Epicatechin mediated modulation of the Nrf2/HO-1 pathway alleviates senile cerebral ischemic/reperfusion injury

doi:10.21203/rs.3.rs-2975278/v1

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Epicatechin mediated modulation of the Nrf2/HO-1 pathway alleviates senile cerebral ischemic/reperfusion injury

https://doi.org/10.21203/rs.3.rs-2975278/v1

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The aging of the global population has resulted in a significant increase in the incidence of stroke, disability, and mortality. Findings from numerous studies suggest that ROS generated during ischemic reperfusion are crucial in subsequent tissue damage. However, the role of nuclear factor-erythroid 2-related factor 2 (Nrf2), which regulates endogenous antioxidative functions, in pathological models of aging is not well understood. Our previous study showed that epicatechin (EC) upregulated brain Nrf2 expression in 2-month-old rats and reduced oxidative stress in cerebral ischemia-reperfusion injury (CIRI). In the present study, EC was administered to a 12-month-old rat model of CIRI (induced by middle cerebral artery occlusion and reperfusion) and a senescent SH-SY5Y cell model subjected to oxygen glucose deprivation/reoxygenation. EC treatment improved cerebral morphology and function; increased p-Nrf2 expression and unclear transportation and HO-1, SOD, and GSH expression; and reduced infarct volume and apoptosis in senescent rats. Moreover, EC enhanced cellular activity and the expression of p-Nrf2, HO-1, and NQO-1 while decreasing the ROS and MDA levels and mitigating apoptosis in senescent SH-SY5Y cells. These effects were attenuated upon si-Nrf2 introduction. The findings of this study offer novel insights into stroke prevention and therapy as well as drug development for the elderly population.

cerebral ischemia-reperfusion injury

oxidative stress

aging

p-Nrf2

antioxidants

Cerebral ischemia-reperfusion injury (CIRI) typically occurs following the restoration of blood flow during stroke treatment. Among various physiological mechanisms, oxidative stress (OS) plays a pivotal role in the pathophysiology of CIRI, as excessive reactive oxygen species (ROS) generation can lead to severe brain damage.

Age is one of the most significant factors that influences stroke incidence [1], resulting in higher mortality rates, a poorer quality of life, and increased financial costs for survivorship care in individuals aged over 65 years [2]. However, despite the potential benefits of antioxidants reported in clinical applications, few benefits have been demonstrated to be effective in treatments for CIRI, particularly in the elderly. Research shows that oxidative stress is higher in older organisms, which leads to weaker ischemia-reperfusion resistance, more severe brain damage, and challenges in the restoration of neurological function [3, 4]. Therefore, aging-specific studies are necessary to understand recessionary endogenic antioxidative functions and explore promising medicines using aging models. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that is sensitive to the redox potential. Nrf2 induces the generation of several antioxidative proteins and antioxidants, which play an important role in combating OS[5, 6]. However, Nrf2 gene and protein expression in the brain declines with age [7]. When CIRI occurs, the combination of pathological ROS production and aging Nrf2 function may result in higher ROS levels in the elderly than in younger individuals. Therefore, identifying natural drugs that modulate the function of the Nrf2 antioxidant pathway and increase the resistance of aging nerves to OS damage is an urgent need.

In our previous study, we demonstrated that epicatechin (EC) can enhance the activity of antioxidant enzymes and upregulate Nrf2 expression, thus ameliorating neuronal damage caused by middle cerebral artery occlusion (MCAO) at 2 months of age8. To add to our findings, we investigated the potential of EC to upregulate Nrf2 expression for alleviating CIRI in aging rats subjected to transient focal cerebral ischemia/reperfusion (MCAO/R) and senescent SH-SY5Y cells subjected to oxygen glucose deprivation/reoxygenation (OGD/R) in vitro.

