pupil and improving aqueous flow. However, high levels of acetylcholine over-contract the iris sphincter, increase the resistance to aqueous drainage, and increase intraocular pressure, which in turn contributes to the development of glaucoma. Given the adverse effects of organophosphorus pesticide exposure on the body, it is particularly important to explore the effects and mechanisms of its main metabolites in the human body. Dimethyl phosphate (DMP) is the primary metabolite of organophosphorus pesticides in the human body. It is usually derived from environmental exposure, agricultural product residues, and occupational exposure, and can reflect an individual's cumulative exposure to OPs over time. Studies have shown that DMP is neurotoxic ( 7 ), and its mechanism involves inducing oxidative stress ( 17 ), and interfering with neurotransmitter function ( 18 ). In addition, DMP can also cause liver toxicity by destroying the antioxidant system, damaging the cell membrane structure and causing enzyme loss. ( 8 ). Although the effects of DMP on eye health are relatively understudied, existing experimental and epidemiological evidence suggests that DMP may increase the risk of glaucoma by inhibiting acetylcholinesterase activity in retinal and optic nerve tissue, leading to impaired neural signaling and abnormal IOP. Through a series of experiments, we clearly confirmed for the first time that DMP exposure promotes the development of glaucoma by inhibiting the PI3K/Akt pathway and promoting the JAK/STAT, NF-κB, Wnt/β-catenin and other pathways to exert toxic effects. The pathogenic logic of this multi-pathway effect may be that the PI3K/Akt pathway is an important survival signal in cells( 19 ). Its inhibition leads to a decrease in the stress resistance of retinal ganglion cells and promotes the initiation of the apoptosis program. As classic inflammatory signaling centers, the JAK/STAT and NF-κB pathways( 20 ), whose sustained activation will promote retinal inflammatory responses. Abnormal activation of the Wnt/β-catenin pathway is often associated with tissue fibrosis and pathological remodeling( 21 ). In glaucoma disease models, it may be involved in fibrotic changes in the trabecular meshwork, thereby increasing aqueous humor outflow resistance. The disruption of these pathways ultimately exacerbates oxidative stress in the retina, forming a vicious cycle that ultimately leads to irreversible apoptosis of retinal ganglion cells. This study provides new laboratory evidence for the potential ocular toxicity of pesticides and provides key data support for the development of stricter pesticide regulatory measures in the future. This study has several significant strengths. First, through a multi-faceted experimental design, we reveal for the first time the effects of DMP on glaucoma progression. Second, by examining inflammation- and apoptosis-related proteins and markers of oxidative stress, this study provides insight into the underlying mechanisms of DMP-induced retinal ganglion cell damage, providing strong evidence supporting the role of environmental pollutants in glaucoma. Finally, molecular biology experiments combined with functional validation experiments demonstrate that DMP exerts its retinal toxicity by modulating multiple signaling pathways. While this study represents significant progress in exploring the effects and mechanisms of DMP in glaucoma, it still has several limitations. First, we did not use primary cultured retinal ganglion cells for in vitro experiments, lacking cell-specific evidence for molecular changes. Second, while this study identified DMP effects on multiple pathways, it remains unclear whether these pathways are upstream and downstream or synergistic. Further rescue experiments will be conducted to identify the dominant upstream pathway. Furthermore, although two DMP concentrations (5 mg/kg and 10 mg/kg) were used, it is unclear whether these concentrations accurately represent actual exposure levels in clinical or environmental settings. Future studies should further investigate the effects of DMP on optic nerve damage at varying concentrations to ensure the physiological relevance of the experimental conditions. Finally, this study was conducted entirely in an animal model. Future studies investigating DMP levels in aqueous humor or serum samples from glaucoma patients and correlating them with disease severity would greatly enhance the clinical significance and impact of this research. 5 Conclusion Our study demonstrates that the organophosphorus metabolite dimethyl phosphate (DMP) promotes glaucoma progression in a rat model by increasing intraocular pressure, reducing optic nerve thickness, and depleting neurotrophic factors (NGF, BDNF) in a dose-dependent manner. Mechanistically, DMP dysregulates multiple signaling cascades, suppressing the PI3K/Akt pathway while activating JAK/STAT, NF-κB and Wnt/β-catenin signaling, thereby amplifying inflammation, oxidative stress and apoptotic responses in retinal tissue; these effects were corroborated by Western blot, ELISA and pharmacological inhibitor experiments. Together, the findings provide novel mechanistic evidence that environmental exposure to DMP can compromise optic nerve homeostasis and drive glaucoma-relevant pathology, underscoring the need for epidemiological investigation of DMP exposure in patients and for consideration of exposure limits in regulatory policy. Future work should define pathway hierarchy through rescue experiments, assess dose relevance using clinically representative exposure levels, and validate associations between DMP concentrations in