Edaravone dexborneol attenuates cognitive impairment in a rat model of vascular dementia by inhibiting hippocampal oxidative stress and inflammatory responses and modulating the NMDA receptor signaling pathway

Exploring the intricate pathogenesis of Vascular Dementia (VD), there is a noted absence of potent treatments available in the current medical landscape. A new brain-protective medication developed in China, Edaravone dexboeol (EDB), has shown promise due to its antioxidant and anti-inflammatory properties, albeit with a need for additional research to elucidate its role and mechanisms in VD contexts. In a research setup, a VD model was established utilizing Sprague-Dawley (SD) rats, subjected to permanent bilateral typical carotid artery occlusion (2VO). Behavioral assessment of the rats was conducted using the Bederson test and pole climbing test, while cognitive abilities, particularly learning and memory, were evaluated via the novel object recognition test and the Morris water maze test. Ensuing, the levels of malondialdehyde (MDA), superoxide dismutase (SOD), IL-1 β , IL-6, IL-4, and tumor necrosis factor-α (TNF-α ) were determined through Enzyme-Linked Immunosorbent Assay (ELISA). Synaptic plasticity-related proteins, synaptophysin (SYP), post-synaptic density protein 95 (PSD-95), and N-methyl-D-aspartate (NMDA) receptor proteins (NR1, NR2A, NR2B) were investigated via Western blotting technique. The findings imply that EDB has the potential to ameliorate cognitive deficiencies, attributed to VD, by mitigating oxidative stress, dampening inflammatory responses, and modulating the NMDA receptor signaling pathway, furnishing new perspectives into EDB ’ s mechanism and proposing potential avenues for therapeutic strategies in managing VD.