Main chemical and reagents

Epicatechin (IE0120, Solarbio, Beijing), Nissl stain solution (Solarbio, Beijing); a malondialdehyde (MDA) assay kit; a glutathione (GSH) assay kit (Beyotime Biotechnology, Beijing); dimethyl sulfoxide (DMSO); BCA kits (Beyotime Biotechnology), rabbit antibodies against anti- heme oxygenase-1 (HO-1, Abcam, USA, ab68477), NQO-1 (ab80588, Abcam), p-Nrf2 (PA5-67520, Thermo Fisher, USA), GAPDH (1:1000, Cell Signaling Technologies, USA, #5174), and β-tubulin (1:1000, Cell Signaling Technologies); a Micro Malondialdehyde (MDA) Assay Kit (BC0025, Solarbio, Beijing); a Micro Reduced Glutathione (GSH) Assay Kit(BC1175, Solarbio, Beijing); an LDH Assay Kit (A039-2, Nanjing Jiancheng, China); a Reactive Oxygen Species Assay Kit (S0033, Beyotime Biotechnology); fetal bovine serum (FBS) (GIBCO 10100147, USA); and deferoxamin mesylate (DFO, APExBIO, Cat.No:B6068, USA) were also purchased.

Experimental Animals and Middle Cerebral Ischemia Reperfusion Modeling

Twelve-month-old (n = 108) and 2-month-old (n = 36) male Sprague-Dawley rats were purchased from the Animal Center of Guangxi Medical University (Nanning, China). All animal procedures were approved by the Animal Ethics Committee of Guangxi Medical University. After acclimatizing the SD rats by feeding them normal rat chow and water (provided ad libitum) under a 12:12 h light-dark cycle for 1 week, the classic middle cerebral artery wire embolization (MCAO) method was used to establish a focal cerebral ischemia-reperfusion model. In the Sham group, the ligation and insertion of pins was not performed. The rats were then randomly divided into the following five groups: model group (MCAO group); positive control group (edaravone group), and three EC-treated groups. Following this, 0.9% saline (NS) and different doses of EC (5, 10, and 20 mg/kg) and edaravone (3 mg/kg) were administered via the femoral vein immediately after resuscitation. Thirty-six hours after resuscitation, the experimental rats were killed by the intraperitoneal injection of excess sodium pentobarbital, and their brain tissues were harvested.

Neurological and morphological evaluation

The Longa-5-Point scale is a frequently-used method for neurological and morphological evaluation. The specific scoring system is as follows: 0 points: no symptom of neurological impairment; 1 point: failure to fully extend the contralateral front paw; 2 points: turn to the opposite side; 3 points: dump to the opposite side; 4 points: inability to walk spontaneously and loss of consciousness. The damage to nerve function is more severe when the scores are high. The volume of cerebral infarction in rats is commonly measured by TTC staining. At 36 h after ischemia-reperfusion, the rat brain tissues were taken and frozen in the refrigerator at -20 ℃ for 15 min. After the removal of parts that were not required, such as the olfactory bulb and brain stem, the brain slices were cut into 2 mm thick brain slices along the coronal plane. Following this, these slices were incubated in 2% TTC solution at 37 ℃ for 15 min and turned once every 5 min. Subsequently, the slices were taken out and immersed in a 10% formalin solution overnight. Finally, the slices were imaged, and the cerebral infarction area was computed using the Image J software (v1.4.3.67, USA). The percentage of area with cerebral infarction on the ischemic part was calculated using the formula (infarct area) / (cerebral area on the ischemic part) × 100%.

Histological Examination and Immunohistochemical Staining

Rats were anesthetized using excess sodium pentobarbital administered via intraperitoneal injection and then transcardially perfused with 0.9% saline (NS) followed by 4% neutralized formalin for in situ perfusion fixation. Brains samples were collected and post-fixed overnight in 10% neutralized formalin. Following this, the tissues were dehydrated routinely, embedded in paraffin, and sectioned into slices of different thicknesses based on experimental requirements.

The slices were stained according to the instructions for HE staining and were then imaged under the microscope. Paraffin-embedded brain sections with a thickness of 5 µm were immersed in 0.05% cresyl violet stain solution at 56°C for approximately 1 h after dewaxing. After rinsing softly with distilled water, the sections were immersed in a Nissl differentiation solution until the desired color was achieved. This was followed by the placement of a cover slip and examination with a light microscope. Five brain sections were randomly selected from each sample, and the data are expressed as the number of Nissl bodies/field.

The apoptosis of neurons in the ischemic part of the brain was observed by TUNEL staining. The experimental procedures were primarily based on the instructions provided in the TUNEL detection kits (Beyotime biotechnology). Following this, the slices were observed under a pathological microscope. Five different visual fields were randomly selected at 40 magnification, and the apoptosis index was calculated according to the formula: apoptosis index = number of apoptotic neurons / total number of neurons × 100%.