biological fluids and glaucoma severity to strengthen translational and public-health implications. Declarations Data Availability Statement: The data supporting the findings of this study are available from the corresponding author upon reasonable request. Conflict of Interest Statement: None of the authors has any conflicts of interest to disclose. Funding: Not applicable Author Contributions: BJC, YMT, SYG, XCW contributed equally in writing the manuscript and design the work. XCS analyzed the experimental data. XCW. provided methodological support. YMT were responsible for writing the original draft. SW reviewed and substantively revised the manuscript. All authors reviewed the manuscript. Acknowledgments: We thank the Department of Ophthalmology of the Second Affiliated Hospital of Anhui Medical University for their collaborative and logistical work. References Bou Ghanem GO, Wareham LK, Calkins DJ. Addressing neurodegeneration in glaucoma: Mechanisms, challenges, and treatments. Prog Retin Eye Res. 2024;100:101261. Zhang Y, Zhao Z, Ma Q, Li K, Zhao X, Jia Z. 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11:53:06","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":67839,"visible":true,"origin":"","legend":"","description":"","filename":"8b2561fa8a534acd8f00ebb40979b28f1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7917001/v1/271ef394f0b6530b854f499f.xml"},{"id":96364769,"identity":"5986f5e9-26a9-44a4-8888-c7d749f38855","added_by":"auto","created_at":"2025-11-20 10:09:37","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":76501,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7917001/v1/b1a2b0275a7f44994dd585c2.html"},{"id":96284208,"identity":"b62de933-8806-4b77-9e05-ca612de1887a","added_by":"auto","created_at":"2025-11-19 11:53:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":566604,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eToxic effects of the organophosphorus insecticide DMP on the optic nerve of rats with glaucoma\u003c/strong\u003e. (A) Intraocular pressure (IOP) of normal control (NC) and glaucoma model (Glu) rats using different concentrations of DMP. (B) Retinal nerve fiber layer (RNFL) thickness in rats using different concentrations of DMP. (C) Expression of nerve growth factor (NGF) in rat optic nerve by ELISA. (D) Expression of brain-derived neurotrophic factor (BDNF) in rat optic nerve using ELISA. Each group consisted of n = 5 rats. Data are presented as mean ± SEM. Glu: glaucoma; Low DMP: 5mg/kg; High DMP: 10mg/kg. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001 compared to control.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7917001/v1/584fd1e3f094e791df9e942a.png"},{"id":96284212,"identity":"63fa33ee-71a7-4a3d-bc66-1d455c2e9fb5","added_by":"auto","created_at":"2025-11-19 11:53:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6961489,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of DMP on the Expression of Inflammation- and Apoptosis-Related Proteins\u003c/strong\u003e. (A-B) Western blot analysis and quantification of inflammation-related proteins (iNOS, TNF-α, IL-6, IL-8, IL-1β) in the optic nerve of glaucoma rats with different concentrations of DMP. (C-D) Western blot analysis and quantitative results of apoptosis-related proteins (Bax, Bcl-2, Caspase-3, Caspase-8) in the optic nerve of glaucoma rats. Each group n = 5 rats. Data are presented as mean ± SEM. Compared with control, * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7917001/v1/a62d513f31469efd2d680167.png"},{"id":96284228,"identity":"a6351a1f-5bcd-42e4-bbd4-4c6c71d4b99b","added_by":"auto","created_at":"2025-11-19 11:53:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10215150,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDMP modulates the expression of critical proteins in PI3K/AKT, JAK/STAT, NF-κB and Wnt/β-catenin signaling pathways within glaucoma model\u003c/strong\u003e. (A-B) Western blot analysis and quantitative results of PI3K/AKT/mTOR pathway proteins (p-PI3K, p-AKT, p-mTOR) in rat optic nerve tissue. (C-D) Western blot analysis and quantitative results of Wnt/β-catenin pathway proteins (Wnt3a, Wnt7a, β-catenin) in rat optic nerve tissue. (E-F) Western blot analysis and quantitative results of NF-κB pathway proteins (p-p65, p-IκBα) in rat optic nerve tissue. (G-H) Western blot analysis and quantification of JAK/STAT pathway proteins (p-JAK1, p-JAK2, p-STAT1, p-STAT3) in rat optic nerve tissue. Each group n = 5 rats. Data are presented as mean ± SEM. Glu: glaucoma; Low DMP: 5mg/kg; High DMP: 10mg/kg. Compared with the control group, * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7917001/v1/d3d59c2f97de6b1c442891f2.png"},{"id":96363699,"identity":"276e7aae-7eac-44a7-94a0-0a743a361aee","added_by":"auto","created_at":"2025-11-20 10:07:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1511122,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDMP Exacerbates Glaucoma Progression by Modulating Multiple Signaling Pathways\u003c/strong\u003e. (A-B) Western blot analysis and quantitative results of PI3K/AKT/mTOR pathway proteins (p-PI3K, p-AKT, p-mTOR) in rat optic nerve tissue. (C-D) Western blot analysis and quantitative results of Wnt/β-catenin pathway proteins (Wnt3a, Wnt7a, β-catenin) in rat optic nerve tissue. (E-F) Western blot analysis and quantitative results of NF-κB pathway proteins (p-p65, p-IκBα) in rat optic nerve tissue. (G-H) Western blot analysis and quantification of JAK/STAT pathway proteins (p-JAK1, p-JAK2, p-STAT1, p-STAT3) in rat optic nerve tissue. Each group n = 5 rats. Data are presented as mean ± SEM. Glu: glaucoma; DMP: 5mg/kg; ZSTK474: PI3K