Mitigation of Oxidative Stress Pathways in the Diabetic Cornea and Lacrimal Glands Contributes to the Rapid Reversal of Diabetic Dry Eye by Naltrexone

Allison K Krebs; David Diaz; Joseph W Sassani; Ian S Zagon; Patricia J McLaughlin

doi:10.1167/iovs.67.2.23

. 2026 Feb 9;67(2):23. doi: 10.1167/iovs.67.2.23

Mitigation of Oxidative Stress Pathways in the Diabetic Cornea and Lacrimal Glands Contributes to the Rapid Reversal of Diabetic Dry Eye by Naltrexone

Allison K Krebs ¹, David Diaz ^1,², Joseph W Sassani ³, Ian S Zagon ¹, Patricia J McLaughlin ^1,^✉

PMCID: PMC12898921 PMID: 41660942

Abstract

Purpose

To determine whether topical naltrexone (NTX) treatment can decrease the elevated reactive oxygen species pathways and select proinflammatory cytokines in the rat cornea and lacrimal glands that are elevated in diabetic dry eye.

Methods

Type 1 diabetic male and female Sprague-Dawley rats were rendered hyperglycemic for 6–8 weeks and then treated topically with NTX eyedrops twice daily for 15 days. Corneal epithelium and lacrimal glands were evaluated for expression of dihydroethidium, C/EBP homologous protein, and NADPH oxidase −2 (NOX-2) as well as the proinflammatory cytokines interleukin (IL) -1β, IL-6, and tumor necrosis factor alpha (TNF-α) to identify pathways targeted in the mitigation of diabetic dry eye.

Results

Type 1 diabetes resulted in dry eye and corneal surface insensitivity, accompanied by increased levels of oxidative stress and inflammation mediators in both corneal epithelium and lacrimal glands. Topical NTX treatment restored tear volume and corneal surface sensitivity, and significantly reduced expression of dihydroethidium, C/EBP homologous protein, and NOX-2, as well as proinflammatory cytokines IL-1β, IL-6, and TNF-α in male and female diabetic rats.

Conclusions

Blockade of the opioid growth factor (OGF)–OGF receptor system with topical NTX rapidly reversed diabetic dry eye and restored corneal surface sensitivity. The mechanism involved downregulating oxidative stress and decreasing proinflammatory cytokines to levels at or below those of nondiabetic rats of the same sex. These findings support a mechanistic role for OGF receptor blockade in the reversal of diabetic dry eye.

Keywords: naltrexone, dry eye, lacrimal gland, corneal epithelium, reactive oxygen species, NOX-2, CHOP, proinflammatory cytokines

The prevalence of diabetes is increasing globally. The most widely recognized ocular complication of diabetes is retinopathy; however, ocular surface disorders including dry eye, poor corneal epithelial wound healing, and decreased corneal sensitivity frequently occur, leading to patient discomfort, vision loss, increased health care costs, and decreased job productivity.¹^,²

An estimated 70% of the 38 million individuals with diabetes in the United States may present with dry eye at some stage in their disease.² Dry eye disease is multifactorial and can be related to the lack of tear production (i.e., aqueous deficient) or increased tear evaporation (evaporative dry eye).²^,³ Environmental factors such as dry air and wind, as well as medications like antidepressants and antihistamines, other autoimmune disorders such as Sjögren syndrome, hormonal fluctuations, age, and genetics can influence the onset and severity of dry eye disease. Inflammation and oxidative stress are also factors that disrupt the homeostasis of tear production.³

Our rat model of diabetic dry eye has been investigated since 2009 and involves the spontaneous development of dry eye.⁴ Onset of the aqueous deficient form follows the development of hyperglycemia and corresponds with the dysregulation of the opioid growth factor (OGF)–OGF receptor (OGFr) pathway.⁵^,⁶ Notably, this model does not require environmental or surgical manipulation. In addition to lower Schirmer scores indicative of decreased tear production and diminished corneal surface sensitivity, increased inflammation of the corneal surface and disruption of the lacrimal functional unit have been reported.⁷ Studies of the conjunctiva, eyelids, and lacrimal glands demonstrated not only decreased tear production in male and female type 1 diabetic (T1D) rats, but also significant decreases in the number and size of lacrimal gland acini, along with decreased expression of aquaporin-1 protein.⁷ The conjunctiva was also morphologically altered with decreases in goblet cell number and size in both sexes of diabetic rats.⁷ Further investigations on the limbus in this animal model revealed dysregulation of the OGF–OGFr pathway, as well as altered limbal cell morphology (reduced diameter and cellular packing density), and decreased expression of cytokeratin-15, a marker of limbal cells, as well as Ki-67, a marker of cellular proliferation.⁸

High glucose levels directly damage corneal nerves, lead to abnormal production of lipids and proteins, and damage DNA in part through elevated levels of reactive oxygen species (ROS), all of which contribute to decreased tear production.⁹^–¹¹ The NOD-like receptor protein-3 inflammasome pathway is associated with ocular surface inflammation and resultant dry eye disease.¹¹ Inflammation may be both a cause and a side effect of dry eye disease, leading to lacrimal gland dysfunction.¹² ROS and inflammation also regulate other physiological processes to maintain corneal surface homeostasis.¹⁰^,¹¹^,¹³

Ocular surface homeostasis depends, in part, on the osmolarity of tears.²^,¹⁰ Hyperosmolar tears, which are frequently related to dry eye disease, promote inflammation on the ocular surface epithelium, leading to stress-induced ROS accumulation and secondary oxidative stress.¹³ Oxidative stress involves the CHOP–NOX-2 pathway, which can be effective in destroying pathogens during an innate immune attack.¹⁴ In the diabetic state, NOX-2 activates signaling pathways to increase ROS, but can also provide some protection during the innate immune response.¹⁴ ROS, in turn, increases the secretion of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6.¹⁴ If untreated, the inflammation increases cell death in the cornea and conjunctiva and may contribute to high levels of ROS on the ocular surface, leading to more permanent dry eye disorders.¹⁴ Zha et al.,¹¹ as well as other investigators,¹⁴^–¹⁶ have reported that reduction of ROS production decreases the NOD-like receptor protein-3 reaction that decreases dry eye disease symptoms. However, the mechanisms involved in the rapid restoration of tear production that occurs after topical NTX treatment are unknown. It is known that diabetes activates innate inflammation and increases cytokines including IL-1β, IL-6, and TNF-α on the corneal surface and lacrimal glands, altering tear formation¹⁴ and diabetic retinal disease. Inflammation also impedes the neuronal signaling in the lacrimal gland, thus contributing to corneal surface dysregulation.⁷^,¹⁴