Assessment of MDA, GSH, SOD and LDH

The activities of MDA, GSH, SOD, and LDH in the brain and SH-SY5Y cells were measured using the assay kits in accordance with the manufacturers’ instructions.

ROS measurement

A fluorescent probe 2’7’-dichlorofluorescein diacetate (DCFH-DA, USA) was procured to monitor intracellular ROS production. SH-SY5Y cells with the indicated treatments were incubated with 20 µM DCFH-DA in the dark for 30 min at 37°C. After washing with PBS, the fluorescence level was measured by flow cytometry. The fluorescence intensity was expressed as a relative value compared with that of the control.

Immunofluorescence and Western Blot detection

Immunofluorescence staining was used to assess protein localization and expression on the tissue slides. The experimental process primarily involved the following. First, antigen repair was performed in the paraffin slices after dewaxing and dehydration. Second, the slices were treated overnight with primary antibodies (anti-HO-1 or anti-Nrf2) at 4°C, followed by treatment with secondary antibodies at 25°C. Third, DAPI was used to counterstain. Lastly, the slices were observed under a fluorescence microscope and images were acquired.

The total protein from ischemic cerebral cortex tissues was extracted using respective kits (Solarbio), and their concentration was measured using a BCA assay kit. Following this, the proteins were segregated using SDS‑PAGE and transferred to polyvinylidene difluoride membranes (PVDF). After sealing with skim milk, the samples were treated with primary antibodies, including anti-HO-1(1:1500), anti-p-Nrf2 (1:1000), and anti-β-tubulin (1:1000) antibodies. Following this, the samples were re-treated with secondary antibodies. The signals from the expressed proteins were measured using an ECL kit. The relative expression level of proteins is equal to the ratio of target protein expression to the expression of the internal reference β-tubulin or GAPDH.

cell culture and treatment

To simulate CIRI conditions in vitro, the OGD of senescent SH-SY5Y cells was performed as described previously [8]. Briefly, SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) at 37 ℃ in a humidified atmosphere with 5% CO2 and 95% O2. First, SH-SY5Y cells were treated with 100 mM D-gal for 48 h to induce senescence. Once the cells achieved 70%~80% confluence, they were subjected to oxygen-glucose deprivation (OGD) for 1 h and then placed in a normal incubator. The glucose-free medium was removed. The cell medium in the OGD/R model group was replaced with fresh complete medium, whereas that in the drug administration group was replaced with medium supplemented with epicatechin at different concentrations (50, 100, and 200 µM) or edaravone (50 µM). After 24 h of incubation, the cells were collected for subsequent experiments.

Nrf2 siRNA Transfection

Senescent SH-SY5Y cells were inoculated into 6-well plates (2 × 105/well). After the cells adhered to the plate, they were washed three times with serum-free and antibody-free Opti MEM cell culture medium, and Opti MEM culture medium was added to each well. siRNA was prepared to a final concentration of 80 nM and diluted with Lipofectamine 2000. The contents were mixed well and left to stand for 10 min. The transfection complex was added to the cells and the cells were incubated for 6 h. The transfected cells were treated with DMEM, EC, or Edaravone for 24 h and then with H2O2 (200 µM) for 24 h. Following this, cell survival was assessed.

Flow cytometric detection of cell apoptosis

Cell apoptosis was assessed using an Annexin V-fluorescein isothiocyanate (FITC)/PI kit (BD Biosciences, San Jose, CA, USA). After the indicated treatments, senescent SH-SY5Y cells were harvested, washed twice with ice-cold PBS, and then suspended in 300 µL of binding buffer. Five microliters of Annexin V-FITC was added to the cells, and the cells were incubated for 15 min in the dark at room temperature and then with 5 µL of PI in the dark for 30 min. The stained cells were immediately analyzed using flow cytometry. The apoptotic cell percentage was calculated.

Statistical analysis

Prism 8.0 software was used to perform experimental data analysis. homogeneity of variance test, One-way analysis of variance (ANOVA) followed by the Kruskal-Wallis test was used to analyze the difference among groups. Values with P < 0.05 were considered statistically significant.