inhibitor, 25 mg/kg; ECT-159: Wnt inhibitor, 10 mg/kg; BAY 11-7082: p65 inhibitor, 5 mg/kg; Baricitinib: Jak inhibitor, 10 mg/kg. Compared with the control group, * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7917001/v1/b5d5955b228b71ee4c7667fe.png"},{"id":96284215,"identity":"559f8036-3e85-4140-8da7-53392bdf6c08","added_by":"auto","created_at":"2025-11-19 11:53:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1114774,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of DMP on oxidative stress levels in different pathways\u003c/strong\u003e. (A) The effects of different DMP concentrations on MDA, SOD, and Gpx expression levels were evaluated using ELISA. (B, C) ELISA was employed to measure MDA, SOD, and Gpx expression levels and IOP changes after DMP and ZSTK474 treatment. (D, E) ELISA was used to assess MDA, SOD, and Gpx expression levels and IOP changes following DMP and BAY 11-7082 administration. (F, G) ELISA was applied to evaluate MDA, SOD, and Gpx expression levels and IOP changes after DMP and Baricitinib treatment. Each group consisted of n = 5 rats. Data are presented as mean ± SEM. Compared with the control group, * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7917001/v1/85bc051379c34986a8705964.png"},{"id":96369306,"identity":"061fb88b-6914-4dd4-8b59-c0e23f57f473","added_by":"auto","created_at":"2025-11-20 10:20:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21205518,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7917001/v1/2ba4e003-4c12-4b66-9c01-f68cfc5f25fe.pdf"},{"id":96284232,"identity":"75402aa4-1410-4e71-8bcc-e275840227f7","added_by":"auto","created_at":"2025-11-19 11:53:08","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":52335416,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryDatasetFile.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7917001/v1/9db3ea89e534371b32f3d0fd.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Organophosphate pesticide exerts toxic effect on the optic nerve of glaucoma rats by promoting oxidative stress and inflammation","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eGlaucoma, a degenerative disease of the optic nerve, is characterized by optic disc depression, apoptosis of retinal ganglion cells, and loss of vision (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The global prevalence of glaucoma is estimated to be approximately 3.5% among people aged 40 to 80 years. With the growing global elderly population, the number of glaucoma patients is expected to surge to 111.8\u0026nbsp;million by 2040, which will make it one of the leading causes of irreversible visual disability worldwide, posing a serious public health challenge (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The main common types of glaucoma are primary open-angle glaucoma (POAG), primary closed-angle glaucoma (PCAG), and normal-tension glaucoma (NTG) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Although the pathophysiologic mechanisms of glaucoma are not fully understood, elevated intraocular pressure (IOP) is recognized as one of the major risk factors for the development of glaucoma. However, many glaucoma patients continue to progress even when treated with IOP-lowering therapy or when IOP itself is not high, prompting researchers to look for other possible interventions to slow or stop further damage in glaucoma (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOrganophosphorus compounds, which are extremely hazardous environmental chemicals, including pesticides used in agriculture and chemical warfare nerve agents (CWNAs), are often exposed to humans through occupational exposure or suicidal ingestion (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). As irreversible inhibitors of cholinesterase, poisoning by organophosphates leads to accumulation of acetylcholine, which in turn triggers hyperactivation of the cholinergic pathway, producing a variety of toxic effects. These acute symptoms include miosis, gastrointestinal and respiratory distress, muscle twitching, and prolonged seizures (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). However, because organophosphorus pesticides (OPPs) are inexpensive, effective, and easy to break down, they have been widely used in agriculture, horticulture, and the home in recent decades and have largely replaced organochlorine pesticides as the basic pesticide. Existing studies have not provided clear evidence of a link between organophosphate exposure and the development of glaucoma.\u003c/p\u003e\u003cp\u003eDimethyl phosphate (DMP) is one of the most important metabolites of organophosphorus pesticides in the human body. It is widely derived from the environmental degradation of common pesticides such as chlorpyrifos and dichlorvos, agricultural product residues, and occupational exposure. In recent years, numerous studies have revealed that DMP has independent toxic effects, the core mechanism of which is closely related to the inhibition of acetylcholinesterase activity and the induction of oxidative stress. In a neurological disease model, Wang et al. demonstrated that DMP exposure impaired children's neurodevelopment(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In addition, in a liver toxicity study, Li et al. found that DMP can destabilize the lipid bilayer of the cell membrane by peroxidizing polyunsaturated fatty acids, ultimately leading to liver cell damage(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Although there is currently no research directly exploring the relationship between DMP and eye diseases, oxidative stress has been proven to be the core pathogenic link of blinding eye diseases such as age-related macular degeneration(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), diabetic retinopathy(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) and glaucoma(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Therefore, based on the clear evidence that DMP promotes oxidative damage in nerve and liver tissues, we propose a scientific hypothesis: DMP may also play an important role in optic nerve degenerative diseases such as glaucoma by disrupting the redox balance in the retina and participating in the damage process of key structures such as retinal ganglion cells.