One regulatory pathway that mediates cellular homeostasis on the cornea surface is the OGF–OGFr pathway.¹⁷^–¹⁹ This pathway becomes dysregulated in hyperglycemia, resulting in elevated levels of the inhibitory neuropeptide OGF, along with its nuclear-associated receptor, OGFr.⁷^,⁸^,¹⁷^–¹⁹ Abnormalities in the OGF–OGFr axis in male and female rats with T1D precede the onset of dry eye and corneal insensitivity and contribute to the development of corneal epithelial defects by repressing cellular proliferation.⁵^,¹⁷^–¹⁹ Naltrexone (NTX) is a general opioid receptor antagonist that blocks OGFr and prevents the inhibitory action of OGF.²⁰ Administration of NTX topically to the cornea rapidly restored tear production within 5 days of treatment initiation in T1D rats.⁷^,²¹ Moreover, short-term treatment with NTX reversed diabetic dry eye and corneal insensitivity, as well as other abnormalities observed with the lacrimal glands and conjunctiva of T1D rats. During this period, aquaporin-5 levels were increased in the lacrimal glands after 15 days of topical NTX treatment.⁷ Based on observations of the rapid reversal of dry eye, we were prompted to examine inflammatory pathways associated with dry eye disease, including stress pathways. Published studies have shown that NTX decreases proinflammatory markers in vitro²² and decreases inflammation in the liver,²³ supporting investigations using pharmacological intervention to resolve inflammation of ROS pathways in diabetic ocular surface disease.²⁴

The current study investigated the mechanisms underlying the rapid reversal of dry eye after 15 or fewer days of topical NTX treatment and focuses on ROS pathways in the corneal epithelium and lacrimal glands of diabetic male and female adult rats. The literature shows that diabetes increases ROS that, in turn, impacts neuronal activity and may contribute to dry eye.¹⁴ Endoplasmic reticulum stress activates the C/EBP homologous protein (CHOP)/phosphorylated PKR-like endoplasmic reticulum kinase (PERK) pathway, leading to increased ATF-4 and altered cell fate. We postulate that one of the mechanisms by which NTX rapidly increases tear volume is by decreasing ROS levels and normalizing CHOP and NOX-2 proteins. NOX-2 protein is found in neutrophils and macrophages, responds to endoplasmic reticulum stress, and produces superoxides that regulate innate inflammation. We began our research investigating the CHOP/PERK/NOX-2 pathway and hypothesize that topical NTX decreases inflammation and decreases the elevated proinflammatory cytokines, enabling tear production to return to normal.

Materials and Methods

Animals and Treatment

The study was approved by the Penn State College of Medicine Institutional Animal Use Committee (protocol 47207) and all experiments conformed to the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the ARRIVE guidelines. Male and female Sprague-Dawley rats weighing approximately 180 g and 160 g, respectively, were purchased from Charles River Laboratories (Wilmington, MA, USA); food and water were available ad libitum. Rats were housed in a humidity- and temperature-controlled environment, with 12-hour light–dark cycles. Two male and two female cohorts of animals were studied independently with three to five rats per sex in each cohort.

Hyperglycemia was induced following previously reported protocols that successfully established high blood glucose levels within days.⁷^,²¹ Briefly, rats were fasted for 4 hours and then given a single intraperitoneal injection of 60 mg/kg streptozotocin (STZ; Sigma-Aldrich, St. Louis, MO, USA) dissolved in citrate buffer, pH 4.5. After 72 hours, tail vein blood was sampled using a glucometer and readings greater than 300 mg/dL indicated successful induction of hyperglycemia. Animals that failed to become hyperglycemic after 5 days were removed from the study. Hyperglycemic rats were housed two per cage to accommodate the frequent cage changes related to increased urination and defecation.

Treatment With NTX to Block the OGFr

Animals were randomly assigned to receive either topical drops of NTX (50 µL, 5 × 10⁻⁵ M) or sterile saline vehicle. Rats that were not injected with STZ were considered normal controls and designated as normals. Hyperglycemic animals receiving vehicle were designated as T1D, and rats receiving NTX were designated as T1D + NTX. Drops were administered only to the right eye twice daily for 10 or 15 days without anesthesia at 0800 to 0900 hours and 1700 to 1800 hours. Normal and some T1D rats were administered drops of sterile vehicle (50 µL) at similar times. Treatment for 10 or 15 days did not result in statistically different measurements of dry eye and data from both treatment schedules were combined. The dosage of NTX was selected based on our previous studies on dry eye as well as on earlier investigations of the efficacy and safety of NTX in promoting corneal re-epithelialization in animal models,²⁵^–²⁸ in addition to reports supporting the tolerability of this topical regimen for humans.²⁹

Physiological Parameters of Diabetes and Dry Eye

Body weights and glucose measurements were collected initially before induction of hyperglycemia and 6 weeks after STZ injections. Tear production and corneal sensitivity were measured following published procedures.⁷^,²¹ Measurements were recorded in both sexes after 6 weeks of hyperglycemia before treatment (baseline) and on days 10 and 15 of treatment before the morning application. All measurements were recorded from unanesthetized animals.

Tear production was measured using Schirmer strips (TearFlo, HUB Pharmaceuticals, Farmington Hills, MI, USA) that were cut to 1 mm wide × 17 mm length and placed in the lower eyelid cul-de-sac in the inferior nasal quadrant for 60 seconds and the wetting distance recorded.⁷^,²¹ Tear production was assessed before corneal surface sensitivity to avoid the premature tearing caused by the aesthesiometer.

Corneal surface sensitivity was recorded using a Cochet-Bonnet aesthesiometer (Boca Raton, FL, USA) following our previous protocols.⁷^,²¹ Sensitivity was calculated from the length of the nylon filament and the amount of force required to elicit a blink reflex. Each measurement was repeated three times and averaged. A greater force (g/mm²) was indicative of corneal insensitivity; values were determined using the manufacturer's scale.

Euthanasia and Tissue Collection

At the conclusion of topical treatments, rats were humanely euthanized using the Euthanex carbon dioxide chamber (Perotech, Chapel Hill, NC, USA) followed by decapitation. Globes and lacrimal glands were excised and rinsed in ice-cold PBS. Tissues were fixed in 2% paraformaldehyde for 1 hour followed by cryoprotection in graded sucrose solutions until saturated, embedded in optimal cutting temperature compound and frozen in 2-methyl-butane cooled by dry ice. Embedded tissue was stored at −80°C until cryosectioned at 10 µm.

Immunofluorescence Detection of ROS and Related Proteins

Immunofluorescent detection followed our published procedures.⁷^,¹⁷^–¹⁹^,²¹ Briefly, frozen sections of corneas were dehydrated, washed, blocked for 1 hour in buffer (1× PBS, 5% normal goat serum, 0.3% Triton X-100), and incubated for 16–18 hours. After primary antibody staining, tissues were washed and stained using appropriate secondary antibodies and DAPI. Original images were obtained using an LSM-Zeiss Examiner.Z1 confocal microscope with 10× and 20× lenses. Images were processed with ZEN3.1 software before being exported to ImageJ.

The method used to assess the presence and relative level of ROS was based on several reports demonstrating that measurements of dihydroethidium (DHE) were effective and reliable.³⁰^,³¹ Briefly, relative levels of ROS were assessed by immediately staining freshly cryosectioned tissue with the DHE dye (ThermoFisher Scientific, Waltham, MA, USA; cat # D11347; 1:1000) diluted in PBS for 15 minutes at 37°C. Upon oxidation, the blue DHE dye is converted to 2-hydroxyethidium, a red fluorescent product with an emission wavelength of 605 nm, which was detected using an LSM-Zeiss Examiner.Z1 confocal microscope. Images were acquired with a resolution of 512 × 512 pixels, a laser power of 0.2%, and a pinhole size of 1 Airy unit. Before image acquisition, laser power, master gain (650 V), detector offset (0), and digital gain (1.0) were optimized with representative control tissue to maximize signal detection. Display settings were maintained at default values. Once optimized, all acquisition parameters were held constant across all samples within the experiment.

