Pharmacological Modulation of ALDH2-SIRT1-Endoplasmic Reticulum Stress Axis in MNU-Induced Retinal Degeneration in C57BL/6 Mice: An In Vivo Study

Summary

The study involved administering methyl-nitrosourea to C57BL/6 mice to induce retinal damage, followed by treatment with ALDH2 activators and inhibitors alongside SIRT1 and endoplasmic reticulum stress (ERS) modulators. The protocol aimed to investigate the protective effects of these treatments on retinal degeneration and their influence on aldehyde metabolism.

Abstract

This study investigated the protective effects of aldehyde dehydrogenase 2 (ALDH2) activation (via Alda-1), ALDH2 inhibition (via Daidzin), and the modulation of sirtuin 1 (SIRT1) and endoplasmic reticulum stress (ERS) on methyl-nitrosourea (MNU)-induced retinal damage in C57BL/6 mice. Seventy-two mice were randomly assigned to eight groups, including a control group, a MNU-alone group, and various treatment combination groups. Mice were administered with intraperitoneal injections of agonists and inhibitors targeting the ALDH2-SIRT1-ERS axis, respectively. Body weight, retinal layer thickness, and aldehyde metabolism biomarkers were assessed on the second and fourth day post-treatment. MNU administration induced retinal degeneration, resulting in significant body weight loss in all groups except the control. Treatment with Alda-1 attenuated retinal damage, while Daidzin exacerbated it. Pharmacological inhibition of SIRT1 (via EX-527) diminished the protective effects of Alda-1, and ERS induction (via TUN) nearly abolished its benefits. Conversely, SIRT1 activation (via SRT1720) mitigated the adverse effects of Daidzin, while ERS inhibition (via 4-PBA) partially alleviated Daidzin's impact. Furthermore, MNU increased levels of aldehyde metabolism markers (malondialdehyde [MDA] and 4-hydroxy-2-nonenal [4-HNE]), with Alda-1 reducing and Daidzin elevating these levels. Besides, pharmacological modulation of the SIRT1/ERS pathways intervened in the regulatory effects of Alda-1 and Daidzin on the aldehyde metabolism. Overall, ALDH2 activation protected retinal structure and regulated aldehyde metabolism, highlighting the critical role of the ALDH2-SIRT1/ERS signaling axis in mitigating retinal degeneration.

Introduction

Retinal degeneration, a leading cause of blindness worldwide, includes conditions such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), and diabetic retinopathy (DR), which collectively affect millions of individuals and impose substantial socioeconomic burdens^1,2,3. However, there is currently a lack of highly effective pharmacological therapies to halt the progression of these blinding retinal diseases in clinical practice. The pathogenesis of retinal degeneration is multifactorial, involving oxidative stress, chronic inflammation, and endoplasmic reticulum stress (ERS), which disrupts retinal cell homeostasis. These stressors are often triggered by internal metabolic dysregulation (e.g., mitochondrial dysfunction), ultimately leading to apoptosis and irreversible retinal damage⁴. Methyl-nitrosourea (MNU), a potent DNA alkylating agent, has emerged as a widely utilized chemical inducer for modeling retinal degeneration⁵. The MNU-induced retinal disease model serves as a valuable tool for investigating disease mechanisms and evaluating potential therapeutic interventions. Mechanistically, MNU induces oxidative stress by generating reactive oxygen species (ROS) and disrupting redox balance, thereby damaging photoreceptor membranes and organelles. This also might affect aldehyde metabolism, impairing the detoxification of lipid peroxidation. The subsequent accumulation of toxic aldehydes might exacerbate oxidative damage, amplifying photoreceptor apoptosis⁶.

Aldehyde dehydrogenase 2 (ALDH2), a mitochondrial NAD⁺-dependent enzyme, plays a critical role in detoxifying toxic aldehydes such as 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) by converting them into less reactive carboxylic acids, thereby preventing oxidative damage⁷. Robust ALDH2 activity is crucial for maintaining redox homeostasis, and impaired ALDH2 function has been linked to accelerated aging and neurodegenerative pathologies⁸. Pharmacological activation of ALDH2, notably via the small-molecule agonist Alda-1, has been demonstrated to exert cytoprotective effects in various models^9,10. Sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide + (NAD+)-dependent deacetylase, regulates stress responses and energy metabolism by modulating transcription factors, including p53 and Nuclear factor kappa B (NF-κB)¹¹. SIRT1 activation has shown promise in promoting mitochondrial function and cell survival, with studies linking its upregulation to reduced oxidative damage and inflammation in retinal degeneration¹². Endoplasmic reticulum stress (ERS), triggered by the accumulation of misfolded proteins, activates the unfolded protein response (UPR), which can become a pro-apoptotic cascade under chronic stress. ERS has been implicated in retinal degeneration, contributing to photoreceptor apoptosis¹³. Despite these findings, the specific interplay between ALDH2, SIRT1, and ERS in the context of the MNU-induced retinal degeneration model remains poorly understood. In addition, the coordinated roles of these interconnected pathways require further systematic investigation.