\u003c/p\u003e\u003cp\u003eThis study aimed to investigate the association between DMP and glaucoma progression. We first examined intraocular pressure and oxidative stress levels after in vivo administration. We then used Western blot and ELISA to assess oxidative stress and apoptosis in animal models and validated the relevant signaling pathways. Furthermore, to verify the association between pathways and DMP treatment, we used pathway inhibitors. Our results demonstrated that DMP treatment promoted increased IOP, providing favorable evidence that DMP accelerates glaucoma progression. This study also offers new insights into clinical diagnosis and treatment.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials and Animals\u003c/h2\u003e\u003cp\u003eHypertonic Saline (Sigma-Aldrich, S9888), RIPA Buffer (Thermo Fisher Scientific, 89900), BCA Protein Assay Kit (Thermo Fisher Scientific, 23225), Primary Antibodies: iNOS, ab15323 (Abcam); TNF-α, ab6671 (Abcam); IL-6, ab9324 (Abcam); IL-8, ab7747 (Abcam); IL-1β, ab9722 (Abcam); Bcl-2, ab32124 (Abcam); Caspase-3, ab13847 (Abcam); Caspase-8, ab25901 (Abcam); p-PI3K, ab182651 (Abcam); p-AKT, ab38449 (Abcam); p-mTOR, ab109268 (Abcam); Wnt3a, ab219412 (Abcam); Wnt7a, ab199252 (Abcam); β-catenin, ab32572 (Abcam); p-p65, ab86299 (Abcam); p-IκBα, ab133462 (Abcam); p-JAK1, ab138005 (Abcam); p-JAK2, ab32101 (Abcam); p-STAT1, ab109461 (Abcam); p-STAT3, ab76315 (Abcam); β-actin, ab8226 (Abcam). Secondary Antibodies (HRP-conjugated): Anti-Rabbit IgG, ab6721 (Abcam); Anti-Mouse IgG, ab6789 (Abcam). MDA (Malondialdehyde) ELISA Kit (Abcam, ab118970), Catalase (CAT) Activity Assay Kit (Abcam, ab83464), Glutathione Peroxidase (GPx) Assay Kit (Abcam, ab102530). Rat NGF (Nerve Growth Factor) ELISA Kit (Abcam, ab99986). Rat BDNF (Brain-Derived Neurotrophic Factor) ELISA Kit (Abcam, ab99978). Tonometer (Icare Finland, TonoLab), Optical Coherence Tomography (OCT) (Heidelberg Engineering).\u003c/p\u003e\u003cp\u003eDMP (Sigma-Aldrich, D16659) is dissolved in saline to achieve the desired concentration (5mg/kg and 10mg/kg). Adult Sprague-Dawley (SD) rats were used in this study. The animals were housed under standard laboratory conditions (12-hour light/dark cycle, 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 50\u0026thinsp;\u0026plusmn;\u0026thinsp;10% humidity) with free access to food and water. The animal experiments involved in this study were approved by the Ethics Committee of Anhui Medical University. All experiments were performed 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 Construction of Animal Models\u003c/h2\u003e\u003cp\u003e All animal experiments were carried out in accordance with the relevant laws and guidelines of the Laboratory Animal Center of Anhui Medical University. Male Sprague\u0026ndash;Dawley rats (8\u0026ndash;10 weeks old, weighing 280\u0026ndash;320 g) were obtained from the Experimental Animal Center of Anhui Medical University and maintained under standard conditions (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 12 h light/dark cycle, free access to food and water).Glaucoma was induced by injecting hypertonic saline into an extrascleral vein to elevate IOP. IOP was measured weekly using an IOP meter to confirm modeling success. Rats with a sustained IOP elevation of \u0026ge;\u0026thinsp;25% compared with baseline were considered successful in modeling (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Twenty-four hours later, low-dose (5 mg/kg) and high-dose (10 mg/kg) DMP were administered via tail vein injection once daily for 28 consecutive days. Grouping: Rats were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;5 per group): Normal Control (NC): Rats that received equal amounts of saline. Glaucoma model (Glu): rats with experimentally induced glaucoma. Glu\u0026thinsp;+\u0026thinsp;DMP (low): Glaucoma model rats receiving low dose of DMP (5mg/kg). Glu\u0026thinsp;+\u0026thinsp;DMP (high): Glaucoma model rats receiving high dose of DMP (10mg/kg). In addition, the dosing regimen of inhibitors for different signaling pathways including ZSTK474 (PI3K inhibitor, 25 mg/kg), ETC-159 (Wnt inhibitor, 10 mg/kg), BAY 11-7082 (p65 inhibitor, 5 mg/kg), Baricitinib (Jak inhibitor, 10 mg/kg). At the same time, the above inhibitors were administered by gavage. This study was approved by the Ethics Committee of Anhui Medical University. The experimental procedures were conducted following the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals: 2020 Edition to ensure humane endpoints and ethical compliance. After all experimental procedures, rats were humanely euthanized via intraperitoneal injection of sodium pentobarbital (\u0026ge;\u0026thinsp;150 mg/kg; Sigma-Aldrich, Cat. No. P3761), which corresponds to approximately three times the anesthetic dose recommended by the AVMA for laboratory rodents. This method induces rapid loss of consciousness followed by respiratory and cardiac arrest through central nervous system depression. Death was confirmed by the absence of heartbeat and respiratory movement, loss of corneal reflex, and the onset of rigor mortis. The average body weight of rats at the time of euthanasia was approximately 300\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g. All procedures were carried out by trained personnel under veterinary supervision.