The CHOP/PERK pathway was evaluated by staining corneal epithelium and lacrimal glands with CHOP antibody (Life Technologies, Invitrogen, Waltham, MA, USA; Cat # PA5104528; 1:100) and NOX-2 polyclonal antibody (Proteintech, Rosemont, IL, USA; cat # 19013-1-AP; 1:200) and measuring immunofluorescence.

Innate inflammation was assessed by staining tissues and measuring optical density (OD) of interleukins (IL-1β, IL-6), and TNF-α.³⁴^–³⁶ Tissues were incubated with antibodies to IL-1β (ThermoFisher Scientific; P420B;1:200), IL-6 (R&D Systems, Minneapolis, MN, USA; #AF501; 1:200), and TNF-α (ThermoFisher Scientific, 1:50) overnight followed by incubation with the secondary antibody Alexafluor 488 (ThermoFisher Scientific; 1:1000). Tissues were imaged within 72 hours.

Data Analysis

Four independent experiments were conducted with male and female rats. Immunofluorescence intensity was measured by OD units using ImageJ software.⁷^,²¹ Background OD measurements were subtracted from each image. Mean gray values were obtained by measuring an area (100 × 25 µm) of corneal epithelium or serous acini in the lacrimal gland. Analyzed densitometric data indicated that there were no differences between experiments and data were combined and analyzed using two-way ANOVAs between sex and condition (normal, T1D_SV, T1D_NTX) with Sidak's post hoc test for multiple comparisons. If sex effects were not noted data were subsequently analyzed using one-way ANOVA with follow-up analysis by Tukey tests for multiple comparisons. Throughout the study, data are presented as means ± SEM. All analyses were performed using GraphPad Prism version 10.0 (GraphPad Software, La Jolla, CA, USA); a P value of less than 0.05 was considered statistically significant.

Results

Clinical Signs of Diabetic Dry Eye

The T1D model was established based on body weights and blood glucose measurements at the time of treatment. Before the induction of hyperglycemia, male rats weighed approximately 172g and female rats weighed approximately 146g. Six weeks later, male and female rats weighed approximately 450g and 250g, respectively, representing a 2.6-fold increase in body weight for males and a 1.7-fold increase for females. Six weeks after STZ injections, hyperglycemic male rats weighed approximately 327g and hyperglycemic female rats weighed approximately 234g, significant decreases from normal animals.

Blood glucose levels for male and female Sprague-Dawley rats averaged 144 mg/dL at baseline. After STZ injection and throughout the 6-week period, blood glucose levels in both male and female rats were consistently greater than 350 mg/dL. No insulin injections were required and no rats died from hyperglycemia during experimentation. Topical NTX did not affect body weight or glucose readings.

Tear production and corneal sensitivity were markedly altered by topical NTX, and values corresponded with those previously published.⁷^,¹⁷^–¹⁹^,²¹ Baseline Schirmer scores ranged between 9 and 10 mm for normal male and female rats. After 6 weeks of hyperglycemia, male and female T1D rats had Schirmer scores of 5.7 ± 0.2 mm and 4.6 ± 0.5 mm, respectively, demonstrating a substantial loss of tear flow relative to normal rats of the same sex. Topical NTX restored the Schirmer scores to 9.7 ± 0.4 mm and 8.6 ± 0.6 mm for T1D + NTX male and female rats, respectively, thus significantly increasing (P < 0.001) tear volume relative to corresponding T1D animals.

Corneal sensitivity was analyzed at the start of treatment and after NTX treatment. Diabetes resulted in significantly reduced corneal sensitivity for both male and female rats, comparable with our previous results.⁷^,²¹ Treatment with topical NTX reduced aesthesiometer readings, indicating increased sensitivity in diabetic animals. Baseline force for normal male and female animals ranged from 0.37 to 0.40g/mm². After 6 weeks of hyperglycemia, T1D rats had a mean corneal sensitivity measurement of 1.44 ± 0.27g/mm². After NTX treatment, the mean measurements for male T1D + NTX rats were 0.54 ± 0.02g/mm², representing nearly a three-fold decrease in force and, thus, increased sensitivity. Baseline corneal sensitivity for normal female rats was 0.38 ± 0.00g/mm², and 6 weeks after receiving STZ, sensitivity was decreased with force measurements of 0.70 ± 0.12g/mm². NTX treatment increased the sensitivity by decreasing the force required to elicit a blink response to a reading of 0.49 ± 0.02g/mm².

Morphology of Cornea Epithelium and Lacrimal Glands and Mediation of the ROS Pathway

The morphology of the corneal epithelium and lacrimal glands in diabetic is presented in Figure 1. Hematoxylin and eosin-stained images revealed that 6 weeks of hyperglycemia altered the morphology of the corneal surface epithelium (Figs. 1A, 1C, top) and lacrimal glands (Figs. 1E, 1G, top) of male and female T1D rats.

Activation of ROS in Diabetes and Mediation of ROS by NTX on the Corneal Surface

Hyperglycemia-induced ROS levels in corneal epithelium (Figs. 1A, 1C, bottom) and lacrimal glands (Figs. 1E, 1G, bottom) of male and female rats were significantly greater relative to those of normal animals. DHE OD readings were elevated for T1D animals relative to normal rats of the same sex, and significantly reduced with topical NTX treatment in comparison with levels in diabetic animals of the same sex (Figs. 1B, 1D). Normal rats. DHE levels in the corneal epithelium of normal male Sprague-Dawley rats were 1.0 ± 0.04 in comparison with mean T1D values of 1.34 ± 0.05 (P < 0.001) and mean T1D + NTX values of 0.74 ± 0.05 (P < 0.01). DHE levels in the corneal epithelium of normal female rats were 0.89 ± 0.05 in comparison with mean OD levels of 1.22 ± 0.09 (P < 0.02) for T1D female rats and 0.67 ± 0.05 OD units (P < 0.001) for T1D + NTX female rats (Figs. 1B, 1D)

Mediation of ROS in the Lacrimal Glands With NTX

Expression of ROS and corresponding OD readings of DHE in the lacrimal glands are presented in Figures 1E–H. ROS levels increased in lacrimal glands (Figs. 1E, 1G, bottom) in diabetic male and female rats. DHE OD levels were significantly increased (P < 0.0001) in the lacrimal glands of T1D males (Fig. 1F) and T1D females (Fig. 1H). Topical NTX reduced levels of ROS significantly (P < 0.0001) such that no differences were noted in lacrimal glands between T1D + NTX animals and their normal counterparts (Figs. 1F, 1H).

