THE PROTECTIVE EFFECT OF APOCYNIN ON A SELENITE-INDUCED CATARACT MODEL

Introduction

Cataract, defined as the opacification of the lens, is the leading cause of reversible blindness, accounting for approximately 51% of all cases worldwide.¹ Although modern cataract surgery is generally regarded as highly safe and remains the only effective treatment for cataracts, it is not always accessible or affordable. Moreover, potential intraoperative and postoperative complications further highlight the need for non-surgical alternatives.² Oxidative stress is a major contributor to cataractogenesis, primarily through the imbalance between reactive oxygen species (ROS) and endogenous antioxidant defence systems such as CAT, superoxide dismutase (SOD), and glutathione peroxidase (GPx).³ The accumulation of ROS promotes lipid peroxidation, protein aggregation, DNA damage, and apoptosis of lens epithelial cells, all of which contribute to lens opacification.^3–5 Recent metabolomic studies also indicate that cataract formation is accompanied by disruptions in glutathione-related pathways and other antioxidant-linked metabolites, further supporting the central role of oxidative imbalance in cataractogenesis.⁶ Given its central role in cataract development, antioxidant therapy has been widely proposed as a promising non-surgical approach for delaying or preventing disease progression.⁷ Various compounds, including sildenafil, melatonin, ascorbic acid, resveratrol, ellagic acid, N-acetylcysteine and rosmarinic acid have demonstrated protective effects in experimental cataract models.^2,4,8–12

Of these models used in previous research, the selenite-induced cataract model in rats is one of the most extensively utilized due to its reproducibility and close resemblance to age-related human cataracts.^2,4 Selenite administration disrupts calcium homeostasis, accelerates proteolysis, and depletes glutathione levels, thereby mimicking the molecular and structural alterations observed in human cataractogenesis.^2,13

Apocynin (4-hydroxy-3-methoxyacetophenone) is a natural NADPH oxidase inhibitor derived from Picrorhiza kurroa and Apocynum cannabinum. It prevents the assembly of the NADPH oxidase complex and consequently reduces reactive oxygen species (ROS) production. Apocynin has been reported to exhibit antioxidant, anti-inflammatory, and cytoprotective effects in several systemic disease models.¹⁴ Previous studies have demonstrated its beneficial roles in cardiovascular disease, renal injury, neuroinflammation, and metabolic disorders, largely attributed to its capacity to suppress oxidative stress and modulate redox-sensitive signaling pathways.¹⁵ Furthermore, apocynin has been reported to enhance endogenous antioxidant defenses, preserve mitochondrial function, and attenuate inflammatory cytokine production in multiple in vivo models.¹⁶ Despite these documented systemic benefits, its potential therapeutic effects in ocular tissues, particularly in cataract formation, remain insufficiently explored, highlighting the need for further investigation.

The aim of this study was to investigate the potential protective effect of apocynin against selenite-induced cataract formation. In addition to assessing cataract severity, the impact on key oxidative stress biomarkers in the lens tissue was evaluated, including malondialdehyde (MDA), catalase (CAT), and glutathione reductase (GR). MDA is a well-established marker of lipid peroxidation and reflects structural damage caused by ROS,¹⁷ whereas CAT and GR are critical enzymatic components of the endogenous antioxidant defence system.¹⁸ These parameters were selected as they have been shown to be sensitive and relevant for the evaluation of redox imbalance in experimental cataract models.¹⁹ Based on this background, we hypothesized that apocynin may exert a protective effect against selenite-induced cataract formation through modulation of oxidative stress in lens tissue. An earlier version of this study was posted as a preprint.²⁰

Materials and Methods

The establishment of the cataract rat model and other experimental procedures were conducted in the Experimental Animals Application and Research Centre of Abant Izzet Baysal University. Biochemical analyses were performed in the Biochemistry Laboratory of Basaksehir Cam and Sakura City Hospital. Prior to the study, ethical approval was obtained from the Animal Experiments Local Ethics Committee of Abant Izzet Baysal University (Date: 9^th June 2021, Approval Number: 2021/19).

Results

Morphological examination of cataract formation

The microscopic images obtained from the rat lenses are presented shown in Figure 1, and the clinical assessment results regarding cataract grading are summarized shown in Table 2. Cataract formation was observed in all rats in Group 1, confirming the success of the selenite-induced cataract model used in this experimental study.