Introduction
Vascular dementia (VD) emerges from a spectrum of cerebral vascular disorders that precipitate either inadequate cerebral perfusion or hemorrhagic cerebral insults, invariably presenting with cognitive impairments.Given the surging prevalence of cerebrovascular diseases, VD has ascended to the position of the second most common dementia etiology, superseded only by Alzheimer's disease (O'Brien and Thomas, 2015).The quest for an effective pharmacological intervention remains ongoing, with no definitive clinical therapeutic drug currently accessible (Kuang et al., 2021;Sun, 2018).Present-day clinical management of VD predominantly employs anticoagulants and antiplatelet aggregation medications (Spence et al., 2020).Nevertheless, these pharmaceutical agents exhibit limitations such as constrained treatment windows, singular therapeutic targets, and an array of side effects upon prolonged administration.Consequently, the clinical arena necessitates the development of novel drugs for VD that not only epitomize efficiency but also manifest minimal adverse effects.
Accumulating evidence has begun to underscore the pivotal roles of oxidative stress and inflammation within hippocampal tissue as substantial risk elements contributing to cognitive deficits observed in VD patients (Wang et al., 2020;Zhang et al., 2022).Upon the event of cerebral ischemia, a surge in free radical release ensues, fostering lipid peroxidation augmentation and neuronal injury, thereby intensifying cognitive dysfunctions.Elevated concentrations of inflammatory markers, such as IL-1β, IL-6, and tumor necrosis factor-α, found in the serum of cognitively impaired patients, substantiate the hypothesis that inflammatory agents can instigate cognitive function diminution.
Inflammation emerges as a cascade subsequent to cerebral ischemia, propelling the progression of secondary cerebral injury (Gogoleva et al., 2019;Liu and Zhang, 2012;Lee et al., 2015).Consequently, focusing therapeutic strategies on antioxidation and anti-inflammation emerges as a potentially viable approach in VD management.
Within mature hippocampal and cortical neurons, NR2A and NR2B are primarily localized at synaptic junctions (Hansen et al., 2017;Vyklicky et al., 2014;Erreger et al., 2007).A growing corpus of evidence sheds light on the pivotal role of NMDA receptor activation in synaptic plasticity, cerebral development, and the modulation of learning and memory through the amplification of pro-survival signaling cascades (Vyklicky et al., 2014;Paoletti et al., 2013;Hansen et al., 2018).Furthermore, aberrations in NMDA receptor signaling pathways have been pinpointed as intermediary factors in numerous neuronal damages and bear a strong correlation with diverse neurological afflictions, such as cerebral ischemia, Alzheimer's disease (Hansen et al., 2017;Surmeier and Schumacker, 2013;Choi et al., 1988).Prevailing research underscores that the induction of oxidative stress or inflammatory cascades may precipitate a decline in NMDA receptor functionality, engendering subsequent modifications in synaptic plasticity and cognitive debilitation (Zong et al., 2019;Kumar et al., 2018).Investigations employing VD rat models have illuminated that although hippocampal NR1 and NR2B expression levels diminish, fostering the expression of NR1 and NR2B can potentiate spatial learning and memory faculties in VD rats (Yin et al., 2022;Zhang et al., 2014).Thus, the modulation of NMDA receptor-mediated synaptic plasticity surfaces as a promising mechanism to mitigate cognitive impairments associated with VD.
Edaravone dexboeol (EDB), recently developed in China as a neuroprotective pharmaceutical, comprises a compound formulation of edaravone and dexboeol, both acknowledged for their efficacy.Experimental pharmacological insights have revealed that edaravone, when utilized in conjunction with dexboeol, showcases a synergistic impact, thereby furnishing augmented protection to cerebral cells (Xu et al., 2019;Zhang et al., 2023;Chen et al., 2022).A specific clinical trial in China delineated that when EDB and edaravone are separately administered within a 48-hour window following Cerebral Ischemic Stroke (CIS), EDB offers superior neurological amelioration at a 90-day checkpoint (Xu et al., 2021).The therapeutic potential of EDB, underscored by a burgeoning volume of evidence, spans numerous neurological predicaments, embracing acute ischemic stroke, subarachnoid hemorrhage, neuroinflammation, and amyotrophic lateral sclerosis (Chen et al., 2022;Zhang et al., 2023;Huang et al., 2022).The intrinsic mechanisms, through which EDB promotes cognitive recuperation in VD patients, warrant additional exploration.Preliminary research proposes that the enhancement of cognitive capabilities through EDB can be ascribed to its antioxidative and anti-inflammatory proficiencies, along with its involvement with various signaling pathways, pivotal to synaptic plasticity (Duan et al., 2022).Consequently, this formulation proposes that EDB's facilitation of cognitive recovery in VD is plausibly linked to its antioxidative and anti-inflammatory actions, and its modulation of the NMDA receptor signaling pathway.
This study indicates that EDB alleviates cognitive impairments observed in rats subjected to bilateral common carotid artery occlusion (2VO), acting notably through the suppression of hippocampal oxidative stress and inflammatory responses while concurrently modulating the NMDA receptor signaling pathway.This study further reveals the reasonable mechanism by which EDB has neuroprotective effects in the treatment of VD.

Animals
Male Sprague-Dawley rats (weighing between 200 and 250 g and aged 1.5-2 months) were acquired from SPF (Beijing) Biotechnology Co. Ltd. (License number SCXK[JING]2019-001) and were hosted at the Experimental Animal Center of Shanxi Medical University, Yingze Campus.In an environment where temperature and humidity were meticulously regulated (22 ± 2 • C and 50 ± 5 % humidity, respectively), these rats were kept under a strict 12-hour light/dark cycle.Furthermore, unobstructed access to food and water was permitted throughout the study period.All experimental procedures were carried out in accordance with the Ethics Committee for Animal Experimentation and Use Committee of China Academy of Chinese Medical Sciences and approved by the Animal Care and Use Committee of the First Hospital of Shanxi Medical University (License number DWLL-2024-004).