Activation of the CHOP and NOX-2 Pathways in Corneal Epithelium of Diabetic Rats and Reversal With NTX

ROS pathways are mediated by the CHOP/PERK pathway and NOX-2. Images of CHOP and Nox-2 expression (Figs. 2A, 2C), as well as OD measurements (Figs. 2B, 2D), in the corneal epithelium of male and female normal and diabetic rats were analyzed by two-way ANOVA. No significant interactions were noted and thus, subsequent analyses were performed using one-way ANOVA. CHOP proteins were significantly increased (P < 0.01) in the corneal epithelium of male and female T1D rats (Figs. 2B, 2D) relative to normals. NOX-2 proteins were significantly elevated (P < 0.0001) for T1D male and female rats relative to normal rats of the respective sex. After topical NTX application to the cornea, CHOP and NOX-2 OD levels were significantly reduced to levels comparable with normals or below normal values (Figs. 2B, 2D).

Figure 2. — CHOP and NOX2 proteins in the ROS pathway were elevated in the corneal epithelium of male (A, B) and female (C, D) diabetic rats and reduced after topical NTX treatment. Representative images of corneal epithelium from male (A) and female (C) rats stained with CHOP or NOX2 antibodies. OD measurements recorded at 40× magnification for corneal epithelium are plotted in (B) and (D) for male and female rats, respectively. Treatment paradigms are detailed in the Figure 1 legend. Histograms represent analysis of OD; data are presented as means ± SEM. Data were analyzed using ANOVA and post hoc Tukey's multiple comparison tests and presented as means ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. NOR, normal rats.

Regulation by NTX of CHOP and NOX-2 in the Lacrimal Glands of Diabetic Rats

Two prominent proteins in the ROS pathway are CHOP and NOX-2 and both were elevated in the lacrimal glands of T1D (Fig. 3). The mean CHOP OD values were 1.04 ± 0.06 for normal male rats and increased to 1.51 ± 0.09 for T1D male rats, a 46% increase (P = 0.001) (Figs. 3A, 3B). Topical NTX rapidly reduced CHOP expression to levels of 1.07 ± 0.04, which was not significantly different from normal male rats (P = 0.93), but significantly lower than T1D male OD readings (P < 0.01) (Figs. 3A, 3B). CHOP expression in the lacrimal glands of female T1D rats was increased by 51% to 1.6 ± 0.1 (Figs. 3C, 3D). Topical NTX treatment reduced CHOP expression to 1.23 ± 0.03 OD units, corresponding with a 23% decrease (P < 0.001) relative to T1D female rats. Although CHOP values for NTX-treated females remained slightly elevated above normal female CHOP levels (P = 0.03), expression was substantially normalized.

Figure 3. — CHOP and NOX2 proteins in the ROS pathway were elevated in the lacrimal glands of male (A, B) and female (C, D) diabetic rats and reduced after topical NTX treatment. Representative images of lacrimal glands from male (A) and female (C) rats stained with CHOP or NOX2 antibodies. OD measurements recorded at 40× magnification for lacrimal glands are plotted in (B) and (D) for male and female rats, respectively. Treatment paradigms are detailed in the Figure 1 legend. Histograms represent analysis of OD values from five rats/treatment group/sex; data are presented as means ± SEM. Data were analyzed using ANOVA and post hoc Tukey's multiple comparison tests and presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. NOR, normal rats.

NOX-2 enzyme, which aids in the destruction of pathogens, is activated during adaptive immunity. In the lacrimal glands of both male and female T1D rats, NOX-2 expression was upregulated (Figs. 3B, 3D) relative to normal levels, suggesting an elevation in oxidative stress. Mean NOX-2 OD values for T1D male rats increased from 0.99 ± 0.03 in normal male animals to 1.26 ± 0.03 OD units, a 28% elevation (P = 0.0001) (Figs. 3A, 3B). Topical NTX treatment significantly (P < 0.01) reduced NOX-2 expression in the lacrimal glands relative to T1D male rats, comparable with normal values (Fig. 3B). In lacrimal glands of female animals, NOX-2 expression rose from 1.14 ± 0.03 OD in normal rats to 1.60 ± 0.04 OD in T1D rats, an approximately 41% increase (P < 0.0001) (Figs. 3C, 3D). After NTX treatment, NOX-2 expression in T1D + NTX female rats decreased to 1.12 ± 0.02, a 30% reduction relative to T1D animals (P < 0.0001) and returned to OD levels comparable with normals. Both CHOP and NOX-2 expression levels were restored near normal within 15 days of treatment.

Mediation of Inflammasome Pathways on the Corneal Surface and Lacrimal Glands by Topical NTX: IL-1β

Three proinflammatory cytokines were densitometrically evaluated in the corneal epithelium and lacrimal glands of male and female diabetic rats (Figs. 4, 5, 6). IL-1β activates immunity, playing a critical role in inflammation, and is elevated in diabetes.³² IL-1β expression and quantitative assessments in our model are presented in Figure 4. The patterns of expression were similar between males and females in the epithelium (Fig. 4A) and lacrimal glands (Fig. 4C). Densitometric analyses of the stained tissues revealed that diabetes increased expression of IL-1β and topical NTX decreased levels after 15 days in the corneal epithelium (Fig. 4B) and lacrimal glands (Fig. 4D). Two-way ANOVA revealed some sex differences for IL-1β in that OD values for normal and T1D female rats were significantly higher (P < 0.01) than males in the corneal epithelium. The mean OD levels of normal males were 0.66 ± 0.02 and 1.33 ± 0.04 in T1D males, representing a two-fold increase (P < 0.0001) (Fig. 4B). After NTX treatment, the values in the corneal epithelium of T1D males were restored to 0.63 ± 0.01 (a 53% reduction relative to T1D levels; P < 0.0001). Similar patterns were noted in female rats, whereby baseline OD values for normal rats were 0.89 ± 0.03 in comparison with T1D females with expression levels of 1.64 ± 0.02, a 1.8-fold increase (P < 0.0001) (Fig. 4B). NTX treatment restored the expression of IL-1β in the corneas of female diabetic rats to 0.91 ± 0.01, corresponding with a decrease of approximately 56% (P < 0.0001).

Figure 4. — Topical NTX reduced the inflammation recorded in the corneal epithelium and lacrimal glands of diabetic rats. Proinflammatory cytokine IL-1β was elevated in the corneal epithelium (A, B) and lacrimal glands (C, D) of diabetic male and female rats and reduced substantially by topical treatment with NTX for 15 days. Tissues from male and female diabetic and rats were stained with anti–IL-1β antibody and OD values were analyzed using a one-way ANOVA and post hoc Tukey's multiple comparison tests; data were presented as means ± SEM. Significantly different at *P < 0.05, ***P < 0.001, ****P < 0.0001; ns, not significant. NOR, normal rats.

Figure 5. — Proinflammatory cytokine IL-6 was elevated in the corneal epithelium (A, B) and lacrimal glands (C, D) of diabetic rats. Topical NTX for 15 days significantly reduced the expression levels in both tissues of male and female diabetic rats. Tissues were stained with anti–IL-6 antibody and OD values were analyzed using a one-way ANOVA and post hoc Tukey's multiple comparison tests; data were presented as means ± SEM. Significantly different at *P < 0.05, ***P < 0.001, ****P < 0.0001; ns, not significant. NOR, normal rats.