Figure 1. Right and left microscopic images of lenses obtained from rats with cataract.

Table 2

Comparison of cataract grades among control, selenite-induced cataract, and treatment groups in the experimental model

NUMBER of LENSES	CONTROL (Saline)	GROUP 1 (Selenite)	GROUP 2 (Selenite+Dmso)	GROUP 3 (Selenite + 10 mg/kg apocynin)	GROUP 4 (Selenite + 20 mg/kg apocynin)
1	0	6	5	1	2
2	0	5	5	1	2
3	0	5	4	2	3
4	0	6	5	1	3
5	0	6	4	1	2
6	0	5	4	2	4
7	0	6	4	2	2
8	0	5	4	2	4
9	0	5	5	1	2
10	0	5	4	1	2
X ± SD Min-Max	0 ± 0 0 – 0	5.40 ± 0.52*** 5 – 6	4.40 ± 0.52*** 4 – 5	1.4 ± 0.52 ## , † 1 – 2	2.60 ± 0.84*, # 2 – 4

All values are given as mean ± standard deviation (X ± SD), minimum-maximum (Min-Max). ***p<0.001 and *p<0.05 vs. Control group (Saline-treated group).

## p<0.001

# p<0.05 vs Group 1(Selenite-induced cataract group).

† p<0.05 vs Group 2 (Selenite + DMSO group). Cataract grades represent ordinal data; mean values are provided for descriptive purposes only, and non-parametric statistical tests were used for group comparisons.

Slit-lamp biomicroscopic evaluation revealed that sodium selenite administration resulted in marked lens opacities in Groups 1 and 2, whereas apocynin treatment significantly reduced cataract severity (p < 0.001, p < 0.05). Specifically, cataract grades were significantly reduced in the apocynin-treated groups compared to the selenite-induced cataract group (Group 1), as also reflected in Table 2. The low-dose apocynin group (Group 3) showed numerically lower cataract grades compared to the high-dose group (Group 4); however, this difference did not reach statistical significance (p > 0.05). The detailed cataract grading of all the groups is shown in Table 2. Table 2 displays individual lens-level cataract grades to ensure transparency of morphological findings; however, all statistical comparisons were conducted at the animal level. Although cataract grades are ordinal by nature, mean values are presented for descriptive comparison across groups, while non-parametric statistical tests were applied for group comparisons, and individual lens-level data are also provided to allow direct interpretation of the ordinal distribution.

Biochemical analyses of lenses

Boxplots were included to visually illustrate the distribution and variability of biochemical parameters across groups and to complement the tabulated summary statistics.

In the selenite (Group 1) and DMSO (Group 2) groups, MDA levels were significantly elevated compared to the control group (p < 0.001). MDA levels in the apocynin-treated groups (Groups 3 and 4) were evaluated relative to the selenite-induced cataract group (Group 1). No significant change in MDA levels was observed in Group 3 (p = 0.99), and Group 4 showed a significant increase in MDA levels (p = 0.04). This finding suggests a potential pro-oxidant effect at the higher dose of apocynin (shown in Figure 2 and Table 3).

Figure 2. Box-plot graph presentation of comparison of malondialdehyde (MDA) levels measured in lenses between the experimental groups.

*p < 0.05, **p < 0.01 and ***p < 0.001 vs the control group.

Table 3.

Comparison of biochemical parameters among control, selenite-induced cataract, and treatment groups

Biochemical parameter	Control	Group 1	Group 2	Group 3	Group 4	P value
MDA (nmol/ml)	0.57 ± 0.05a	0.88 ± 0.09b	0.80 ± 0.18b	0.84 ± 0.07b	1.07 ± 0.06c	<0.0001
	0.49 – 0.62	0.79 – 0.99	0.62 – 1.09	0.74 – 0.92	1.00 – 1.15
Catalase (ng/ml)	64.94 ± 3.46d	29.56 ± 11.66e	41.99 ± 16.31e,f	57.44 ± 6.71d	44.79 ± 6.76f	0.0002
	59.88-68.33	20.47 – 49.77	25.53 – 64.79	46.51 – 63.72	36.63 – 53.49
GR (ng/ml)	18.88 ± 2.73g	11.12 ± 2.41h	13.31 ± 2.49h	18.01 ± 2.51g	20.61 ± 2.54g	<0.0001
	15.52–21.33	7.78 – 13.87	9.91 – 16.86	15.37 – 20.87	17.26 – 23.14

All values are given as mean ± standard deviation, minimum-maximum values. MDA: Malondialdehyde, GR: Glutathione reductase

a-h: Different superscript letters indicate statistically significant differences between groups (p < 0.05, one-way ANOVA followed by Tukey post hoc test). In addition, comparisons relative to the control group and the selenite-induced cataract group (Group 1) were specifically evaluated to improve interpretability of treatment effects.