Animal model preparation
The establishment of the rat VD model was achieved through employing permanent bilateral common carotid artery occlusion (2VO) surgery, as per established protocols (Tuo et al., 2021;Speetzen et al., 2013).Sprague-Dawley rats, within the weight range of 200-250 g, underwent anesthesia through an intraperitoneal injection of pentobarbital sodium, administered at a dosage of 40-50 mg/kg while positioned supine.Following the removal of neck fur and sterilization of the designated surgical area, meticulous separation of the bilateral common carotid arteries from the vagus nerve ensued, ensuring the avoidance of thyroid and surrounding tissue damage.Ligation was performed with 5-0 silk sutures, followed by the careful suturing and disinfection of the incision.Vigilant monitoring of the respiratory and heart rates of the rats persisted throughout the surgical procedure.Subsequent to the operation, each rat was singularly housed until visible incision healing was noted.Meanwhile, sham-operated group animals were subjected to all procedural steps with the exception of the carotid artery ligation.

Groups and drug administration
Sprague-Dawley male rats were allocated randomly across four distinct groups, each subjected to varying surgical and treatment regimens.The Sham group experienced a surgical procedure identical in nature to the experimental setup but did not undergo bilateral common carotid artery occlusion (2VO), receiving daily intraperitoneal administrations of a volume-matched physiological saline for a period of four weeks.Contrastingly, the 2VO group not only underwent the 2VO surgery but was also subjected to daily intraperitoneal injections of a comparable volume of physiological saline for an identical four-week span.For the 2VO + EDB group, designated as the treatment cohort, rats experienced 2VO surgery and were administered intraperitoneal injections of EDB (3.75 mg/kg) on a daily basis for a duration of four weeks.In the EDB group, rats were subjected to the consistent surgical process, excluding 2VO, and were administered daily intraperitoneal injections of EDB (3.75 mg/kg) for the stipulated four-week duration.The mortality rate of rats undergoing 2VO surgery is approximately 18 %.EDB (Lot No: 181-221004) was procured from Simcere Pharmaceutical Co., Ltd.(Nanjing, China), with dosages and administration methodologies adhering to previously conducted research and initial experiments (Zhang et al., 2023;Xu et al., 2022).A comprehensive depiction of the study design is illustrated in Fig. 1, with a graphical abstract elucidated in Fig. 2.

Neurobehavioral tests
The order in which each rat undergoes behavioral testing is Bederson test,Pole climbing test,Morris water maze test,Novel object recognition test.After each test, all rats had sufficient rest time.

Bederson test
The application of the Bederson test facilitated a thorough examination of the neurological functionality within each rat group (Tuo et al., 2021).Evaluation through Bederson scoring encompassed an analysis of forelimb flexion, resistance to lateral pushing, and behavioral rotation in H. Guo et al. the subjects.The criteria for scoring, as detailed in Table 1, operated under the principle that an escalation in score directly correlated with an intensification of neurological compromise within the subjects.

Pole climbing test
The pole-climbing test was utilized to examine the limb coordination capabilities of rats across all groups (Bieber et al., 2019).Rats were positioned atop a pole, oriented with their heads directed downward, and the nature of their descent was carefully observed.The criteria for    scoring are elucidated in Table 2, with an elevated score being indicative of diminished limb coordination capacities within the rats.

Morris water maze test
Following a period of one month involving sustained administration, an evaluation using the Morris water maze test commenced.The MWM, as described in earlier reports, serves as a tool for conducting behavioral evaluations pertinent to both learning and memory (Vorhees and Williams, 2006).
The experiment unfolded in two primary phases: a phase dedicated to hidden platform training and a subsequent spatial probe test.Within the training phase, rats participated in four training trials daily, one per quadrant, for a total of five successive days, with the aim of locating the concealed platform.Rats were afforded a ceiling of 90 s to discover the hidden platform; failure to locate it within this time frame resulted in trial termination, recording of the maximum time of 90 s, and guiding of the rat to the platform where it was permitted to remain for a duration of 10 s.
Subsequent to the quintet of training days, the spatial probe test unfolded on day six, a point in time at which the concealed platform was absent from the pool.Rats experienced the liberty to navigate the aquatic environment for a duration of 90 s, after which they were extracted from the water.
Metrics, including the frequency of crossing the previous platform locale and the average duration spent in the designated target quadrant, were meticulously documented.Evaluation of the learning and memory faculties of the rats within each grouping was performed utilizing the derived data.Indicative of enhanced learning and memory capacities in the rats were diminished escape latency, an amplified count of crossings over the erstwhile platform locale, and protracted duration within the target quadrant, with the converse also being true (Lissner et al., 2021).