Figure 6. — Expression of TNF-α was increased in the corneal epithelium (A, B) and lacrimal glands (C, D) of diabetic rats and substantially decreased following 15 days of topical NTX. Tissues from the corneal epithelium (A) and lacrimal glands (C) from male and female diabetic and normal rats were stained with antibodies to anti–TNF-α and OD values (B, D) were analyzed using a one-way ANOVA and post hoc Tukey's multiple comparison tests; data are presented as means ± SEM. Significantly different at *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. NOR, normal rats.

Expression levels of IL-1β in lacrimal gland tissue were elevated for male and female diabetic rats (Figs. 4C, 4D). Topical NTX for 15 days reduced the expression of IL-1β in lacrimal glands of these animals. One-way ANOVA analyses of IL-1β OD levels revealed 36.5% elevations in male T1D rats and 45.5% increases in female T1D rats relative to values in normal animals. Topical NTX was effective in reducing expression levels of IL-1β in lacrimal glands by 17.9% in male T1D + NTX rats and by 24.2% in female T1D-NTX animals relative to diabetic rats of the same sex (Fig. 4D).

Mediation of Inflammasome Pathways on the Corneal Surface and Lacrimal Glands by Topical NTX: IL-6

IL-6 is a widely dispersed proinflammatory marker that is related to insulin resistance in both T1D and type 2 diabetes. Concerning its function in the development of dry eye, reports have shown that increased levels of IL-6 decreased corneal homeostasis and accelerated dry eye disease.³³^,³⁴ In Figure 5, IL-6 expression in the corneas of normal male and female rats was 0.76 ± 0.02 and 0.99 ± 0.02, respectively (Fig. 5B). Six weeks of hyperglycemia increased IL-6 levels in the corneal epithelium to 1.67 ± 0.07 (a 164% increase; P < 0.0001) and 2.26 ± 0.07 (a 133% increase; P < 0.0001) in T1D male and female rats, respectively. Topical NTX for 15 days resulted in significant reductions in expression of IL-6 in the corneal epithelium to OD levels of 82 ± 0.02 in the corneal epithelium of male T1D + NTX rats (a 58% decrease vs T1D; P < 0.0001) and a lesser decrease in females to 1.33 ± 0.03 (a 41% decrease vs T1D; P < 0.0001). Interestingly, two-way ANOVA indicated significant effects of diabetes and treatment on IL-6 (P < 0.0001 for condition and sex factors) in the corneal epithelium and a significant interaction between sex and treatment (P < 0.01); females had higher OD readings than male rats in all cohorts.

In the lacrimal glands (Figs. 5C, 5D), IL-6 values were significantly increased (P < 0.0001) in T1D rats relative to the normals of the same sex, being 80.7% and 84.7% higher in T1D males and females, respectively. A two-way ANOVA revealed that baseline levels for IL-6 differed between male and female diabetic rats, with female rats having higher IL-6 values; however, the differences were not significant. Topical NTX treatment rapidly reduced (P < 0.0001) cytokine expression in lacrimal glands from T1D animals by at least 32% in males and 36% in females (Fig. 5D). IL-6 OD values in the treated rats were comparable with normal female rats and slightly elevated over normal male rats.

Mediation of Inflammasome Pathways on the Corneal Surface and Lacrimal Glands by Topical NTX: TNF-α

TNF-α regulates inflammation and initiates immune responses; high TNF-α levels can also decrease cell survival and reduce cell proliferation, a hallmark of dry eye symptoms in the lacrimal glands.³⁵

Expression levels of TNF-α were elevated on the corneal epithelium (Figs. 6A, 6B) and lacrimal glands (Figs. 6C, 6D) for both male and female diabetic rats and decreased by short-term topical exposure to NTX. No differences were noted between male and female measurements for animals in any cohort. Regarding the corneal epithelium, TNF-α values for normal rats were similar between sexes (male 0.91 ± 0.09, female 0.94 ± 0.07) (Fig. 6B). Hyperglycemia for 6 weeks resulted in elevated TNF-α expression in the cornea. OD values for male T1D rats increased to 1.35 ± 0.02 OD (a 49% increase; P = 0.0004) and for T1D females to 1.39 ± 0.1 OD (a 48% increase, P = 0.002). NTX treatment reduced TNF-α expression in the cornea by 21% relative to T1D males and 25% relative to T1D females (P < 0.01) (Fig. 6B).

Topical NTX for 15 days mitigated the elevated expression of TNF-α in lacrimal glands of both male and female T1D rats (Figs. 6C, 6D). Normal values were comparable for male and female rats. Diabetic animals exhibited elevated levels by 49.9% in males (T1D, 1.79 ± 0.03) and 52.1% in females (T1D, 1.82 ± 0.04). Topical NTX for 15 days reduced TNF-α expression by 21.1% relative to T1D males or 17.2% in T1D female lacrimal glands, respectively (male T1D + NTX, 1.48 ± 0.05 OD; female T1D + NTX, 1.44 ± 0.07 OD). The mitigation by NTX was least effective on the expression of TNF-α and more effective on IL-1β and IL-6 expression levels.

Discussion

In our model, T1D rats treated with topical NTX demonstrated rapid restoration of tear production.⁷^,²¹ The rapid reversal of dry eye appears to be associated with both increased cell proliferation and reduced corneal surface inflammation.²¹ Examination of nerves in the trigeminal ganglion indicated an increase in calcitonin gene-related peptide–positive neurons after NTX treatment, suggesting that diabetes impacts this pathway.²¹ The current study targeted ROS pathways and proinflammatory cytokines and demonstrated that the topical application of NTX to the corneal surface rapidly decreased ROS expression by decreasing CHOP and NOX-2 and reduced expression of cytokines in both the corneal epithelium and lacrimal glands of T1D rats.

Diabetes increased ROS expression in both sexes and topical NTX reduced DHE, CHOP and NOX-2 levels in both sexes of T1D + NTX animals, often to values below normal rats. The duration of NTX treatment may have influenced the partial reduction of IL-1β and IL-6, because cytokine levels decreased in diabetic animals, but remained above levels for normal rats in some cohorts.

The complete mechanistic pathway is still being elucidated to ascertain how NTX works rapidly to reverse dry eye, but mitigation of inflammation is one factor that will be studied. Application of NTX has been shown to reduce inflammation by blockade of receptors.³⁶^,³⁷ Reduction of cellular fibrosis, neovascularization, and inflammation by kinase inhibitors may also provide new directions in the treatment of corneal complications.³⁸ Whether topical NTX exerts its effects through other pathways such as the Toll-like receptor-4 signaling or uses the OGF–OGFr axis as the primary mechanism to restore tear production remains unknown. Notably, hyperglycemia has been shown to dysregulate the OGF–OGFr pathway in diabetes¹⁷^–¹⁹^,²¹ and may render the pathway dysfunctional in prediabetic or early hyperglycemic conditions.

These exploratory studies were limited by the absence of complementary assays assessing oxidative stress and inflammatory mediators, including Western blotting and ELISA assays on tears, to more fully characterize modulation of the CHOP/NOX-2 pathway and proinflammatory cytokines by NTX. Given that OGFr knockout mice are lethal, elucidation of the specific pathways utilized by NTX may require in vitro approaches with overexpression and underexpression studies in corneal epithelial cell lines. Further investigation is also warranted to determine the mechanisms that may address sex differences. Finally, research is necessary to establish whether the cessation of NTX treatment results in the recurrence of oxidative stress, dysregulation of the OGF–OGFr pathway in ocular tissue, and resultant corneal surface insensitivity and/or dry eye symptoms.