Changes in the oxidative damage status in young and aging rats after I/R

The neurological deficit scores in each group were determined after 36 h of developing the models. No neurological deficits were observed in rats from the Sham group at 2 and 12 months of age, and rats from both MCAO/R groups showed varying degrees of elevated scores. In MCAO/R, the damage increased in the 12-month group compared with that in the 2-month group (Fig. 1A). According to the TTC staining results, no infarct focus was observed in the Sham groups, whereas the middle cerebral artery embolization (MCAO/R) groups showed infarcts with a large area. The white infarct volume in the 12-month MCAO/R group was greater than the infarct volume in the 2-month-old group. HE staining showed morphological changes in rats after CIRI. As shown in Fig. 1B, the rats in the Sham group had abundant cells that were closely arranged, round, and clear, with the nucleoli positioned centrally. While the MCAO/R groups showed a reduction in the number of cells, the cells were arranged in a dispersed manner, their morphology was irregular, and some cells showed nuclear pyknosis and pathological damage. Compared with the 12-month MCAO/R groups, the 2-month groups showed elevated cells with regular morphology. Neurological function scores are used to assess the dysfunction in neurons after MCAO/R to an extent. As shown in Fig. 1C, the MCAO/R (12-month) groups had higher scores than the MCAO/R (2-month) groups. This implies that neurological functions were more severely affected. Besides, TUNEL staining is used to observe apoptosis after CIRI. After TUNEL staining, the nuclei of normal cells were blue, and those of apoptotic cells were brown. The results showed that apoptotic nuclei were barely present in the Sham group, whereas the number of apoptotic cells increased markedly in the MCAO/R group (Fig. 1D). Compared with that in the MCAO/R groups, the apoptosis rate was greater in the 12-month group than in the 2-month group. Generally, Nissl bodies are stored in surviving neurons. As expected, the Sham group had the maximum number of Nissl bodies, and the MCAO /R group had the least (Fig. 1E). Interestingly, the number of Nissl bodies in the 12-month group was lesser than the number of Nissl bodies in the 2-month group. As expected, we also measured the MDA (Fig. 1F) and LDH levels (Fig. 1G). Compared with rats in the Sham group, rats both significate increases. However, the MDA and LDH content was significantly higher in the infarction penumbra in the 12-month group than in the 2-month group. The above findings suggested that age attenuated oxidative damage and MCAO/R-induced neuronal death.

Differences in antioxidant capacity between young and aging rats after I/R

First, we investigated whether the expression level of p-Nrf2 reflects the level of Nrf2 phosphorylation and antioxidant capacity. We measured the changes in the p-Nrf2 levels in each group using immunofluorescence. After MCAO/R, p-Nrf2 expression was significantly downregulated in the 12-month group compared with that in the 2-month group (Fig. 2A). The same result was observed in the western blotting experiment (Fig. 2C). HO-1 expression is an important antioxidant index. HO-1 levels decreased to a greater extent in the 12-month group than in the 2-month group after MCAO/R (Fig. 2B). To better understand the changes in the Nrf2 pathway in 2-month and 12-month rats, we performed western blotting using brain tissues. The same result was verified, as shown in Fig. 2C. These results were consistent with findings from previous studies, suggesting that the Nrf2 pathway is a marker for aging. As evidence from multiple independent studies often shows the association between GSH expression and antioxidative index, we next focused on GSH expression. As shown, the GSH levels decreased after MCO/R, with a greater reduction observed in the 12-month group than in the 2-month model group (Fig. 2D). SOD serves as a marker for antioxidative potential in the brain. We thus checked the changes in the expression of SOD. The SOD content was greater in the 2-month group than in the 12-month model group (Fig. 2E). These data suggested that Nrf2 expression is age-associated, and along with antioxidant proteins, Nrf2 might play a functional role in physiological CIRI processes with aging.

Figure 2 Differences in antioxidant capacity between young and aging rats after I/R.(A) immunofluorescence staining of p-Nrf2.(B) immunofluorescence staining of HO-1.(C) WB analysis of p-Nrf2, HO-1.(D-E) GSH content(D) and SOD Activity.(E) of the cerebral cortex at 36 h after I/R. All data above are represented as the mean ± SEM and analyzed by the Kruskal-Wallis test. n = 3 per group. ** p < 0.01 ,*** p < 0.005vs. the Sham group. # p < 0.05 vs. the MCAO/R group.