CAT activity was significantly reduced in the selenite and DMSO groups (Groups 1 and 2) (p < 0.01). In Group 3, CAT activity improved significantly compared to the selenite-induced cataract group (Group 1) (p = 0.003), and a moderate increase was observed in Group 4, not of statistical significance (p > 0.05) (Shown in Figure 3 and Table 3).

Figure 3. Box-plot graph presentation of comparison of catalase levels measured in lenses between the experimental groups.

*p < 0.05 and ***p < 0.001 vs the control group.

GR levels were significantly decreased in the selenite group. A statistically significant increase in GR activity was observed in both Group 3 and Group 4 compared to the selenite-induced cataract group (Group 1) (p < 0.05) (shown in Figure 4 and Table 3).

Figure 4. Box-plot graph presentation of comparison of glutathione reductase (GR) levels measured in lenses between the experimental groups.

*p < 0.05 and **p < 0.01 vs the control group.

Following statistically significant overall group differences, post-hoc pairwise comparisons were performed as specified in the Methods section, and corresponding p-values are reported for the relevant group comparisons. In these analyses, apocynin-treated groups (Groups 3 and 4) demonstrated significantly more favorable oxidative stress profiles compared with the selenite-only (Group 1) and DMSO (Group 2) groups.

Discussion

The results of this study demonstrated that apocynin, particularly at a low dose (10 mg/kg), exerted protective effects against selenite-induced cataractogenesis in rats. These effects were supported by reduced lens opacification and restoration of antioxidant enzyme activities. Low-dose apocynin significantly increased catalase (CAT) and glutathione reductase (GR) activities to levels comparable with those of the control group. Moreover, the oxidative stress marker malondialdehyde (MDA) did not increase and remained similar to control levels, indicating effective prevention of lipid peroxidation.

Oxidative stress plays a central role in cataract pathogenesis. The selenite-induced cataract model is widely used due to its rapid onset, simplicity, reproducibility, and strong resemblance to human age-related cataracts, particularly in terms of glutathione depletion, lipid peroxidation, and insoluble lens protein accumulation.^2,4,13 In the current study, the model validity was confirmed by the development of dense nuclear opacities in Group 1, together with elevated MDA levels and reduced CAT and GR activities, indicating oxidative stress.

Despite inducing a partial enhancement in antioxidant enzyme activities (demonstrated by selective enhancement of GR activity, with minimal influence on CAT levels), high-dose apocynin (20 mg/kg) was found to increase MDA levels. This paradox suggests that the antioxidant benefits of apocynin may be counteracted by dose-dependent toxicity, resulting in a pro-oxidant response. This interpretation is consistent with literature indicating that excessive suppression of physiological ROS signaling can disrupt cellular homeostasis.^21,22

Similar dual effects have been reported for other redox-active compounds such as resveratrol and quercetin, which show cytoprotective effects at low doses but may induce oxidative stress, DNA damage, and apoptosis at higher concentrations.^23,24 Similar antioxidant protection has also been demonstrated for curcumin in rabbit cataract models, where curcumin reduced oxidative stress and delayed lens opacification.²⁵ Although curcumin and apocynin act through different upstream pathways, both compounds converge on modulating oxidative damage, supporting the broader relevance of redox-targeted therapeutic strategies in cataract prevention. In addition, the potential contribution of DMSO as a vehicle to these effects should not be overlooked, as it has been shown to exert cytotoxic and pro-oxidant effects under certain conditions.²⁶ The findings of this study highlight the importance of carefully optimizing both the dose and duration when evaluating antioxidant compounds in preclinical studies. Additionally highlight the need for careful dose optimization in human applications, as dose-dependent shifts between antioxidant and pro-oxidant activity may also occur in clinical settings.