Acknowledgments

Funded by NIH NEI grant EY029223.

Disclosure: A.K. Krebs, None; D. Diaz, None; J.W. Sassani, intellectual property on the composition and use of NTX for treatment of dry eye and has licensed this IP; I.S. Zagon, intellectual property on the composition and use of NTX for treatment of dry eye and has licensed this IP; P.J. McLaughlin, intellectual property on the composition and use of NTX for treatment of dry eye and has licensed this IP

References

1. American Diabetes Association. Eye health: dry eye with diabetes. 2023. Available at: https://diabetes.org/sites/default/files/2023-09/EyeHealth_Resource_Dry-Eye_rev-1.pdf.
2. Sapra A, Bhandari P.. Diabetes. 2023 Jun 21. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025. PMID: 31855345. [PubMed] [Google Scholar]
3. Zeppieri M, Capobianco M, Visalli F, Musa M, et al.. Beyond the surface: revealing the concealed effects of hyperglycemia on ocular surface homeostasis and dry eye disease. Medicina. 2025; 61: 1992–2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
4. Zagon IS, Klocek MS, Sassani JW, McLaughlin PJ. Dry eye reversal and corneal sensation restoration with topical naltrexone in diabetes mellitus. Arch Ophthalmol. 2009; 127: 1468–1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
5. Zagon IS, Sassani JW, Purushothaman I, McLaughlin PJ. Dysregulation of the OGF-OGFr pathway correlates with elevated serum OGF and ocular surface complications in the diabetic rat. Exp Biol Med. 2020; 245: 1414–1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
6. Zagon IS, Sassani JW, Purushothaman I, McLaughlin PJ. Blockade of the opioid growth factor receptor (OGFr) delays the onset and reduces the severity of diabetic ocular surface complications Exp Biol Med . 2021; 246(5): 629–636. [DOI] [PMC free article] [PubMed] [Google Scholar]
7. Diaz D, Sassani JW, Zagon IS, McLaughlin PJ. Topical naltrexone increases aquaporin 5 production in the lacrimal gland and restores tear production in diabetic rats. Exp Biol Med . 2024; 249: 10175. [DOI] [PMC free article] [PubMed] [Google Scholar]
8. McLaughlin PJ, Sassani JW, Diaz DP, Zagon IS. Elevated opioid growth factor alters the limbus in type 1 diabetic rats. J. Diabetes Clin Res. 2023; 5: 1–10. [DOI] [PMC free article] [PubMed] [Google Scholar]
9. Volpe CMO, Villar-Delfino PH, do Anjos PMF, Nogueira-Machado JA. Cellular death, reactive oxygen species (ROS) and diabetic complications. Cell Death Disease. 2018; 9: 119. [DOI] [PMC free article] [PubMed] [Google Scholar]
10. Zhuang D, Misra SL, Mugisho OO, et al.. NLRP3 Inflammasome as a potential therapeutic target in dry eye disease. Int J Mol Sci. 2023; 24: 10866. [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Zha Z, Xiao D, Liu Z, et al.. Endoplasmic reticulum stress induces ROS production and activates NLRP3 inflammasome via the PERK-CHOP signaling pathway in dry eye disease. Invest Ophthal Vis Sci. 2024; 65: 34. [DOI] [PMC free article] [PubMed] [Google Scholar]
12. Rao SK, Mohan R, Gokhale N, et al.. Inflammation and dry eye disease-where are we? Int J Ophthalmol. 2022; 15: 820–827. [DOI] [PMC free article] [PubMed] [Google Scholar]
13. Wan L, Bai X, Zhou Q, et al.. The advanced glycation end-products (AGEs)/ROS/NLRP3 inflammasome axis contributes to delayed diabetic corneal wound healing and nerve regeneration. Int J Biol Sci. 2022; 18: 809–825. [DOI] [PMC free article] [PubMed] [Google Scholar]
14. Meier DT, De P, Souza J, Donath MY. Targeting the NLRP3 inflammasome-IL-1β pathway in type 2 diabetes and obesity. Diabetologia. 2025; 68: 3–16 [DOI] [PMC free article] [PubMed] [Google Scholar]
15. Niu L, L ZS, Wu J, Chen L, Wang Y. Upregulation of NLRP3 inflammasome in the tears and ocular surface of dry eye patients. PloS One. 19(5): e0126277. [DOI] [PMC free article] [PubMed] [Google Scholar]
16. Seen S, Tong L. Dry eye disease and oxidative stress. Acta Ophthalmol. 2018:96: e412–e420. [DOI] [PubMed] [Google Scholar]
17. Zagon S, Purushothaman McL. Blockade of the opioid growth factor receptor (OGFr) delays the onset and reduces the severity of diabetic ocular surface complications. Exp Biol Med. 2021; 246(5): 629–636. [DOI] [PMC free article] [PubMed] [Google Scholar]
18. Purushothaman I, Sassani JW, Zagon IS, McLaughlin PJ. Ocular surface complications result from dysregulation of the OGF-OGFr signaling pathway in female diabetic rats. Exp Ther Med. 2021; 22: 687. [DOI] [PMC free article] [PubMed] [Google Scholar]
19. Purushothaman I, Zagon IS, Sassani JW, et al.. Sex differences in the magnitude of diabetic ocular surface complications: role of serum OGF. Physiol Behav. 2021; 237: 113436. [DOI] [PMC free article] [PubMed] [Google Scholar]
20. McLaughlin PJ, Zagon IS. Duration of opioid receptor blockade determines biotherapeutic response. Biochem Pharmacol . 2015; 97: 236–246. [DOI] [PubMed] [Google Scholar]
21. Diaz D, Sassani JW, Zagon IS, McLaughlin PJ. Reversal of diabetic dry eye by topical opioid receptor blockade follows dual pathways. Invest Ophthalmol Vis Sci. 2025; 66: 24. [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Cant R, Dalgleish AG, Allen RL. Naltrexone Inhibits IL-6 and TNFα production in human immune cell subsets following stimulation with ligands for intracellular toll-like receptors. Front Immunol. 2017; 8: 809. [DOI] [PMC free article] [PubMed] [Google Scholar]
23. Yang P, Xiao L, Zhao F, et al.. Effects of naltrexone on expression of lipid metabolism-related proteins in liver steatosis induced by endoplasmic reticulum stress in mice. Contrast Media Mol Imaging. 2022; 2022: 6572499. [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Bennici G, Almahasheer H, Alghrably M, et al.. Mitigating diabetes associated with reactive oxygen species (ROS) and protein aggregation through pharmacological interventions. Royal Soc Chem. 2024; 14: 17448–17460. [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Zagon IS, Sassani JW, McLaughlin PJ. Re-epithelialization of the rat cornea is accelerated by blockade of opioid receptors. Brain Res. 1998;798: 254–260. [DOI] [PubMed] [Google Scholar]
26. Zagon IS, Sassani JW, McLaughlin PJ. Reepithelialization of the human cornea is regulated by endogenous opioid. Invest Ophthalmol Vis Sci. 2000; 41: 73–81. [PubMed] [Google Scholar]
27. Zagon IS, Klocek MS, Sassani JW, Mauger DT, McLaughlin PJ. Corneal safety of topically applied naltrexone. J Ocul Pharmacol Therap. 2006; 22: 377–387. [DOI] [PubMed] [Google Scholar]
28. Klocek MS, Sassani JW, McLaughlin PJ, Zagon IS. Topically applied naltrexone restores corneal reepithelialization in diabetic rats. J Ocul Pharmacol Ther. 2007; 23: 89–102. [DOI] [PubMed] [Google Scholar]
29. Liang D, Sassani JW., McLaughlin PJ, Zagon IS. Topical application of naltrexone to the corneal surface of healthy volunteers: a safety and tolerability study. J Ocul Pharmacol Ther. 2016;32: 127–132. [DOI] [PubMed] [Google Scholar]
30. Kumar R, Gullapalli RR. High throughput screening assessment of reactive oxygen species (ROS) generation using dihydroethidium (DHE) fluorescence dye. J Vis Exp . 2024; 203: 66238. [DOI] [PMC free article] [PubMed] [Google Scholar]
31. Sabitha S, Hegde SV, Agarwal SV, et al.. Biomarkers of oxidative stress and their clinical relevance in type 2 diabetes mellitus patients: a systematic review. Cureus. 2024; 16: e66570. [DOI] [PMC free article] [PubMed] [Google Scholar]
32. Solomon A, Dursun D, Lu Z, Xie Y, Macri A, Pflugfelder SC. Pro-and anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry-eye disease. Invest Ophthalmol Vis Sci. 2001; 42: 2283–2292. [PubMed] [Google Scholar]
33. Kristiansen OP, Mandrup-Poulsen T. Interleukin-6 and diabetes. The good, the bad, or the indifferent? Diabetes. 2005; 54: S114–S124. [DOI] [PubMed] [Google Scholar]
34. Ortiz G, Blanco T, Singh RB, et al.. IL-6 induces Treg dysfunction in desiccating stress-induced dry eye disease. Exp Eye Res. 2024; 246: 110006. [DOI] [PMC free article] [PubMed] [Google Scholar]
35. Jang D, Lee A, Shin H, et al.. The role of tumor necrosis factor alpha (TNF-α) in autoimmune disease and current TNF-α inhibitors in therapeutics. Int J Mol Sci. 2021; 22: 2719. [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Choubey A. Girdhar K, Kar AK, et al.. Low-dose naltrexone rescues inflammation and insulin resistance associated with hyperinsulinemia. J Biol Chem. 2020; 295: 16359–16369. [DOI] [PMC free article] [PubMed] [Google Scholar]
37. Rodriguez S, Sharma S, Tiarks G, et al.. Neuroprotective effects of naltrexone in a mouse model of post-traumatic seizures. Sci Rep. 2024; 14: 13507. [DOI] [PMC free article] [PubMed] [Google Scholar]
38. Fink MK, Gupta S, Kumar R, Sinha P, et al.. Topical Rho-associated protein kinase inhibit HA1077 reduces rabbit corneal fibrosis and neovasculariation in vivo. J Ocul Pharmacol Ther. 2025. Dec 18. doi: 10.1177/10807683251405622. Online ahead of print. [DOI] [PubMed] [Google Scholar]