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Measurement of IOP and Retinal Nerve Fiber Layer (RNFL) Thickness\u003c/h2\u003e\u003cp\u003eIOP was measured weekly using a rebound tonometer. The average of three readings was recorded for each eye. RNFL thickness was assessed using optical coherence tomography (OCT) at the end of the experiment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Western Blot Analysis\u003c/h2\u003e\u003cp\u003eRetinal and optic nerve tissues were collected and homogenized in RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using the BCA assay. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against iNOS, TNF-α, IL-6, IL-8, IL-1β, Bcl-2, Caspase-3, Caspase-8, p-PI3K, p-AKT, p-mTOR, Wnt3a, Wnt7a, β-catenin, p-p65, p-IκBα, p-JAK1, p-JAK2, p-STAT1, and p-STAT3. β-actin was used as a loading control. Band intensities were quantified using ImageJ software and normalized to β-actin.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 ELISA Analysis\u003c/h2\u003e\u003cp\u003eThe retinal tissue were homogenized and the supernatant were collected for ELISA. MDA, GSH and GPx levels were measured using the corresponding ELISA kits according to the instructions. The concentration of target proteins were measured at specific wavelength using the Microplate Reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e\u003cp\u003eAnimal experimental data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical significance was analyzed by one-way or two-way ANOVA followed by Tukey's post hoc test. We used GraphPad Prism 8.0 for statistical analysis and graphing. p values less than 0.05 were considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 DMP Exacerbates Optic Nerve Injury and Reduces Neurotrophic Factors in rats with glaucoma\u003c/h2\u003e\u003cp\u003eFirstly, we validated the toxic effects of DMP in animal models. The results showed that IOP was significantly elevated in the glaucoma model group (Glu) compared to the normal control (NC) group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). With the increase of DMP concentration, the IOP of glaucoma rats was further elevated in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In addition, the thickness of the optic nerve fiber layer was significantly reduced in the glaucoma group, with significant difference compared with NC group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). DMP treatment further exacerbated the reduction of the optic nerve fiber layer thickness, especially in the high-dose group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). We examined the expression of optic neurotrophic factors in rats by ELISA.The expression of both NGF and BNDF were significantly lower in the Glu group than in the NC group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The DMP intervention further reduced the expression level of optic neurotrophic factors, which was relatively lowest in the high-dose group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 DMP Promotes Inflammation and Apoptosis of Optic Nerve in vivo\u003c/h2\u003e\u003cp\u003eWe found that the expression of inflammation-related proteins (iNOS, TNF-α, IL-6, IL-8, IL-1β) in the optic nerve of glaucoma rats were significantly higher than that of the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Moreover, inflammatory proteins were increased about 1-fold in the Glu\u0026thinsp;+\u0026thinsp;DMP(high) group compared to the Glu group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). DMP also promoted the expression of tissue apoptotic proteins Bax, Caspase-3, and Caspase-8 in glaucomatous rats, as well as significantly down-regulated the level of anti-apoptotic protein Bcl-2 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 DMP affects multiple signaling pathways in optic nerve damage in glaucomatous rats\u003c/h2\u003e\u003cp\u003eWB analysis showed that DMP affected the expression of key proteins related to PI3K/AKT/mTOR, Wnt/β-catenin, NF-κB, and JAK/STAT pathways in rat optic nerve tissues. In the glaucoma group, the phosphorylation levels of PI3K, AKT, and mTOR were significantly reduced compared with NC, and DMP intervention further inhibited the expression of these phosphorylated proteins (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Similarly, the expression of Wnt3a, Wnt7a, and β-catenin in the Wnt/β-catenin pathway was significantly downregulated in the Glu group, and their low expression was exacerbated by DMP exposure (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), suggesting that optic nerve regeneration and repair may be impaired (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In addition, the NF-κB pathway was overactivated in the Glu group, as evidenced by increased phosphorylation levels of p65 and IκBα (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). In the JAK/STAT pathway, the phosphorylation levels of JAK1, JAK2, STAT1, and STAT3 were elevated in the Glu group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), but the phosphorylated proteins were higher in the DMP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn addition, we verified the corresponding inhibitors for the mechanism pathway verified above. The experimental results showed that after the action of ZSTK474, the phosphorylation levels