Epicatechin reduces oxidative damage in cerebral ischemia-reperfusion

MCAO/R leads to neuronal injury, thus aggravating brain damage and cerebral infarction. To assess the cerebral protective effect of epicatechins, we evaluated neuronal damage using TTC staining. We observed no infarction in the Sham group. MCAO/R significantly increased the infarct volume ratio in the model group, and the Epicatechin treatment group significantly decreased the infarct volume while increasing the EC dose (Fig. 3A). This indicated serious neurological deficits in the MCAO/R model. HE staining showed that the number of neurons reduced considerably after MCAO/R, whereas it increased in response to epicatechin treatment (Fig. 3B). Neurological function scores were assessed at 36 h after MCAO/R. Rats with MCAO/R showed the highest score among all groups. Epicatechin treatment significantly reduced the neurological scores compared with those in the MCAO/R group and ameliorated the mental status of rats subjected to MCAO/R (Fig. 3C). Nissl staining is used to assess dysfunction in neuronal protein synthesis. As shown in Fig. 3D, compared with that in the Sham group, a significant loss of neurons, faint staining of nuclei, and swelling and vacuolar changes were observed in the MCAO/R groups. The number of Nissl bodies increased upon treatment with epicatechin and showed deeper staining as a whole, especially in the EC 20 mg/kg group. In the brain tissues, CIRI induced apoptosis. Cells with a brown stain are apoptotic cells in TUNEL staining. The number of apoptotic cells decreased significantly with an increasing dose in the EC group compared with that in the MCAO/R group (Fig. 3E). Based on the data described above, we further confirmed the damage at the biochemical level. The MDA and LDH content in brain tissues was measured. As shown in Fig. 3F, MDA expression increased significantly in rats subjected to MCAO/R, and epicatechin treatment reduced the LDH and MDA expression in a dose-dependent manner compared with that in the MCAO/R group (P < 0.05). The results suggested that epicatechin treatment could protect the brain from MCAO/R-induced oxidative damage.

Epicatechin enhances the antioxidant potential of aging rats

To test the mechanism of action of epicatechin, we assessed the expression/activation of the Nrf2 pathway in vivo. Nrf2 signaling is the key endogenous antioxidant signaling cascade. Once activated, Nrf2 will separate from Keap1, which will lead to Nrf2 phosphorylation. To determine how epicatechin exerts a neuroprotective effect, we studied the antioxidant protein p-Nrf2. The level of p-Nrf2 is indicated in Fig. 4A by the overlapping red and blue fluorescence. The greater the overlapping region, the higher the p-Nrf2 infiltration into the nucleus. We observed that the p-Nrf2 level in the cytoplasm increased after MCAO/R compared with that in the Sham group, and the fluorescence intensity of p-Nrf2 in the nucleus was significantly enhanced after epicatechin treatment vs. that in the MCAO/R group. We verified this result simultaneously using western blotting (Fig. 4C). HO-1 plays an important role in the antioxidant defense system. Green fluorescence increased with the EC dose (Fig. 4B). The same result was verified in Fig. 4C. The improvement of the antioxidant capacity of the organism by epicatechin was also investigated, and the levels of GSH and SOD in fresh brain samples were measured using corresponding kits. The GSH content reduced significantly after MCAO/R. Epicatechin boosted GSH content, especially in the 20 mg/kg group (Fig. 4D). The SOD activity decreased after MCAO/R, whereas epicatechin treatment increased its activity (Fig. 4E). These results suggest that Epicatechin treatment can obviously suppress oxidative stress induced by MCAO/R and facilitate the activation of the Nrf2/HO-1 signaling pathway in vivo.

Epicatechin protects senescent SH-SY5Y cells from OGD/R-induced cytotoxicity and oxidative stress