[bib1] 1. American Diabetes Association. Eye health: dry eye with diabetes. 2023. Available at: https://diabetes.org/sites/default/files/2023-09/EyeHealth_Resource_Dry-Eye_rev-1.pdf.

[bib2] 2. Sapra A, Bhandari P.. Diabetes. 2023 Jun 21. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025. PMID: 31855345. [PubMed] [Google Scholar]

[bib3] 3. Zeppieri M, Capobianco M, Visalli F, Musa M, et al.. Beyond the surface: revealing the concealed effects of hyperglycemia on ocular surface homeostasis and dry eye disease. Medicina. 2025; 61: 1992–2008. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib4] 4. Zagon IS, Klocek MS, Sassani JW, McLaughlin PJ. Dry eye reversal and corneal sensation restoration with topical naltrexone in diabetes mellitus. Arch Ophthalmol. 2009; 127: 1468–1473. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib5] 5. Zagon IS, Sassani JW, Purushothaman I, McLaughlin PJ. Dysregulation of the OGF-OGFr pathway correlates with elevated serum OGF and ocular surface complications in the diabetic rat. Exp Biol Med. 2020; 245: 1414–1421. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib6] 6. Zagon IS, Sassani JW, Purushothaman I, McLaughlin PJ. Blockade of the opioid growth factor receptor (OGFr) delays the onset and reduces the severity of diabetic ocular surface complications Exp Biol Med . 2021; 246(5): 629–636. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib7] 7. Diaz D, Sassani JW, Zagon IS, McLaughlin PJ. Topical naltrexone increases aquaporin 5 production in the lacrimal gland and restores tear production in diabetic rats. Exp Biol Med . 2024; 249: 10175. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib8] 8. McLaughlin PJ, Sassani JW, Diaz DP, Zagon IS. Elevated opioid growth factor alters the limbus in type 1 diabetic rats. J. Diabetes Clin Res. 2023; 5: 1–10. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib9] 9. Volpe CMO, Villar-Delfino PH, do Anjos PMF, Nogueira-Machado JA. Cellular death, reactive oxygen species (ROS) and diabetic complications. Cell Death Disease. 2018; 9: 119. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib10] 10. Zhuang D, Misra SL, Mugisho OO, et al.. NLRP3 Inflammasome as a potential therapeutic target in dry eye disease. Int J Mol Sci. 2023; 24: 10866. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib11] 11. Zha Z, Xiao D, Liu Z, et al.. Endoplasmic reticulum stress induces ROS production and activates NLRP3 inflammasome via the PERK-CHOP signaling pathway in dry eye disease. Invest Ophthal Vis Sci. 2024; 65: 34. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib12] 12. Rao SK, Mohan R, Gokhale N, et al.. Inflammation and dry eye disease-where are we? Int J Ophthalmol. 2022; 15: 820–827. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib13] 13. Wan L, Bai X, Zhou Q, et al.. The advanced glycation end-products (AGEs)/ROS/NLRP3 inflammasome axis contributes to delayed diabetic corneal wound healing and nerve regeneration. Int J Biol Sci. 2022; 18: 809–825. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib14] 14. Meier DT, De P, Souza J, Donath MY. Targeting the NLRP3 inflammasome-IL-1β pathway in type 2 diabetes and obesity. Diabetologia. 2025; 68: 3–16 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib15] 15. Niu L, L ZS, Wu J, Chen L, Wang Y. Upregulation of NLRP3 inflammasome in the tears and ocular surface of dry eye patients. PloS One. 19(5): e0126277. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib16] 16. Seen S, Tong L. Dry eye disease and oxidative stress. Acta Ophthalmol. 2018:96: e412–e420. [DOI] [PubMed] [Google Scholar]