of PI3K, AKT, and mTOR were significantly increased compared with Glu and Glu\u0026thinsp;+\u0026thinsp;DMP groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Moreover, We found that after applying ETC-159, the protein levels of Wnt3a, Wnt7a and β-catenin were significantly recreased compared with Glu and Glu\u0026thinsp;+\u0026thinsp;DMP groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Meanwhile, The experimental results of BAY 11-7082 and Baricitinib were similar to those of inhibitor 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-H), which further indicated that organophosphate exposure accelerated the progression of glaucoma through the above pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Pathway-Dependent Modulation of Oxidative Stress and Intraocular Pressure by DMP\u003c/h2\u003e\u003cp\u003eBased on experimental validation of the aforementioned pathways, we further investigated the effects of DMP on oxidative stress levels and IOP in the presence of different pathway inhibitors. After application of ZSTK474, MDA, SOD and IOP levels were significantly reduced, and Gpx expression was significantly increased, compared to the Glu group and the Clu\u0026thinsp;+\u0026thinsp;DMP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Similar results were observed with BAY 11-7082 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D) and Baricitinib (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F), indicating that organophosphate exposure significantly affects oxidative stress levels through the aforementioned pathways.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThis study revealed the relationship between the organophosphorus pesticide metabolite DMP and glaucoma progression. Experimental results showed that in a glaucoma rat model, DMP increased intraocular pressure, reduced optic nerve thickness, and decreased NGF and BNDF levels in a concentration-dependent manner. Western blot analysis revealed that DMP significantly increased the levels of inflammation- and apoptosis-related proteins in the glaucoma rat model, inhibited the PI3K/Akt pathway, and promoted the JAK/STAT, NF-κB, and Wnt/β-catenin pathways. Functional validation experiments, including ELISA, demonstrated that DMP significantly increased oxidative stress levels in the retina of glaucomatous rats through these pathways. These findings suggest that DMP promotes oxidative stress and apoptosis by regulating multiple signaling pathways, thereby driving the development and progression of glaucoma. This discovery provides new experimental evidence for a deeper understanding of the effects and mechanisms of organophosphorus pesticide exposure on the body.\u003c/p\u003e\u003cp\u003eGlaucoma is a progressive optic neuropathy characterized by retinal ganglion cell degeneration and resulting optic nerve head changes (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In addition to oxidative stress (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), retinal ischemia-reperfusion injury (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) and hypertension (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), organophosphorus pesticide exposure is increasingly being considered as a risk factor for glaucoma. Organophosphorus pesticides inhibit the enzyme acetylcholinesterase, causing hyperexcitability of the nervous system, which may impede the regulation of intraocular pressure, which in turn affects glaucoma (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Organophosphorus pesticide exposure inhibits acetylcholinesterase (AChE) at synapses and neuromuscular junctions in the cholinergic pathway, and some organophosphorus pesticides even cause permanent inactivation of phosphorylated AchE, leading to accumulation of acetylcholine and post-synaptic hyperexcitability of muscarinic and nicotinic receptors (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Low concentrations of acetylcholine can lower intraocular pressure by shrinking the pupil and improving aqueous flow. However, high levels of acetylcholine over-contract the iris sphincter, increase the resistance to aqueous drainage, and increase intraocular pressure, which in turn contributes to the development of glaucoma. Given the adverse effects of organophosphorus pesticide exposure on the body, it is particularly important to explore the effects and mechanisms of its main metabolites in the human body.\u003c/p\u003e\u003cp\u003eDimethyl phosphate (DMP) is the primary metabolite of organophosphorus pesticides in the human body. It is usually derived from environmental exposure, agricultural product residues, and occupational exposure, and can reflect an individual's cumulative exposure to OPs over time. Studies have shown that DMP is neurotoxic (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), and its mechanism involves inducing oxidative stress (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), and interfering with neurotransmitter function (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). In addition, DMP can also cause liver toxicity by destroying the antioxidant system, damaging the cell membrane structure and causing enzyme loss. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Although the effects of DMP on eye health are relatively understudied, existing experimental and epidemiological evidence suggests that DMP may increase the risk of glaucoma by inhibiting acetylcholinesterase activity in retinal and optic nerve tissue, leading to impaired neural signaling and abnormal IOP. Through a series of experiments, we clearly confirmed for the first time that DMP exposure promotes the development of glaucoma by inhibiting the PI3K/Akt pathway and promoting the JAK/STAT, NF-κB, Wnt/β-catenin and other pathways to exert toxic effects. The pathogenic logic of this multi-pathway effect may be that the PI3K/Akt pathway is an important survival signal in cells(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Its inhibition leads to a decrease in the stress resistance of retinal ganglion cells and promotes the initiation of the apoptosis program. As classic inflammatory signaling centers, the JAK/STAT and NF-κB pathways(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), whose sustained activation will promote retinal inflammatory responses. Abnormal activation of the Wnt/β-catenin pathway is often associated with tissue fibrosis and pathological remodeling(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In glaucoma disease models, it may be involved in fibrotic changes in the trabecular meshwork, thereby increasing aqueous humor outflow resistance. The disruption of these pathways ultimately exacerbates oxidative stress in the retina, forming a vicious cycle that ultimately leads to irreversible apoptosis of retinal ganglion cells. This study provides new laboratory evidence for the potential ocular toxicity of pesticides and provides key data support for the development of stricter pesticide regulatory measures in the future.\u003c/p\u003e\u003cp\u003eThis study has several significant strengths. First, through a multi-faceted experimental design, we reveal for the first time the effects of DMP on glaucoma progression. Second, by examining inflammation- and apoptosis-related proteins and markers of oxidative stress, this study provides insight into the underlying mechanisms of DMP-induced retinal ganglion cell damage, providing strong evidence supporting the role of environmental pollutants in glaucoma. Finally, molecular biology experiments combined with functional validation experiments demonstrate that DMP exerts its retinal toxicity by modulating multiple signaling pathways.\u003c/p\u003e\u003cp\u003eWhile this study represents significant progress in exploring the effects and mechanisms of DMP in glaucoma, it still has several limitations. First, we did not use primary cultured retinal ganglion cells for in vitro experiments, lacking cell-specific evidence for molecular changes. Second, while this study identified DMP effects on multiple pathways, it remains unclear whether these pathways are upstream and downstream or synergistic. Further rescue experiments will be conducted to identify the dominant upstream pathway. Furthermore, although two DMP concentrations (5 mg/kg and 10 mg/kg) were used, it is unclear whether these concentrations accurately represent actual exposure levels in clinical or environmental settings. Future studies should further investigate the effects of DMP on optic nerve damage at varying concentrations to ensure the physiological relevance of the experimental conditions. Finally, this study was conducted entirely in an animal model. Future studies investigating DMP levels in aqueous humor or serum samples from glaucoma patients and correlating them with disease severity would greatly enhance the clinical significance and impact of this research.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eOur study demonstrates that the organophosphorus metabolite dimethyl phosphate (DMP) promotes glaucoma progression in a rat model by increasing intraocular pressure, reducing optic nerve thickness, and depleting neurotrophic factors (NGF, BDNF) in a dose-dependent manner. Mechanistically, DMP dysregulates multiple signaling cascades, suppressing the PI3K/Akt pathway while activating JAK/STAT, NF-κB and Wnt/β-catenin signaling, thereby amplifying inflammation, oxidative stress and apoptotic responses in retinal tissue; these effects were corroborated by Western blot, ELISA and pharmacological inhibitor experiments. Together, the findings provide novel mechanistic evidence that environmental exposure to DMP can compromise optic nerve homeostasis and drive glaucoma-relevant pathology, underscoring the need for epidemiological investigation of DMP exposure in patients and for consideration of exposure limits in regulatory policy. Future work should define pathway hierarchy through rescue experiments, assess dose relevance using clinically representative exposure levels, and validate associations between DMP concentrations in biological fluids and glaucoma severity to strengthen translational and public-health implications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement:\u0026nbsp;\u003c/strong\u003eNone of the authors has any conflicts of interest to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eBJC, YMT, SYG, XCW contributed equally in writing the manuscript and design the work. XCS analyzed the experimental data. XCW. provided methodological support. YMT were responsible for writing the original draft. SW reviewed and substantively revised the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eWe thank the Department of Ophthalmology of the Second Affiliated Hospital of Anhui Medical University for their collaborative and logistical work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBou Ghanem GO, Wareham LK, Calkins DJ. Addressing neurodegeneration in glaucoma: Mechanisms, challenges, and treatments. Prog Retin Eye Res. 2024;100:101261.\u003c/li\u003e\n\u003cli\u003eZhang Y, Zhao Z, Ma Q, Li K, Zhao X, Jia Z. Association between dietary calcium, potassium, and magnesium consumption and glaucoma. PLoS One. 2023;18(10):e0292883.\u003c/li\u003e\n\u003cli\u003eLiu Z, Hu Y, Wang Y, Xu B, Zhao J, Yu Z. 