The production of NADPH oxidase–driven reactive oxygen species (ROS) production plays a critical role in cataractogenesis by promoting protein aggregation, lipid peroxidation, and lens opacification.²⁷ Given that oxidative stress is a key mechanism in selenite-induced cataract formation,^4,9 targeting ROS-generating pathways has therapeutic relevance. Therefore, apocynin, which is an inhibitor of NADPH oxidase, was selected for its potential to suppress intracellular ROS production and enhance endogenous antioxidant defence systems, including catalase and glutathione reductase.^28,29 Consistent with previous reports of oxidative stress-related disease models the current study results demonstrate that apocynin exerts protective effects on lens tissue, most likely through both antioxidant and anti-inflammatory mechanism.^28–30 In line with earlier findings in studies of retinal injury and diabetic retinopathy,^31,32 apocynin treatment was found to reduce cataract severity and modulate oxidative biomarkers in the current study model.

Importantly, this study is among the first to have compared different apocynin doses in a cataract model. The findings indicate that low-dose apocynin is more effective than a high dose in reducing oxidative damage, thereby emphasizing the importance of dose optimization to achieve therapeutic benefit while minimizing toxicity.

The biochemical analysis results further support these outcomes. Elevated malondialdehyde (MDA) levels in the selenite group confirmed increased lipid peroxidation, while apocynin treatment, particularly at low doses, reduced MDA levels. Similarly, catalase and glutathione reductase (GR) activities were preserved or enhanced with apocynin, indicating improved redox balance. As disruption of antioxidant enzymes is associated with lens protein denaturation and cataract progression,^33,34 the ability of apocynin to stabilize these enzymes may underlie its protective mechanism.

The results of this study also support a strategic approach for the non-surgical prevention of cataract. Although cataract surgery remains the most effective treatment, access to surgical care is limited in many low- and middle-income countries due to resource constraints and economic burden.^35,36 Pharmacological interventions based on natural compounds may offer a safe, cost-effective, and preventive alternative for early-stage cataracts. Given its pharmacokinetic profile and pleiotropic effects, apocynin emerges as a strong candidate for further preclinical and clinical investigations.

Unlike other antioxidant agents such as ascorbic acid, resveratrol, melatonin, ellagic acid, N-acetylcysteine, rosmarinic acid, and sildenafil, apocynin directly targets the NADPH oxidase complex, a key enzymatic source of ROS generation, thereby representing a mechanistically distinct approach to modulating oxidative stress at the level of ROS generation.¹⁴ This unique mechanism may confer broader and more sustained protective effects in lens tissue.

This study had several limitations that should be acknowledged. First, although the results suggest beneficial effects of apocynin, there was no assessment of the molecular mechanisms underlying its modulation of redox signaling in the lens. Key antioxidant pathways such as Nrf2/ARE, thioredoxin, and glutaredoxin systems, as well as apoptotic markers, were not evaluated.²¹ A second limitation was that the biochemical analysis was limited to ELISA-based measurements, and more detailed molecular techniques such as Western blotting or RT-PCR were not performed. In addition, apocynin was administered exclusively via the intraperitoneal route. Therefore, the comparative efficacy of alternative delivery methods, such as oral, topical, or intravitreal administration, remains unknown. The treatment duration was short-term, and the effects of long-term administration were not investigated. Finally, the absence of a positive control group using a well-characterized antioxidant agent limits the broader interpretability and comparability of the findings.

Conclusion

The results of this study demonstrated that apocynin, particularly at a low dose (10 mg/kg), exerts a significant protective effect against selenite-induced cataract formation in a rat model. This beneficial outcome is believed to be mediated through the enhancement of endogenous antioxidant defence mechanisms in the lens, as evidenced by increased catalase and glutathione reductase activities and the absence of elevated MDA levels. In contrast, high-dose apocynin (20 mg/kg) was associated with increased lipid peroxidation, suggesting a potential dose-dependent pro-oxidant effect. These findings emphasize the importance of dose optimization in the therapeutic application of antioxidant compounds. Although the results are promising, they remain preliminary and require further validation through comprehensive histopathological and molecular studies. If confirmed by future research, apocynin may represent a promising pharmacological candidate for preventing or delaying cataractogenesis through modulation of redox signaling in the lens.

Statement of ethics

The procedures applied to all animals involved in the study comply with the standards outlined in the Universal Declaration of Animal Rights, proclaimed at UNESCO in Paris in 1978. Prior to the study, ethical approval was obtained from the Animal Experiments Local Ethics Committee of Abant Izzet Baysal University (Date: 9th June 2021, Approval Number: 2021/19).