(T1230-03-20) A Novel Trifunctional Donor Enhances Nrf2 Activation and Preserves Retinal Integrity Under Oxidative Stress

Purpose: Glaucoma, a leading cause of irreversible blindness worldwide, is a multifactorial neurodegenerative disorder characterized by progressive degeneration of retinal ganglion cells (RGCs) and their axons. Oxidative stress has been implicated in the progression of glaucoma, especially through its contribution to mitochondrial dysfunction. Current research suggests that oxidative stress can serve both as a trigger and as a consequence of mitochondrial dysfunction. It forms a self-perpetuating cycle that contributes to RGC degeneration (Böhm et al., 2023). Nrf2 activation plays a pivotal role in maintaining redox homeostasis in response to physiological stressors that include aging, chronic inflammation, and exposure to light (Zhang et al., 2023). Inadequate activation of Nrf2 has been found to be related to various oxidative stress-related diseases, including glaucoma (Song et al., 2014). Diminished activity of Nrf2 weakens the antioxidant shield, rendering RGCs more vulnerable to oxidative damage (Rohowetz et al., 2018). Hence, modulating the Nrf2 signaling either through molecular or pharmacological means represents a promising strategy in the management of glaucoma. Such an approach holds the potential to mitigate both oxidative and inflammatory damage in glaucomatous degeneration (Batliwala et al., 2017). In this context, certain endogenous gaseous molecules, known as gas transmitters, such as nitric oxide (NO) and hydrogen sulfide (H₂S), have attracted interest for their therapeutic potential in glaucoma, owing to their ability to modulate oxidative stress pathways and influence neuroprotective mechanisms (Han et al., 2021). Therefore, the purpose of this study was to evaluate the potential of AMP, a novel trifunctional compound capable of donating NO and H₂S, to activate the Nrf2 pathway and preserve retinal integrity against oxidative damage induced by hydrogen peroxide (H₂O₂).

Methods: Eyeballs were obtained from a local slaughterhouse (Nebraska beef LLC, Omaha, NE) within 30 minutes of slaughter and transported in a freezer container to the lab in 15 minutes. Eyeballs were injected intravitreally with 50 µl of AMP (10^-11 M and 10-¹²M) or H₂O₂ (10^-3 and 10^-4) in the presence and absence of AMP (10^-11 Mand 10^-12 M). Eyeballs were incubated in oxygenated Kreb’s buffer solution (37°C) for 1 hour, then dissected, and retinas isolated and fixed in formalin for morphological studies using light microscopy. Some retinal tissues were used for western blotting analysis to quantify Nrf2 (p-Nrf2) protein expression in different treatment groups as follows: control (No treatment), AMP (10^-11 & 10^-10) only, H₂O₂ 10^-3 & 10^-4only, and H₂O₂ (10^-3 & 10^-4) in the presence of AMP (10^-11 & 10^-10). Protein lysates were normalized, separated by SDS-PAGE, and probed with Anti-Nrf2 (phospho S40) antibody (Abcam), the membrane was stripped and re-probed for loading control with anti-beta Actin antibody (Abcam), followed by appropriate secondary detection. Bands developed were quantified using ImageJ software.

Results: H&E staining revealed that AMP (10^-11 & 10^-10)-treated retinas [Fig 1B (b) & (c)] were similar to the control group [Fig A (a) & (b)], suggesting that the trifunctional compound preserved retinal architecture and maintained clearly distinguishable retinal layers. In corroboration, in Western blot analysis, AMP treatment significantly (p < 0.001) activated Nrf2 signaling from 0.1 (control) to 1.48 and 1.72 [Fig 2(a)], suggesting its potential role in priming retinal cells by enhancing their basal antioxidant defense mechanism even in the absence of external oxidative stress. Exposure to H₂O₂ stimuli induced loss of the outer segment layer and loss of retinal laminar organization, suggesting oxidative damage to retinal tissues [Fig B (a) & (b)]. Similarly, H₂O₂ treatment (10⁻³ M and 10⁻⁴ M) [Fig 2 (b)] led to a reduction in total Nrf2 expression, suggesting suppression of the endogenous antioxidant pathway under oxidative stress conditions. However, the presence of AMP (10^-11 & 10^-10) in H₂O₂-treated tissues elicited a significant (p < 0.0001) upregulation of Nrf2 expression from 0.390 (H₂O₂) to 1.84 (371.8%) and 1.98 (407.7%), respectively [Fig 2(c)], compared to the H₂O₂-treatment group [Fig 2(b)], suggesting that the trifunctional compound activated the antioxidant defense pathways. Quantification of Nrf2 bands (normalized to β-actin) showed a significant increase in protein level in the AMP-treated group.