Novel object recognition test
Employing the Novel object recognition test facilitated an in-depth examination of how rats navigate and interact with novel items within an environment to which they have previously been acclimated, shedding light on critical aspects of cognitive functionality and memory aptitude (Antunes and Biala, 2012).
The experimental framework was delineated into three distinct phases: habituation, the training period, and the evaluative testing stage.Throughout the initial habituation period, rats underwent a process of adaptation to an open-field enclosure, spending 10-minute sessions each day for a bi-daily duration, with the deliberate absence of objects within the environment.
In the phase dedicated to training, a pair of identical items, denoted as A and B, were strategically positioned within the box in symmetrically congruent locations.Rats interacted and navigated around these items for a duration of 10 min.Following an interlude of 6 h, item B underwent replacement with an alternative object, denoted as C, maintaining a comparable texture and dimension yet exhibiting a distinctive shape.Item A retained its position within the box.A subsequent exploratory period of 5 min was allotted to the rats among these items.
The respective exploration time devoted to each item was recorded, enabling the computation of a discrimination index as [(time spent exploring the new object -time spent exploring the old object) / total time spent exploring both objects] × 100 % to elucidate the recognition and memory capabilities of the rats.In scenarios where a rat exhibited compromised cognitive capacities, no variance in exploration of the novel and familiar objects would be discerned.In contrast, a rat with unimpaired cognitive function would demonstrate a proclivity for exploring the new object for an extended duration relative to the old object (Lissner et al., 2021).

Hippocampal tissues preparation
Following neurobehavioral evaluations, the rats were euthanized through inhalation of carbon dioxide, with the subsequent collection of hippocampal tissue intended for the examination of markers associated with oxidative stress and inflammation.Preservation of the hippocampal tissue specimens was accomplished at − 80 • C.

Malondialdehyde content and superoxide dismutase activities determination
Employing the respective assay kits for MDA and SOD, obtained from Wuhan Myhalic Biotechnology Co.Ltd., measurements were conducted to ascertain the concentrations of MDA and SOD within the hippocampal regions of the rats.

Enzyme-Linked Immunosorbent assay (ELISA) for inflammatory cytokines levels
The quantifications of IL-1β, IL-6, TNF-α, and IL-4 were conducted utilizing ELISA assay kits sourced from Wuhan Myhalic Biotechnology Co.Ltd.The optical density (OD) values were recorded at a wavelength of 450 nm utilizing a microplate reader (AMR-100).
On the subsequent day, secondary antibodies (1:5000; Boster Biological Technology Co.Ltd.) were introduced and a 2-hour incubation period ensued.The membrane subsequently encountered a 30-second incubation with ECL (Boster Biological Technology Co.Ltd.) and imagery was obtained through an imaging system.This entire experimental procedure was executed on three separate occasions.

Statistical analysis
GraphPad Prism 9.5 software facilitated all statistical evaluations.The examination of variances among multiple groups employed a oneway analysis of variance (ANOVA).The F value is calculated as MSB / MSE.Escape latency was analyzed using repeated measures ANOVA (through SPSS software).Post hoc test were analyzed using the tukey test.All data undergo presentation as mean ± standard error, and statistical significance garnered consideration when a p-value was beneath 0.05.

EDB alleviates neurobehavioral impairments in Bederson test and pole climbing test
To ascertain the potential efficacy of EDB in ameliorating the neurobehavioral manifestations in VD rats, two evaluative methods, the Bederson Test and the Pole Climbing Test, were utilized.Fig. 3 vividly illustrates the neurobehavioral scores attributed to rats within each group.Observations revealed that, relative to the Sham group, the 2VO group manifested a notable escalation in both Bederson scores and pole climbing test scores (p < 0.001 in each instance).When juxtaposed with the 2VO group, the group receiving treatment displayed a marked reduction in both aforementioned scores (p < 0.001 for each).Furthermore, no substantial disparities were discernible in Bederson scores and pole climbing test scores when comparing the EDB group with the Sham group (p > 0.05 in both situations).Cumulatively, these findings propose that EDB may serve to mitigate neurobehavioral detriments observed in VD rats.