Therefore, the current study aimed to investigate the effects of key components of the ALDH2-SIRT1-ERS axis—ALDH2 (via Alda-1 or Daidzin), SIRT1 (via SRT1720 or EX527), and ERS (via 4-PBA or TUN)—on MNU-induced retinal damage in C57BL/6 mice. Using histopathological and biochemical analyses, we examined how targeting this axis affected photoreceptor survival and aldehyde metabolism. We hypothesized that coordinated activation of ALDH2, enhancement of SIRT1, and suppression of ERS would synergistically mitigate MNU-induced retinal degeneration, thereby providing a rationale for targeting this axis in retinal degenerative diseases.

Protocol

Ethical statement
All experimental protocols were reviewed and approved by the Experimental Animal Care and Use Committee of Fuzong Clinical Medical College of Fujian Medical University and conducted in strict compliance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.

Animals and group allocation
A total of 72 male C57BL/6 mice (8–10 weeks old, body weight 20–25 g) were obtained from the certified supplier, adhering to national standards for laboratory animal breeding. The mice were housed in individually ventilated cages under standardized environmental conditions, including a 12-h light/dark cycle, controlled temperature (22 ± 2 °C), and relative humidity (50% ± 10%), with ad libitum access to food and water. To investigate the modulatory effects of ALDH2, SIRT1, and ERS pathways on MNU-induced retinal damage, the mice were randomly allocated into eight experimental groups (n = 9 per group; n = 5 for the second day group; n = 4 for the fourth day group) using a computer-generated randomization sequence to minimize selection bias. The graphic abstract of the study was shown in Figure 1.

Reagents preparation and treatment regimens
The experimental reagents used in this study were listed in the Table of Materials, with detailed information on preparation and dosage as follows. (1) MNU (50 mg/kg): 100 mg of MNU was dissolved in 2 mL of normal saline to prepare a 50 mg/mL solution. Then, 1.4 mL of this solution was diluted with 5.6 mL of normal saline to a final concentration of 10 mg/mL for animal administration. (2) Alda-1 (10 mg/kg): 50 mg of Alda-1 powder was dissolved in 1 mL of dimethyl sulfoxide (DMSO) to prepare a 50 mg/mL solution, which was diluted with 24 mL of normal saline to a final concentration of 2 mg/mL. (3) Daidzin (10 mg/kg): 50 mg of Daidzin powder was dissolved in 1 mL of DMSO to prepare a 50 mg/mL solution, which was then diluted with 24 mL of normal saline to a final concentration of 2 mg/mL. (4) EX-527 (10 mg/kg): 5 mg of EX-527 powder was dissolved in 0.1 mL of DMSO to prepare a 50 mg/mL solution, which was diluted with 2.4 mL of normal saline to a final concentration of 2 mg/mL. (5) SRT1720 (10 mg/kg): 5 mg of SRT1720 powder was dissolved in 0.1 mL of DMSO and mixed well. Then, 400 µL of PEG300, 50 µL of Tween-80, and 450 µL of normal saline were added to prepare a 2 mg/mL working solution. (6) TUN (10 mg/kg): 5 mg of TUN powder was dissolved in 0.1 mL of DMSO to prepare a 50 mg/mL solution, which was then diluted with 2.4 mL of normal saline to a final concentration of 2 mg/mL. (7) 4-Phenylbutyrate (4-PBA; 10 mg/kg): 2 mg of 4-PBA powder was dissolved in 1 mL of normal saline to prepare a 2 mg/mL solution. All the above administration volumes were 5 × M milliliters (where M = animal body weight in kg). After that, the mice were divided into eight groups with the following treatment protocols:

Control group: A single intraperitoneal injection of normal saline.
MNU group: A single intraperitoneal injection of MNU.
MNU + Alda-1 group: Intraperitoneal injection of Alda-1, followed by a single administration of MNU 2 h later. Alda-1 was then administered intraperitoneally once daily.
MNU + Daidzin group: Intraperitoneal injection of Daidzin, followed by a single administration MNU injection 2 h later. Daidzin was then administered intraperitoneally once daily.
MNU + Alda-1 + EX-527 group: Intraperitoneal injection of Alda-1, followed by EX-527 injection 1 h later, and MNU injection 1 h after that (single administration). Alda-1 was then administered intraperitoneally once daily.
MNU + Alda-1 + TUN group: Intraperitoneal injection of Alda-1, followed by TUN injection 1 h later, and MNU injection 1 h after that. Alda-1 was then administered intraperitoneally once daily.
MNU + Daidzin + SRT1720 group: Intraperitoneal injection of Daidzin, followed by SRT1720 injection 1 h later, and MNU injection 1 h after that. Daidzin was then administered intraperitoneally once daily.
MNU + Daidzin + 4-PBA group: Intraperitoneal injection of Daidzin, followed by 4-PBA injection 1 h later, and MNU injection 1 h after that. Daidzin was then administered intraperitoneally once daily.

Body weight monitoring
Systemic toxicity and treatment-related physiological effects were monitored by recording the body weight of each mouse daily for four consecutive days following MNU administration. Weights were measured using a precision digital scale with an accuracy of ±0.1 g, and data were documented to track trends in weight loss or gain across groups, which served as indirect indicators of treatment tolerance and systemic stress.

Retinal histopathology examination
At the timepoints of the second and fourth days post-experiment, mice were euthanized by cervical dislocation after anesthesia with intraperitoneal (i.p.) injection of sodium pentobarbital at a dose of 50–60 mg/kg body weight, strictly adhering to institutional euthanasia guidelines. The eyes, specifically the left eyes, were immediately enucleated. Prior to enucleation, a small incision was made at the nasal limbus to mark the orientation. The globes were then immersed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS, pH 7.4) for 24 h at 4 °C for fixation. The fixed eyes were then dehydrated through a graded ethanol series (70%, 80%, 95%, and 100%), cleared in xylene, and embedded in paraffin blocks for histological processing. During embedding, the orientation was standardized by positioning the marked nasal side downward.

Serial sections (5 µm thick) were prepared using a rotary microtome through the optic nerve head (ONH) to ensure representative sampling of the central retinal region. Specifically, sections were cut from the superior aspect toward the marked nasal point, ensuring that all sections passed through the ONH. Sections were then mounted on positively charged glass slides. Hematoxylin and eosin (HE) staining was performed according to standard protocols. Retinal morphology was evaluated by light microscopy at 400× magnification. Measurements were systematically taken from the temporal retina at predetermined distances from the ONH: 200, 400, 600, 800, 1000, and 1200 µm. At each location, total retinal thickness, inner and outer segment (IS/OS) length, outer nuclear layer (ONL) thickness, outer plexiform layer (OPL) thickness, and inner nuclear layer (INL) were measured.

To ensure objectivity and minimize measurement bias, a rigorous quality control protocol was implemented. Specifically, each of the eight experimental groups was subdivided into five or four subgroups (e.g., Subgroup 1, 2, 3, 4, 5). Five or four independent observers, blinded to group assignments, were assigned to measure the same corresponding subgroup across all experimental groups (e.g., Observer A always measured Subgroup 1 from every group). At each measurement point, three readings were taken within a ~5 µm range (e.g., at 200 µm, 195 µm, and 205 µm) and averaged to minimize technical error. Quantitative measurements were performed using ImageJ software (version 1.54g), and the mean thickness value for each point was calculated to ensure reproducibility.