In the previous part of the experiment, we observed that epicatechin reduced oxidative stress in vivo. Thus, we speculated whether epicatechin could promote an anti-oxidative effect in vitro. The age-dependent accumulation of senescent cells in the nervous system accelerates apoptosis. In addition, previous studies have shown the induction of apoptosis by OGD/R. We measured the apoptosis-related parameters to determine whether preconditioning with epicatechin can prevent apoptosis induced by OGD/R. Apoptosis in rats was detected using flow cytometry (Fig. 5A). To validate these observations, we also evaluated the LDH content using a kit (Fig. 5B). To evaluate lipid peroxidation expression, we tested their oxidation products MDA to determine their ability to induce preconditioning against OGD/R (Fig. 5C). Cells subjected to OGD/R showed an increase in MDA levels. Cell viability was assessed using a CCK8 assay kit. EC treatment significantly restored the cell viability, which was suppressed by OGD/R, in senescent SH-SY5Y cells (Fig. 5D). As shown in Fig. 5E and 5F, the levels of SOD and GSH in the OGD/R group were significantly increased compared with those in the control group. In contrast, epicatechin reduced the levels of LDH and MDA in a concentration-dependent manner. These results indicate that Epicatechin can protect senescent SH-SY5Y cells from OGD/R-induced cytotoxicity and oxidative stress.

si-Nrf2 reversed the effect of epicatechin on OGD/R-induced oxidative injury in senescent SH-SY5Y cells

Next, we explored whether Nrf2 was involved in OGD/R-induced oxidative stress. OGD-R stimulation in neuronal cells induced profound ROS generation and oxidative stress, causing cell death and apoptosis. The findings of the flow cytometry assay demonstrated that OGD/R induced more ROS generation than that in the control group. However, epicatechin substantially decreased OGD/R-triggered ROS generation. In contrast, we did not observe significant changes in the si-Nrf2 + OGD/R group compared with that in the si-Nrf2 + OGD/R + EC group (Fig. 6A). To further explore the Nrf2-mediated targeting of damaged neurons in OGD/R, the expression of NQO-1 and HO-1 in senescent SH-SY5Y cells and ODG-treated senescent SH-SY5Y cells was investigated by western blotting (Fig. 6B), as these are well-known downstream targets of Nrf2. The NQO-1 and HO-1 levels significantly increased in response to OGD/R exposure and were further increased upon epicatechin treatment. Next, we executed Nrf2 knockdown using siRNA-Nrf2 to verify whether epicatechin could downregulate the expression of HO-1 and NQO-1. As shown in Fig. 6B, an increase in the levels of NQO-1 indicated that epicatechin failed to protect against OGD/R-induced cell death as compared with that in cells transfected with control siRNA (p > 0.05). Collectively, the findings from the two experimental approaches support a direct correlation between the expression of NQO-1 and HO-1 and the preconditioning against OGD/R-induced cell death.

To explore whether Nrf2 was involved in OGD/R-induced oxidative stress, the concentration of SOD was measured in senescent SH-SY5Y cells, because SOD generation increased and subsequent oxidative activation occurred in epicatechin-treated cells. Next, we determined whether SOD played a role in the results observed in the si-Nrf2 group. Epicatechin has been shown to exert neuroprotective and neuro-restorative effects. However, these findings were not observed in the si-Nrf2 group (Fig. 6C). This suggests that epicatechin enhances antioxidant capacity via Nrf2 in OGD/R. The concentration of GSH was assessed in senescent SH-SY5Y cells. The GSH levels were lower in ODG-treated SH-SY5Y cells but increased in response to epicatechin treatment (Fig. 6D). This indicated that epicatechin effectively promoted the antioxidant potential in OGD-treated cells. However, these alterations were reversed upon si-Nrf2 introduction. These findings support the fact that epicatechin has neuroprotective effects that are potentially mediated by Nrf2. Conversely, EC + Nrf2 group were not lesser in preconditioning SH-SY5Y cells (Fig. 6C,D). These results revealed that the abatement of OGD/R-induced oxidative injury in SH-SY5Y cells by melatonin partly relies on the activation of the Nrf2 pathway.

The findings of this study demonstrate that epicatechin can alleviate senile CIRI by improving the anti-oxidative function of the Nrf2 system.

First, we compared CIRI in 2-month-old and 12-month-old SD rats and found that aging attenuated Nrf2 function while increasing OS and increasing the severity of CIRI. Results showed that p-Nrf2 and HO-1 expression was lower in the 12-month-old Sham group than in the 2-month-old Sham group, indicating a decline in Nrf2 function with age. The increased expression of p-Nrf2 and HO-1 in the 2-month-old MCAO/R group, when compared with that in the Sham group, may indicate a negative feedback response to CIRI triggered by high levels of ROS. In contrast, rats in the 12-month-old MCAO/R group exhibited lower SOD activity and GSH content than those in the 2-month-old MCAO/R group, which resulted in increased MDA content and heightened cellular damage, as indicated by elevated levels of LDH and apoptosis. Consequently, rats from this group exhibited more severe structural and neurological damage in the brain. These findings align with previous reports of higher stroke mortality rates and poorer functional outcomes in the elderly. Aging animals exhibit earlier disruption of the blood-brain barrier, larger infarct volumes, greater neuronal degeneration, and poorer neurological outcomes in ischemic stroke relative to younger individuals [3, 7, 9].