EDB mitigates learning and memory impairments in the Morris water maze test
In an effort to verify the potential of EDB to mitigate learning and memory deficits, the Morris water maze test was utilized.Fig. 4 delineates the outcomes derived from both the training and probe segments of the experiment.Throughout the training segment, every group of rats underwent a training span of five days, witnessing a consistent decrease in escape latency (Fig. 4A).From day 2 through day 5, a marked escalation in escape latency was observed in the 2VO group when juxtaposed with the Sham group (P < 0.001).Conversely, a notable diminution in escape latency was exhibited by the treatment group when assessed against the 2VO group (P < 0.001).No considerable disparities in escape latency were discernible when comparing the EDB group with the Sham group (P > 0.05).The derived outcomes suggest that while the 2VO group experienced a hindrance in spatial learning capabilities, EDB significantly alleviated this deficit.
Outcomes from the spatial probe phase are displayed in Fig. 4B-E.A notable diminution in both duration within the target quadrant and the count of platform crossings was evidenced by the 2VO group when contrasted with the Sham group (P < 0.001).Alternatively, an appreciable enhancement in these parameters was displayed by the treatment group relative to the 2VO group (P < 0.001).Comparisons between the EDB group and the Sham group yielded no significant divergences in the aforementioned parameters (P > 0.05).Additionally, no substantial distinctions were observed in the mean swimming velocity among all experimental groups (P > 0.05).Hence, these findings infer that EDB possesses the capacity to augment learning and memory faculties in VD rats.

EDB improves short-term memory in the NOR test
To validate the potentiality of EDB in enhancing short-term memory, the novel object recognition test was administered, with Fig. 5 delineating both the discrimination index and the aggregate exploration time.A discernible diminution in the discrimination index was evidenced in the 2VO group when benchmarked against the Sham group (P < 0.001).Conversely, compared to the 2VO group, the treatment group manifested a statistically significant recuperation in the discrimination index (P < 0.001).A comparative analysis between the EDB group and the Sham group did not reveal substantial discrepancies in the discrimination index (P > 0.05).Moreover, no marked differences were noted in the cumulative exploration time across all groups (P > 0.05).Consequently, these findings substantiate the hypothesis that EDB has the Sham group: Bilateral neck artery ligation was not performed, and an equivalent amount of physiological saline was injected; 2VO group: Bilateral neck artery ligation was performed, and an equivalent amount of physiological saline was injected; 2VO + EDB group: Bilateral neck artery ligation was performed, and EDB was injected (daily dose of 3.75 mg/kg); EDB group: Bilateral neck artery ligation was not performed, and EDB was injected (daily dose of 3.75 mg/kg).When compared to the Sham group, *** P＜0.001; when compared to the 2VO group, ### P＜0.001.Fig. 4. The impact of EDB on the learning and memory abilities of VD rats.A: The average escape latency in the first 5 days for each group of rats was measured.B: The time taken by each group of rats to enter the target quadrant on the 6th day was recorded.C: The number of times each group of rats crossed the platform area on the 6th day was counted.D: The average swimming speed of each group of rats was calculated.E: The swimming trajectories of the rats in each group on the 6th day were plotted.All values are presented as mean ± SEM. *** P＜0.001 compared to the Sham group; ### P＜0.001 compared to the 2VO group; "ns" indicates no significant difference.capability to augment short-term memory in VD rats.