Aldehyde metabolism markers
To assess oxidative stress and aldehyde metabolism, serum samples were analyzed for MDA and 4-HNE levels, which are key biomarkers of lipid peroxidation and aldehyde stress. Specifically, all mice were anesthetized via intraperitoneal (i.p.) injection of sodium pentobarbital at a dose of 50–60 mg/kg body weight. After the mice were deeply anesthetized, euthanasia was performed by cervical dislocation. Immediately following euthanasia, the eyeballs were enucleated, and retrobulbar blood was rapidly collected. The samples were allowed to clot at room temperature for 30 min, followed by centrifugation at 3,000 × g for 10 min. The serum fraction was carefully transferred to enzyme-free microcentrifuge tubes and stored at -80 °C until subsequent analysis. Serum MDA levels were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer's protocols. In brief, standards and samples were added to precoated microplates, incubated with specific primary antibodies, washed to remove unbound materials, and reacted with horseradish peroxidase (HRP)-conjugated secondary antibodies. Tetramethylbenzidine (TMB) substrate was then added, and the reaction was terminated with sulfuric acid. Optical density (OD) values were read at 450 nm using a microplate reader, and MDA/4-HNE concentrations were determined by comparing the OD values to standard curves generated using known concentrations of the analytes.

Statistical analysis
All data were presented as mean ± standard error (SE). Given the small sample size (n = 5 for the second day group; n = 4 for the fourth day group), the Bootstrap test statistical methods were employed. Specifically, the ​​Bootstrap resampling method​​ (1,000 iterations with replacement) was used to estimate 95% confidence intervals (CI) for group means and to compare differences between groups. For each outcome (e.g., weight, retinal thickness, MDA/4-HNE levels), Bootstrap samples were drawn from the original dataset, and the mean was calculated for each resample. The 95% CI was derived from the 2.5^th and 97.5^th percentiles of the bootstrap distribution. The significance was indirectly assessed through the following approach by examining the confidence interval. If the CI of the Bootstrap results did not include zero, this typically indicated that the difference was significant (assuming a comparison of means between two or more groups). Figures were generated using the Graphpad software (version 5.01).

Results

Body weight changes
Before the experiment, there were no significant differences in body weight among the groups. On the second day after the experiment, except for the Control group, which showed an increase in body weight from 17.0 ± 0.5 g to 17.4 ± 0.4 g, the body weight of mice in all other groups significantly decreased. Specifically, the MNU group mice decreased from 17.4 ± 0.3 g to 16.2 ± 0.2 g, MNU + Alda-1 group from 17.3 ± 0.3 g to 16.7 ± 0.4 g, MNU + Daidzin group from 17.3 ± 0.3 g to 16.2...

Discussion

Previous studies suggested that ALDH2 agonists could offer protective effects against MNU-induced retinal degeneration¹⁴. However, the intervention effects of its inhibitor, along with potential downstream pathways such as SIRT1 and ERS, had not been explored in detail. There have been studies indicating the involvement of SIRT1 and ERS in retinal degeneration models, including light-induced retinal degeneration and genetically modified mice with retinal degeneration^15...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4HNE Kit	Elabscience,China	E-EL-0128	For Serum 4HNE levels measurement
4-Phenylbutyric acid (4-PBA)	MCE, US	HY-A0281	Reagent for ERS inhibition
Alda-1	MCE, US	HY-18936	ALDH2 activator for research
Daidzin	MCE, US	HY-N0018	ALDH2 inhibitor for research
Fluorescein sodium	Guangzhou	N/A	Used for  acquired using a fluorescein angiography system. The angiographic images were analyzed for the presence of any leakage, non-perfused areas, or changes in retinal vessel morphology.
ImageJ software	National Institutes of Health, USA	Version 1.53t
MDA Kit	Beyotime, China	S0131S	For Serum MDA levels measurement
N-Nitroso-N-methylurea (MNU)	MCE, US	HY-34758	DNA alkylating agent for inducing retinal degeneration
Paraformaldehyde	Sigma-Aldrich, US	30525-89-4	Used for sections of retinal tissue
Precision digital scale	Electronic scale , China	SF-400	CAPACITY: 5000 g ´ 1 g/177ozX0.10z,used for recording the body weight of each mouse
Prism	GraphPad	Version 5.01	Statistical analysis software
Rotary microtome	LEICA, Germany	RM2016	Used for Retinal sections preparation
Selisistat (EX-527)	MCE, US	HY-15452	SIRT1 inhibitor for research
Sodium Pentobarbital	Sigma-Aldrich, US	1.93248	Used for anesthetized for later researches via intraperitoneal (IP) injection
SRT1720	MCE, US	HY-10532	SIRT1 activator for research
Sumianxin	Jilin Shengda Animal Pharmaceutical Co	N/A	Used for anesthetized for later researches via intraperitoneal (IP) injection
Tunicamycin (TUN)	MCE, US	HY-A0098	Reagent for ERS induction