In the second part of our study, we investigated the anti-CIRI effects of epicatechin in 12-month-old rats. Epicatechin is a plant polyphenol abundant in the human diet [10]. It has two aromatic rings and an oxygenated heterocycle [11], along with multiple phenolic hydroxyl groups that can scavenge ROS both directly and indirectly by modulating pathways that regulate the expression of ROS-scavenging compounds and enzymes [12]. The presence of hydroxyl groups and double bonds allows epicatechin to interact with proteins and lipids that form structural membrane lipids, which are associated with the redox state of cells and the activation of redox signaling pathways[13]. These unique pharmacological properties of epicatechin distinguish it from other flavanols. Additionally, pharmacokinetic studies have shown that epicatechin can penetrate the blood-brain barrier when administered intravenously in rats [14]. Epicatechin has been shown to exhibit neuroprotective effects as well [15, 16]. To investigate its effects on oxidative homeostasis, we evaluated the GSH and MDA contents and SOD activity in brain tissues. A gradual increase in SOD activity and GSH content were observed in the epicatechin group as the epicatechin concentration increased and the MDA content decreased. GSH can scavenge free radicals by binding to oxygen radicals via non-enzymatic reactions, thus protecting cell membranes from peroxide and free radical damage and reducing LDH levels[17, 18]. These results suggest that epicatechin plays a role in reversing the pathological redox imbalance in the brains of aged animals.

The restoration of GSH content and increase in SOD activity were found to be associated with improved Nrf2 function in response to epicatechin treatment. In a reperfusion experiment conducted 60 min after MCAO in mice, Nrf2 knockout mice demonstrated more pronounced neurological deficits and significantly larger infarct areas than wild-type mice[19]. Conversely, Nrf2 upregulation resulted in a reduction in brain injury. Lan et al. established an MCAO model in rats and observed increased Nrf2 expression, improved neurological function, and reduced infarct size[20]. The experimental findings of the present study are consistent with the findings reported in the studies cited above[21, 22].

To investigate the effect of epicatechin in vitro, an oxidative test was conducted on senescent SH-SY5Y cells treated with varying doses of epicatechin. The aim was to explore the function of Epicatechin in the OGD/R model. The results indicate that EC improves the endogenous antioxidant capacity of senescent cells by upregulating GSH levels and SOD activity, reducing MDA production, and decreasing apoptosis. As a result, epicatechin reduces OGD/R injury. Fernando observed that epicatechin reduces ROS-induced apoptosis in MCF-7 cells[23]. Moreover, epicatechin can reduce cellular OGD/R-induced apoptosis in neuronal cells by enhancing mitochondrial function [24]. The results of the present study are consistent with the findings reported in the studies cited above.

To investigate whether epicatechin enhances endogenous antioxidant function by regulating Nrf2 expression, we conducted an Nrf2 silencing experiment and observed changes in HO-1 and NQO-1 expression. The results showed that in the non-silencing Nrf2 group, OGD/R stimulated the expression of HO-1 and NQO-1, and epicatechin further increased their expression. In contrast, Nrf2 silencing reversed the effect of epicatechin on HO-1 and NQO-1 expression. Our results suggest that epicatechin promotes the activation of phosphorylated Nrf2, which further regulates downstream HO-1 and NQO-1 expression in response to OGD/R-induced oxidative stress after Nrf2 silencing. Consistent with our findings, Zhang et al. reported that Nrf2 knockout mice exhibited lower HO-1 and NQO-1 expression [25] .