Conclusion: The trifunctional compound, AMP, preserved retinal morphology and enhanced antioxidant responses by activating Nrf2 (p-Nrf2), affirming its potential to counteract oxidative damage in ocular neuropathies such as glaucoma. These preliminary findings lay the groundwork for a new therapeutic strategy that targets neurodegeneration in glaucoma and related optic neuropathies.

References: Batliwala, S., Xavier, C., Liu, Y., Wu, H., Pang, I.-H., 2017. Involvement of Nrf2 in Ocular Diseases. Oxid. Med. Cell. Longev. 2017, 1703810. https://doi.org/10.1155/2017/1703810
Böhm, E.W., Buonfiglio, F., Voigt, A.M., Bachmann, P., Safi, T., Pfeiffer, N., Gericke, A., 2023. Oxidative stress in the eye and its role in the pathophysiology of ocular diseases. Redox Biol. 68, 102967. https://doi.org/10.1016/j.redox.2023.102967
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Song, J., Kang, S.M., Lee, W.T., Park, K.A., Lee, K.M., Lee, J.E., 2014. Glutathione Protects Brain Endothelial Cells from Hydrogen Peroxide-Induced Oxidative Stress by Increasing Nrf2 Expression. Exp. Neurobiol. 23, 93–103. https://doi.org/10.5607/en.2014.23.1.93
Zhang, J., Zhang, T., Zeng, S., Zhang, X., Zhou, F., Gillies, M.C., Zhu, L., 2023. The Role of Nrf2/sMAF Signalling in Retina Ageing and Retinal Diseases. Biomedicines 11, 1512. https://doi.org/10.3390/biomedicines11061512

Acknowledgements: Financial support from School of Pharmacy and Health Professions, Creighton University.

Funding: This study was funded by NIH NEI 1R15EY032275-01A1. Conflict of Interest Statements: We confirm that there are no conflicts of interest associated with this work, and there has been no significant financial support for this work that could have influenced its outcome.

Figure 1A: Effect of AMP on retinal morphology in bovine ex vivo model (H&E staining).
Representative H&E-stained retinal sections are shown for:
(a) No Treatment (NT/control) (b) AMP

Figure 1B: Effect of H2O2 on retinal morphology in bovine ex vivo model (H&E staining).
Representative hematoxylin and eosin (H&E) stained retinal sections are shown for:
(a) No Treatment (NT/control) (b) H₂O₂.

Figure 2 (a): AMP treatment alone (10⁻¹¹ M and 10⁻¹⁰ M) significantly increased p-Nrf2 levels, indicating activation of Nrf2 signaling via phosphorylation, even in the absence of oxidative in (n = 3; AMP 10⁻¹¹ M, p < 0.1; AMP 10⁻¹⁰ M, p < 0.05 vs. control).

Figure 2 (b): Treatment with hydrogen peroxide (H₂O₂; 10⁻³ M and 10⁻⁴ M) led to a reduction in total Nrf2 expression, suggesting suppression of the endogenous antioxidant pathway under oxidative stress conditions.

Figure 2 (c): Co-treatment with AMP (10⁻¹¹ M and 10⁻¹⁰ M) and H₂O₂ (10⁻³ M) restored and significantly enhanced total Nrf2 expression compared to the H₂O₂-only group, demonstrating that the trifunctional compound counteracts oxidative damage by upregulating the Nrf2-mediated antioxidant defense mechanism (n = 3; p < 0.00001 for both AMP concentrations vs. H₂O₂ alone).