EDB can suppress oxidative stress in the hippocampus of VD rats
Oxidative stress establishes a crucial linkage with the progression of cognitive deficits observed in VD patients, as identified in previous research (Liu and Zhang, 2012;Wang et al., 2021).In the pursuit to decipher whether EDB augments learning and memory by mitigating oxidative stress, MDA and SOD levels in the rat hippocampus were scrutinized,as illustrated in Fig. 6A and B. A noteworthy escalation in MDA levels was observed in the 2VO group when juxtaposed with the Sham group (P < 0.001).Conversely, the treatment group, when compared with the 2VO group, demonstrated a substantial diminution in MDA levels (P < 0.01).Concurrently, an appreciable decline in SOD levels was detected in the 2VO group relative to the Sham group (P < 0.001).On the opposite spectrum, the treatment group manifested a notable elevation in SOD levels in contrast to the 2VO group (P < 0.001).Thus, these findings infer that EDB may attenuate oxidative stress in VD rats, enhancing their learning and mnemonic capabilities.

EDB can alleviate the inflammatory response in the hippocampus of VD rats
Inflammatory responses are posited to exert a substantial influence on the evolution of cognitive deficits in VD patients (Wang et al., 2020).Endeavoring to ascertain whether EDB is capable of ameliorating cognitive deterioration in VD rats through the modulation of inflammatory reactions, the study employed ELISA to quantify proinflammatory (IL-1β, IL-6, TNF-α) and anti-inflammatory (IL-4) factor levels within the hippocampus of the rats, which are depicted in Fig. 6C through 6F.A salient elevation in IL-1β (P < 0.001), IL-6 (P < 0.001) and TNF-α (P < 0.001), coupled with a decline in IL-4 (P < 0.001) were discerned in the 2VO group relative to the Sham group.In a parallel comparison with the 2VO group, the treatment group manifested marked diminutions in IL-1β (P < 0.001), IL-6 (P < 0.01), TNF-α (P < 0.05), and an augmentation in IL-4 (P < 0.01).No substantive disparities in IL-1β, IL-6, TNF-α, and IL-4 levels were observed between the EDB and Sham groups (P > 0.05).Thus, the findings suggest that EDB may mitigate the inflammatory response in VD rats, thereby facilitating an enhancement in cognitive functioning.

EDB enhances synaptic plasticity and NMDA receptor expression in VD rats
In an endeavor to elucidate the underlying mechanisms through which EDB augments learning and mnemonic capabilities, this investigation employed the WB method to evaluate the hippocampal expression levels of proteins related to synaptic plasticity, notably synaptophysin (SYP) and postsynaptic density protein 95 (PSD-95), as illustrated in Fig. 7A, C, and D. When contrasted with the Sham group, a diminution in the levels of SYP (P < 0.001) and PSD-95 (P < 0.05) was evident in the 2VO group.Moreover, the treatment group, when compared to the 2VO group, manifested elevated levels of SYP (P < 0.01) and PSD-95 (P < 0.05).No consequential disparities in the expression levels of SYP and PSD-95 were discerned between the EDB and Sham groups (P > 0.05).Consequently, the findings suggest that EDB has the potential to amplify synaptic plasticity in rats afflicted with VD, thereby enhancing their learning and mnemonic capabilities.
The NMDA receptor signaling pathway remains not merely integrally connected to synaptic plasticity and cognitive functionality (Hunt and Castillo, 2012;Diering and Huganir, 2018) but also critically influences VD (Li et al., 2020).The current study, aiming to explore the effects of EDB on the NMDA receptor signaling pathway, utilized the WB technique to assess the expression of three subunits within the hippocampus of rats, specifically NR1, NR2A, and NR2B, as delineated in Fig. 7B, E, F,  and G.A diminution in expression levels of NR1 (P < 0.05) and NR2B (P < 0.05) was observed in the 2VO group when juxtaposed with the Sham group.Conversely, the treatment group revealed an augmentation in the expression levels of NR1 (P < 0.05) and NR2B (P < 0.05) relative to the 2VO group.No substantive discrepancies were identified in the expression levels of NR1 and NR2B between the EDB and Sham groups (P > 0.05).NR2A expression did not demonstrate noteworthy statistical significance.Thus, the findings imply that EDB has the capacity to modulate the NMDA receptor signaling pathway expression in rats experiencing VD, thereby potentially enhancing cognitive function.

