The Effects of Caffeic Acid Phenethyl Ester on Retina in a Diabetic Rat Model

Abstract

Purpose: We aimed to investigate the effect of caffeic acid phenethyl ester (CAPE) on retinal apoptosis and oxidative stress parameters in a streptozotocin (STZ)-induced diabetic rat model.

Methods: This study included three groups: control, STZ, and STZ+CAPE. The rats in the STZ and STZ+CAPE groups were injected with STZ (35 mg/kg, i.p.) to induce diabetes. In the STZ+CAPE group, 10 mmol/kg of CAPE was administered intraperitoneally for four weeks. Control and STZ groups were given only intraperitoneal vehicle (saline). Rats were anesthetized and sacrificed at the fourth week of the experiment. Total antioxidant status (TAS) and total oxidant status (TOS) were measured in the dissected retinal tissues. Oxidative stress index (OSI) was also calculated. Fellow eyes were used for histopathologic evaluation with caspase-3, matrix metalloproteinase-2 (MMP-2), and MMP-9 assessment.

Results: TAS levels were similar between groups. However, CAPE treatment prevented the elevation of TOS in the STZ+CAPE group compared to the STZ group. OSI was also significantly lower in the STZ+CAPE group than in the STZ group. Retinal caspase-3 staining, MMP-2, and MMP-9 scores were not different between groups.

Conclusion: The present study demonstrated that CAPE treatment may decrease oxidative stress in the retina in STZ-induced diabetic rats. However, apoptosis was not observed in the retina, likely due to the short duration of diabetes.

Introduction

Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia due to defects in insulin secretion and/or action. Hyperglycemia causes dysfunction of organs such as the brain, heart, kidneys, and eyes. One of the major complications of diabetes is diabetic retinopathy, a microvascular disorder that can cause vision loss. While treatment options exist, preventing the retina from the damaging effects of hyperglycemia is of greater importance.

Phytotherapeutic agents have long been used for various conditions, including diabetes. Propolis is one such agent historically used in folk medicine. Caffeic acid phenethyl ester (CAPE), a flavonoid-like compound, is an active component of propolis. CAPE has been extensively studied and is known to have immunomodulatory, anti-inflammatory, antioxidant, antidotal, antiangiogenic, anticancer, and antiviral properties.

CAPE’s protective effects have been shown in diabetic animal models. It reduces hepatic glucose output, improves insulin resistance, and decreases pro-inflammatory cytokines. Previous studies have also shown that CAPE reduces nitrosative and oxidative stress in diabetic rat retina. In this study, we aimed to assess the effect of CAPE on retinal oxidative stress and apoptosis in STZ-induced diabetic rats.

Materials and Methods

Chemicals

Streptozotocin was obtained from Sigma-Aldrich and diluted in citrate-buffered saline (pH 4.5) at a concentration of 35 mg/kg. CAPE was also purchased from Sigma-Aldrich, dissolved in dimethylsulfoxide, and diluted in saline to a final concentration of 10 mmol/kg.

Animals and Treatment

This study was approved by the Institutional Animal Ethical Committee. Four-month-old male Sprague Dawley rats weighing 250 ± 50 g were used. The rats were housed under standard laboratory conditions and fed with standard rat chow and water ad libitum.

Rats were randomly assigned to three groups: non-diabetic control (Control), diabetic control (STZ), and diabetic rats receiving CAPE (STZ+CAPE). Diabetes was induced in STZ and STZ+CAPE groups via a single intraperitoneal injection of STZ (35 mg/kg). Blood glucose levels were measured 48 hours post-injection, and rats with glucose levels over 250 mg/dL were considered diabetic.

CAPE was administered at 10 μmol/kg/day intraperitoneally for four weeks in the STZ+CAPE group. The control and STZ groups received only saline. At the end of four weeks, all rats were anesthetized and sacrificed. One eye from each rat was processed for histopathological evaluation, and the fellow eye was dissected for biochemical analysis.

Measurements of Biochemical Parameters

Retinal tissues were dissected, weighed, and stored at -80°C. Homogenates were prepared and centrifuged. TAS and TOS levels were measured using colorimetric methods. OSI was calculated as the ratio of TOS to TAS.

Preparation of Histopathological Samples

Eyes were fixed in formalin for two days and embedded in paraffin. Four-micrometer thick sections were cut, deparaffinized, rehydrated, and prepared for immunohistochemical and immunofluorescent staining.

Rat MMP-2 and MMP-9 Immunofluorescent Staining and Imaging

Sections were washed, blocked, and treated to prevent nonspecific binding and autofluorescence. Labelled antibodies against MMP-2 and MMP-9 were applied. Fluorescent imaging was performed using appropriate filters.

Rat Active Caspase-3 Immunohistochemical Staining

Sections were blocked, treated for endogenous peroxidase activity, and incubated with antibodies against active caspase-3. HRP-conjugated secondary antibodies were used, followed by DAB substrate for visualization. Sections were counterstained with hematoxylin and evaluated under light microscopy.

Statistical Analyses

Statistical analyses were conducted using SPSS 11.5. Data were expressed as mean ± standard deviation. The Kruskal–Wallis test was used for overall group comparisons, and the Mann–Whitney U test was used for post hoc analysis. A p-value < 0.05 was considered significant.

Results

The Retinal TAS and TOS Levels

TAS levels did not differ significantly among groups. However, TOS levels were significantly lower in the STZ+CAPE group compared to the STZ group. OSI was also significantly reduced in the STZ+CAPE group compared to STZ, indicating reduced oxidative stress with CAPE treatment.

The MMP-2 and MMP-9 Levels of the Retina

There were no statistically significant differences in MMP-2 and MMP-9 levels between the groups.

Retina Caspase-3 Immunohistochemistry

Caspase-3 staining was comparable across all groups. No significant differences were observed.

Discussion

This study found that retinal oxidative stress is increased in STZ-induced diabetic rats and that CAPE treatment effectively reduced this oxidative stress. However, MMP-2, MMP-9, and caspase-3 levels did not differ significantly, possibly due to the short duration of diabetes in this model.

Hyperglycemia leads to the production of free radicals and oxidative damage. Antioxidant therapy has been suggested to help mitigate diabetic complications. CAPE is known to reduce oxidative stress and inflammation in various tissues. In our study, CAPE decreased TOS and OSI without affecting TAS levels, consistent with previous reports.

Matrix metalloproteinases are markers of inflammation and tissue damage. Though MMP-2 and MMP-9 levels are typically elevated in diabetic tissues, their levels were not increased in our short-term diabetic model. Caspase-3, an apoptosis marker, also showed no increase, which may reflect the need for a longer diabetic duration to detect such changes.

Given the duration of our diabetes model corresponds to approximately three years in humans, it is plausible that retinal apoptosis had not yet occurred. This mirrors clinical observations where retinopathy does not always appear in early diabetes.

Conclusion

CAPE treatment may mitigate oxidative stress in retinal tissues of diabetic rats. The absence of increased apoptosis and MMP levels in our study suggests a need for longer-duration models to evaluate chronic effects. Further research is required to validate CAPE’s protective role in diabetic retinopathy over extended periods.