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Neurotoxic effects of organophosphorus pesticides and possible association with neurodegenerative diseases in man: A review. Toxicology. 2018;410:125-31.\u003c/li\u003e\n\u003cli\u003eHai DQ, Varga IS, Matkovics B. Effects of an organophosphate on the antioxidant systems of fish tissues. Acta Biol Hung. 1995;46(1):39-50.\u003c/li\u003e\n\u003cli\u003eAndroutsopoulos VP, Hernandez AF, Liesivuori J, Tsatsakis AM. A mechanistic overview of health associated effects of low levels of organochlorine and organophosphorous pesticides. Toxicology. 2013;307:89-94.\u003c/li\u003e\n\u003cli\u003eMo MS, Li HB, Wang BY, Wang SL, Zhu ZL, Yu XR. PI3K/Akt and NF-kappaB activation following intravitreal administration of 17beta-estradiol: neuroprotection of the rat retina from light-induced apoptosis. Neuroscience. 2013;228:1-12.\u003c/li\u003e\n\u003cli\u003eBanerjee S, Biehl A, Gadina M, Hasni S, Schwartz DM. JAK-STAT Signaling as a Target for Inflammatory and Autoimmune Diseases: Current and Future Prospects. Drugs. 2017;77(5):521-46.\u003c/li\u003e\n\u003cli\u003eSanges D, Romo N, Simonte G, Di Vicino U, Tahoces AD, Fernandez E, et al. Wnt/beta-catenin signaling triggers neuron reprogramming and regeneration in the mouse retina. Cell Rep. 2013;4(2):271-86.\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":"Glaucoma, Organophosphate exposure, Retinal ganglion cell apoptosis, Oxidative stress, Neurodegeneration","lastPublishedDoi":"10.21203/rs.3.rs-7917001/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7917001/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eGlaucoma, a leading cause of irreversible blindness, involves progressive retinal ganglion cell (RGC) loss. Beyond intraocular pressure (IOP), environmental risk factors like pesticide exposure are increasingly implicated. Dimethyl phosphate (DMP), a key metabolite of organophosphorus pesticides, accumulates in the body and exhibits systemic toxicity. However, its direct role and mechanism in glaucoma pathogenesis remain entirely unexplored.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe investigated the impact of DMP on glaucoma progression using a rat glaucoma model. Animals were subjected to DMP exposure at varying concentrations. We assessed IOP, optic nerve thickness, and expression of neurotrophic factors (NGF, BDNF). Molecular mechanisms were elucidated via Western blotting for key signaling pathways and apoptosis/inflammation markers, complemented by ELISA for oxidative stress. Functional validation was performed using specific pathway agonists and inhibitors.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eDMP exposure exacerbated core glaucomatous pathology in a concentration-dependent manner, significantly elevating IOP, reducing optic nerve thickness, and downregulating NGF/BDNF. Mechanistically, DMP concurrently inhibited the pro-survival PI3K/Akt pathway while activating the pro-inflammatory JAK/STAT and NF-κB pathways, and the fibrotic Wnt/β-catenin pathway. This multi-pathway disruption synergistically amplified retinal oxidative stress and triggered RGC apoptosis. Rescue experiments confirmed that the modulation of these specific pathways directly influenced the observed oxidative injury and cellular damage.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThis study provides the first evidence that dimethyl phosphate (DMP), a common organophosphorus metabolite, promotes glaucoma progression by regulating a multi-pathway signaling network that ultimately converges on oxidative stress and apoptosis. This work redefines DMP as a novel environmental risk factor for glaucoma, reveals its complex mechanism of action, and offers new perspectives for optic nerve protection strategies and environmental risk assessment.\u003c/p\u003e","manuscriptTitle":"Organophosphate pesticide exerts toxic effect on the optic nerve of glaucoma rats by promoting oxidative stress and inflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-19 11:53:01","doi":"10.21203/rs.3.rs-7917001/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-11T05:41:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-27T11:04:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-20T16:36:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"114686716223824569654689931179260524083","date":"2026-01-10T08:58:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247746083425964453848188684282143128400","date":"2026-01-07T15:54:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272971417547673542373970026862884828752","date":"2025-11-10T15:39:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-10T10:52:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-04T10:30:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-04T05:27:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-30T14:03:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-30T14:00:01+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":"e7a18665-38fa-4007-b5a5-85d37e9cc839","owner":[],"postedDate":"November 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":57771699,"name":"Biological sciences/Cell biology"},{"id":57771700,"name":"Health sciences/Diseases"},{"id":57771701,"name":"Biological sciences/Drug discovery"},{"id":57771702,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-05-05T05:40:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-19 11:53:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7917001","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7917001","identity":"rs-7917001","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}