Discussion
The present study investigated the potential therapeutic effects of EDB in a rat model of VD and explored the underlying mechanisms involved.The findings provide valuable insights into the potential role of EDB in mitigating cognitive impairment in VD and suggest possible mechanisms of action.
The results of this study demonstrated that EDB treatment improved neurobehavioral impairments in VD rats, as indicated by the Bederson test and pole climbing test.Furthermore, EDB enhanced learning and memory abilities, as evidenced by reduced escape latency in the Morris water maze test and increased discrimination index in the novel object recognition test.
Oxidative stress and inflammatory responses are known to contribute to cognitive deficits in VD (Liu and Zhang, 2012;Rosenberg, 2018).Previous studies have shown that EDB can inhibit neuroinflammatory responses and inhibit cell apoptosis and autophagy in VD rats (Zhang et al., 2023).Consistent with previous research, we found that EDB attenuated the inflammatory response by reducing the levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and increasing the level of the anti-inflammatory cytokine IL-4 in the hippocampus.Meanwhile, we also proved that EDB treatment effectively reduced oxidative stress by decreasing malondialdehyde (MDA) levels and increasing superoxide dismutase (SOD) levels in the hippocampus of VD rats and EDB may also be involved in regulating synaptic plasticity.We explored other pathways for the therapeutic effect of EDB.
The modulation of the NMDA receptor signaling pathway is implicated in synaptic plasticity and cognitive function.The study found that EDB treatment enhanced synaptic plasticity by increasing the expression levels of synaptophysin (SYP) and postsynaptic density protein 95 (PSD-95) in the hippocampus.Furthermore, EDB modulated the expression of NMDA receptor subunits (NR1, NR2B), suggesting its potential role in regulating NMDA receptor-mediated synaptic plasticity.
These findings suggest that EDB may improve cognitive impairment in VD rats by mitigating oxidative stress, attenuating inflammatory responses, and modulating the NMDA receptor signaling pathway.The multifaceted effects of EDB provide a promising approach for the management of VD.
It is important to note that this study was conducted in a rat model, and further research is needed to validate these findings in humans.Additionally, the precise mechanisms underlying the effects of EDB in VD require further investigation.Nevertheless, the results of this study contribute to our understanding of the potential therapeutic strategies for VD and highlight EDB as a promising candidate for further exploration in clinical settings.
Emerging evidence indicates a significant association between NMDA receptors and VD, characterized by reduced expression of NMDA receptors in the hippocampus of VD rats.Previous studies have shown that enhancing the expression of synaptic plasticity-related proteins, specifically the NR1 and NR2B subunits of NMDA receptors, improves spatial learning and memory in VD rats (Li et al., 2018;Zhang et al., 2015;Busse et al., 2014).Consistent with these findings, our study observed decreased expression levels of synaptic plasticity-associated proteins (SYP and PSD95) and NMDA receptor subunits (NR1 and NR2B) in the hippocampus of VD rats, highlighting the involvement of the NMDA receptor signaling pathway in the cognitive dysfunction observed in VD.Importantly, our study demonstrated that EDB administration increased the expression levels of SYP, PSD95, NR1, and NR2B in the hippocampus of VD rats, suggesting a potential role of the NMDA receptor signaling pathway in the cognitive improvement mediated by EDB in VD.
Previous interventions targeting oxidative stress in VD animal models have focused on traditional Chinese acupuncture and pharmacological approaches.Acupuncture studies have shown that electroacupuncture can enhance antioxidant enzyme activities and improve learning and cognitive abilities in ischemic rat models (Hou et al., 2015).Pharmacological interventions, such as Timosaponin B-II (TB-II) and N-Palmitoylethanolamine-oxazoline (PEA-OXA), have also demonstrated antioxidant and anti-inflammatory effects, resulting in improved cognitive function in VD animal models (Zhao et al., 2016;Impellizzeri et al., 2019).Additionally, compounds like α-lipoic acid and resveratrol, combined with aerobic exercise, have been shown to mitigate oxidative stress and enhance learning and memory in VD rats (Li et al., 2022).
Consistent with previous studies, our findings support the notion that increasing superoxide dismutase (SOD) levels and reducing malondialdehyde (MDA) levels to mitigate oxidative stress damage can improve learning and memory abilities in rodent models.In contrast to previous acupuncture and pharmacological approaches, our study investigated the therapeutic potential of EDB, which is known for its ability to reduce hippocampal oxidative stress and inflammatory responses (Liu et al., 2023;Hu et al., 2022;Liu et al., 2023).EDB intervention may provide novel therapeutic targets for the management of VD.Furthermore, our study revealed a significant relationship between the NMDA receptor signaling pathway and the neuroprotective effects of EDB, shedding light on the mechanism of action of EDB and offering insights into potential future treatment strategies.
While our research provides valuable insights, it also has certain limitations.Firstly, each rat participated in all behavioral tests, and the pressure of each test may affect the results of other tests.Second,despite elucidating the protective responses of EDB against oxidative stress and inflammation, there may be additional unidentified effects of EDB that contribute to its therapeutic actions in VD.Further investigations are needed to explore the specific mechanisms underlying the protective effects of EDB in VD through additional experimental analyses.