In addition to HO-1 and NQO-1, SOD and GSH are downstream antioxidants regulated by Nrf2, which induces the expression of SOD and GSH synthases [26]. GSH biosynthesis is tightly controlled by the rate-limiting enzyme glutamate-cysteine, which is transcriptionally regulated by Nrf2 [27]. In this study, we found that SOD and GSH expression was lower in the si-Nrf2 group than in the non-si-Nrf2 group, and both were further reduced in response to OGD/R treatment. However, epicatechin significantly upregulated the expression of SOD and GSH in the non-si-Nrf2 group, thereby preventing OGD/R-induced MDA and ROS production, whereas this effect was not observed in the si-Nrf2 group. The result suggests that EC mitigated oxidative stress-induced ROS accumulation and associated oxidative damage. Additionally, evidence from recent studies demonstrates the relevance of Nrf2 in SOD and GSH expression. Nrf2 expression is impaired with age, which causes organisms to enter a chronic oxidative state[28]. The susceptibility of mice to oxidative damage increases when Nrf2 is knocked out[29]; Nrf2 upregulates SOD and GSH by binding to antioxidant response elements. si-Nrf2 suppresses the antioxidant function of Nrf2[30]; The expression of antioxidant enzymes decreases in the skeletal muscle of Nrf2 knockout mice. Additionally, the GSH levels were reduced in Nrf2-deficient (Nrf2-/-) liver tissues and fibroblasts [31]. Our findings showed that Nrf2 silencing decreased the antioxidant potential and increased apoptosis and ROS production, possibly owing to the impairment of the Nrf2/HO-1 pathway. Mechanistically, epicatechin has been implicated in the regulation of Nrf2 phosphorylation via p-Nrf2 expression and nuclear transportation, which in turn elevates HO-1 and NQO-1 protein levels and reduces ROS accumulation.

Our study design had certain limitations, as we only used a 12-month-old SD rat model of focal cerebral ischemia/reperfusion injury in the animal experiments. In future investigations, we intend to include observations of the cerebral protective effects of epicatechin in rats at 18 and 24 months of age.

In summary, in this study, we investigated the neuroprotective effects and underlying mechanisms of action of epicatechin in a 12-month-old MCAO rat model and an OGD/R model of senescent cells. Our findings shed light on the mechanism of action of epicatechin in reducing oxidative stress-induced damage through the upregulation of the Nrf2/ARE system, indicating that epicatechin has potential as a therapeutic target for age-related stroke.

Epicatechin has been shown to improve the functional decline of Nrf2 associated with aging, thereby mitigating oxidative stress and apoptosis during cerebral ischemic reperfusion. Our results suggest that this plant-derived compound may have therapeutic potential in senile cerebral ischemia/reperfusion injury.

Data Availability

Data will be available upon request from the corresponding author.

Conflicts of Interest

The author declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Authors Contributions

All authors have made substantial contributions to the article. Chang-yue Jiang and Xiang-zhen Zhuge are responsible for the analysis and interpretation of data, drafting the article；De-li Li and Meng-Hua Chen for the acquisition of data；Lu Xie and Wan-xiang Hu for the conception and design of the study and final approval of the version to be submitted. Chang-yue Jiang and Xiang-zhen Zhuge contributed equally to this work.There are no conflicts of interest to declare.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 82160372,81860333),Natural Science Foundation of Guangxi (No.2018GXNSFAA050153) and Youth research foundation of Guangxi Medical University（GXMUYSF202127）.

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Epicatechin mediated modulation of the Nrf2/HO-1 pathway alleviates senile cerebral ischemic/reperfusion injury

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Abstract

Figures

Introduction

Material and Methods

Experimental Animals and Middle Cerebral Ischemia Reperfusion Modeling

Neurological and morphological evaluation

Histological Examination and Immunohistochemical Staining

Assessment of MDA, GSH, SOD and LDH

ROS measurement

Immunofluorescence and Western Blot detection

cell culture and treatment

Nrf2 siRNA Transfection

Flow cytometric detection of cell apoptosis

Statistical analysis

Results

Changes in the oxidative damage status in young and aging rats after I/R

Differences in antioxidant capacity between young and aging rats after I/R

Epicatechin reduces oxidative damage in cerebral ischemia-reperfusion

Epicatechin enhances the antioxidant potential of aging rats

Epicatechin protects senescent SH-SY5Y cells from OGD/R-induced cytotoxicity and oxidative stress

si-Nrf2 reversed the effect of epicatechin on OGD/R-induced oxidative injury in senescent SH-SY5Y cells

Disscussion

Conclusions

Declarations

References

Additional Declarations

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