[bib17] 17. Zagon S, Purushothaman McL. Blockade of the opioid growth factor receptor (OGFr) delays the onset and reduces the severity of diabetic ocular surface complications. Exp Biol Med. 2021; 246(5): 629–636. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib18] 18. Purushothaman I, Sassani JW, Zagon IS, McLaughlin PJ. Ocular surface complications result from dysregulation of the OGF-OGFr signaling pathway in female diabetic rats. Exp Ther Med. 2021; 22: 687. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib19] 19. Purushothaman I, Zagon IS, Sassani JW, et al.. Sex differences in the magnitude of diabetic ocular surface complications: role of serum OGF. Physiol Behav. 2021; 237: 113436. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib20] 20. McLaughlin PJ, Zagon IS. Duration of opioid receptor blockade determines biotherapeutic response. Biochem Pharmacol . 2015; 97: 236–246. [DOI] [PubMed] [Google Scholar]

[bib21] 21. Diaz D, Sassani JW, Zagon IS, McLaughlin PJ. Reversal of diabetic dry eye by topical opioid receptor blockade follows dual pathways. Invest Ophthalmol Vis Sci. 2025; 66: 24. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib22] 22. Cant R, Dalgleish AG, Allen RL. Naltrexone Inhibits IL-6 and TNFα production in human immune cell subsets following stimulation with ligands for intracellular toll-like receptors. Front Immunol. 2017; 8: 809. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib23] 23. Yang P, Xiao L, Zhao F, et al.. Effects of naltrexone on expression of lipid metabolism-related proteins in liver steatosis induced by endoplasmic reticulum stress in mice. Contrast Media Mol Imaging. 2022; 2022: 6572499. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib24] 24. Bennici G, Almahasheer H, Alghrably M, et al.. Mitigating diabetes associated with reactive oxygen species (ROS) and protein aggregation through pharmacological interventions. Royal Soc Chem. 2024; 14: 17448–17460. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib25] 25. Zagon IS, Sassani JW, McLaughlin PJ. Re-epithelialization of the rat cornea is accelerated by blockade of opioid receptors. Brain Res. 1998;798: 254–260. [DOI] [PubMed] [Google Scholar]

[bib26] 26. Zagon IS, Sassani JW, McLaughlin PJ. Reepithelialization of the human cornea is regulated by endogenous opioid. Invest Ophthalmol Vis Sci. 2000; 41: 73–81. [PubMed] [Google Scholar]

[bib27] 27. Zagon IS, Klocek MS, Sassani JW, Mauger DT, McLaughlin PJ. Corneal safety of topically applied naltrexone. J Ocul Pharmacol Therap. 2006; 22: 377–387. [DOI] [PubMed] [Google Scholar]

[bib28] 28. Klocek MS, Sassani JW, McLaughlin PJ, Zagon IS. Topically applied naltrexone restores corneal reepithelialization in diabetic rats. J Ocul Pharmacol Ther. 2007; 23: 89–102. [DOI] [PubMed] [Google Scholar]

[bib29] 29. Liang D, Sassani JW., McLaughlin PJ, Zagon IS. Topical application of naltrexone to the corneal surface of healthy volunteers: a safety and tolerability study. J Ocul Pharmacol Ther. 2016;32: 127–132. [DOI] [PubMed] [Google Scholar]

[bib30] 30. Kumar R, Gullapalli RR. High throughput screening assessment of reactive oxygen species (ROS) generation using dihydroethidium (DHE) fluorescence dye. J Vis Exp . 2024; 203: 66238. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib31] 31. Sabitha S, Hegde SV, Agarwal SV, et al.. Biomarkers of oxidative stress and their clinical relevance in type 2 diabetes mellitus patients: a systematic review. Cureus. 2024; 16: e66570. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib32] 32. Solomon A, Dursun D, Lu Z, Xie Y, Macri A, Pflugfelder SC. Pro-and anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry-eye disease. Invest Ophthalmol Vis Sci. 2001; 42: 2283–2292. [PubMed] [Google Scholar]

[bib33] 33. Kristiansen OP, Mandrup-Poulsen T. Interleukin-6 and diabetes. The good, the bad, or the indifferent? Diabetes. 2005; 54: S114–S124. [DOI] [PubMed] [Google Scholar]

[bib34] 34. Ortiz G, Blanco T, Singh RB, et al.. IL-6 induces Treg dysfunction in desiccating stress-induced dry eye disease. Exp Eye Res. 2024; 246: 110006. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib35] 35. Jang D, Lee A, Shin H, et al.. The role of tumor necrosis factor alpha (TNF-α) in autoimmune disease and current TNF-α inhibitors in therapeutics. Int J Mol Sci. 2021; 22: 2719. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib36] 36. Choubey A. Girdhar K, Kar AK, et al.. Low-dose naltrexone rescues inflammation and insulin resistance associated with hyperinsulinemia. J Biol Chem. 2020; 295: 16359–16369. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib37] 37. Rodriguez S, Sharma S, Tiarks G, et al.. Neuroprotective effects of naltrexone in a mouse model of post-traumatic seizures. Sci Rep. 2024; 14: 13507. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib38] 38. Fink MK, Gupta S, Kumar R, Sinha P, et al.. Topical Rho-associated protein kinase inhibit HA1077 reduces rabbit corneal fibrosis and neovasculariation in vivo. J Ocul Pharmacol Ther. 2025. Dec 18. doi: 10.1177/10807683251405622. Online ahead of print. [DOI] [PubMed] [Google Scholar]

PERMALINK

Mitigation of Oxidative Stress Pathways in the Diabetic Cornea and Lacrimal Glands Contributes to the Rapid Reversal of Diabetic Dry Eye by Naltrexone

Allison K Krebs

David Diaz

Joseph W Sassani

Ian S Zagon

Patricia J McLaughlin

Abstract

Purpose

Methods

Results

Conclusions

Materials and Methods

Animals and Treatment

Treatment With NTX to Block the OGFr

Physiological Parameters of Diabetes and Dry Eye

Euthanasia and Tissue Collection

Immunofluorescence Detection of ROS and Related Proteins

Data Analysis

Results

Clinical Signs of Diabetic Dry Eye

Morphology of Cornea Epithelium and Lacrimal Glands and Mediation of the ROS Pathway

Figure 1.

Activation of ROS in Diabetes and Mediation of ROS by NTX on the Corneal Surface

Mediation of ROS in the Lacrimal Glands With NTX

Activation of the CHOP and NOX-2 Pathways in Corneal Epithelium of Diabetic Rats and Reversal With NTX

Figure 2.

Regulation by NTX of CHOP and NOX-2 in the Lacrimal Glands of Diabetic Rats

Figure 3.

Mediation of Inflammasome Pathways on the Corneal Surface and Lacrimal Glands by Topical NTX: IL-1β

Figure 4.

Figure 5.

Figure 6.

Mediation of Inflammasome Pathways on the Corneal Surface and Lacrimal Glands by Topical NTX: IL-6

Mediation of Inflammasome Pathways on the Corneal Surface and Lacrimal Glands by Topical NTX: TNF-α

Discussion

Acknowledgments

References

ACTIONS

PERMALINK

RESOURCES

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