Conclusion
In a comprehensive review of the findings, this investigation substantiates the theoretical proposition that EDB possesses the capacity to mitigate cognitive deteriorations observed in VD-afflicted rats.Intriguingly, the research illuminated that this compound achieves such an impact by concurrently modulating oxidative stress inhibition, mitigating inflammatory responses, and fine-tuning the NMDA receptor signaling pathway, thereby enhancing the cognitive deficits characteristically induced by VD.Consequently, this paves the way for envisioning a novel trajectory regarding the prospective utility of EDB in therapeutic strategies aimed at VD management in future medical practices.
Data Availability: Data will be made available on request.

Fig. 3 .
Fig. 3.The Impact of EDB on Neurobehavior in VD Rats.A: The Bederson Test was employed.B: The Pole Climbing Test was conducted.Each group had a sample size of 10.Statistical analysis utilized a one-way analysis of variance.Sham group: Bilateral neck artery ligation was not performed, and an equivalent amount of physiological saline was injected; 2VO group: Bilateral neck artery ligation was performed, and an equivalent amount of physiological saline was injected; 2VO + EDB group: Bilateral neck artery ligation was performed, and EDB was injected (daily dose of 3.75 mg/kg); EDB group: Bilateral neck artery ligation was not performed, and EDB was injected (daily dose of 3.75 mg/kg).When compared to the Sham group, *** P＜0.001; when compared to the 2VO group, ### P＜0.001.

Fig. 5 .
Fig. 5.The impact of EDB on the short-term memory abilities of VD rats.A: Recognition index; B: Total exploration time of objects; C: Exploration trajectory maps of rats in each group (upper left for new objects, upper right for old objects).All values are presented as mean ± SEM. *** P＜0.001 compared to the Sham group; ### P＜ 0.001 compared to the 2VO group; "ns" indicates no significant difference.

Fig. 7 .
Fig. 7.The impact of EDB on the expression of synaptic plasticity-related proteins and NMDA receptors in the hippocampus of VD rats.A:The expression levels of SYP proteins; B:The expression levels of PSD-95 proteins; C:The expression levels of NR1 proteins; D:The expression levels of NR2A proteins; E:The expression levels of NR2B proteins.All values are presented as mean ± SEM (n = 3 per group).*P＜0.05,*** P＜0.001 compared to the Sham group; # P＜0.05, ## P＜0.01compared to the 2VO group; "ns" indicates no significant difference.
Score Standard 0 No neurological deficits 1 Any flexion component of the forelimb is not accompanied by other abnormal conditions 2 The lateral thrust resistance decreased,accompanied by forelimb buckling, and no circling behavior 3 Same level 2 behavior, accompanied by spontaneous rotation

Table 2
Pole Test.
Score Standard 0Step by step, down the pole 1 Slide down the pole 2 Can't get a grip on the pole 3Loss of the righting reflex H.Guo et al.