Resveratrol Modulates FABP5 to Reduce Neuronal Apoptosis Following Ischemic Stroke

Resveratrol Modulates FABP5 to Reduce Neuronal Apoptosis Following Ischemic Stroke | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 5 April 2025 V1 Latest version Share on Resveratrol Modulates FABP5 to Reduce Neuronal Apoptosis Following Ischemic Stroke Authors : Bingfeng Xing , Min Hong 0009-0007-0048-5748 [email protected] , Xin Zhou , Weihao Lin , and Changqin Xiang Authors Info & Affiliations https://doi.org/10.22541/au.174383649.96992746/v1 Published Open Life Sciences Version of record Peer review timeline 342 views 154 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Abstract Background: Fatty acid-binding proteins (FABPs) influence cellular energy metabolism through the regulation of fatty acid kinetics and play a crucial role in neuronal apoptosis following cerebral infarction. Resveratrol (RSV) has been shown to mediate neuroprotective effects in ischemic stroke; however, its regulatory impact on FABPs and associated pathways remains to be elucidated. Methods & Results: WGCNA analysis reveals that FABP5 was significantly enriched in fatty acid metabolism-related pathways in middle cerebral artery occlusion (MCAO) rats. Modulating FABP5 expression may influence post-infarction neuronal recovery. Molecular docking experiments demonstrated that resveratrol (RSV) exhibits strong binding affinity with FABP5. After administering different doses of RSV to MCAO rats, the cerebral infarct area significantly decreased, and neurological function improved with increasing doses. Concurrently, the expression of FABP5 and NSE in brain tissue was downregulated, while BDNF expression was upregulated, and neuronal morphology improved. Further experiments using FABP5 overexpression and inhibition models revealed that FABP5 overexpression exacerbated neuronal apoptosis and suppressed AMPK protein expression, whereas FABP5 inhibition reduced neuronal apoptosis and enhanced AMPK protein expression. Conclusion:RSV down-regulates FABP5 expression in cerebral infarction tissues and potentially mediates the AMPK-related pathway to ameliorate neuronal apoptosis. Introduction Ischemic stroke (IS) is an acute neurological disorder caused by thrombosis or embolism leading to the interruption of cerebral blood flow. It is the second leading cause of death globally. According to statistics, IS results in approximately 5.9 million deaths and 102 million cases of disability annually, with an incidence rate of 45 per 100,000 people in 2019 1 . During the onset of IS, obstruction of blood supply to the brain through the carotid and vertebral artery systems leads to devastating brain damage and severe neurological deficits 2 . Free fatty acids (FAs) in neuronal cells play a crucial role in signal sensing and transduction. In recent years, FA signaling pathways and their associated proteins related to neuronal protection and repair in IS have garnered increasing attention in the academic community. In the cytoplasm, free fatty acids (FAs) are taken up by fatty acid-binding proteins (FABPs) and transported to specific subcellular structures or metabolic pathways 3 . As important protein markers in fatty acid metabolism pathways related to neuronal injury and repair in IS, FABPs regulate mitochondrial energy metabolism, oxidative stress responses, and blood-brain barrier damage in the brain 4,5 . Previous studies have confirmed that FABPs possess the ability to bind and transport fatty acids, exacerbating central nervous system cell injury by inhibiting cellular energy supply and downstream lipid oxidation and stress responses 6,7 . Among the subtypes of FABPs, FABP3, FABP5, and FABP7 have received considerable attention in ischemic stroke research [8]. As biomarkers for ischemic stroke, fatty acid-binding proteins have been widely studied by researchers in recent years 9,10,11 . Resveratrol (RSV) is a natural polyphenolic compound widely found in medicinal plants and has been demonstrated to possess various effects, including neuroprotection, cognitive improvement, and immune regulation 12,13 . Recent meta-analyses have shown that RSV can effectively reduce cerebral ischemic infarction and edema volume, mitigate blood-brain barrier damage, and improve neurological function, making it a promising neuroprotective agent following cerebral infarction 14 . However, the neuroprotective mechanisms of RSV have not yet been fully elucidated. RSV also plays a significant role in regulating fatty acid metabolism. Studies indicate that RSV can modulate the lipid order and organization of the cytoplasmic membrane, which is closely related to critical cellular functions such as membrane sorting and transport 15 . Whether the neuroprotective mechanisms of RSV in ischemic stroke (IS) neurons are mediated through the modulation of FABPs remains to be further investigated and validated. In this study, we utilized a middle cerebral artery occlusion (MCAO) model and employed biosignal analysis methods to screen for FABP subtypes associated with neuronal repair following cerebral infarction. By administering resveratrol (RSV) via oral gavage to the MCAO rat model, we observed changes in ischemic stroke (IS) neuronal cells and investigated the effects of FABP overexpression and inhibition on key proteins involved in fatty acid metabolism, as well as their relationship with neuronal injury. This approach aims to explore the potential mechanisms by which RSV protects IS neurons through the regulation of fatty acid metabolism. Materials and Methods This study consists of two parts: biosignal analysis and animal experiments, with the latter further divided into two subparts.Part 1: Animal Experiment.Specific pathogen-free (SPF) male Sprague-Dawley (SD) rats were selected and divided into five groups: sham surgery group, MCAO group, RSV5 group, RSV10 group, and RSV20 group. The RSV5, RSV10, and RSV20 groups were administered resveratrol (RSV) at doses of 5 mg/kg, 10 mg/kg, and 20 mg/kg, respectively, via oral gavage.Part 2: Animal Experiment.Rats were divided into the MCAO group, FABP5 overexpression group, and FABP5 inhibition group. Both the FABP5 overexpression and FABP5 inhibition groups were administered RSV at a dose of 10 mg/kg via oral gavage.A total of 64 SPF male SD rats weighing 180–200 g were used in this study. After one week of acclimatization, the rats were randomly divided into a sham surgery group (n=8) and an MCAO model group (n=56). The MCAO model group was further subdivided into the MCAO group, RSV5 group, RSV10 group, RSV20 group, FABP5 overexpression group, and FABP5 inhibition group.This study was supported by the Guangdong Medical Science Research Foundation (Grant No.: C2024079) and approved by the Ethics Committee of the First Affiliated Hospital of Guangdong Pharmaceutical University (Approval Nos.: GYFYDWSY2024029). MCAO and Sham Surgery group rat modeling MCAO Group:The MCAO model group was anesthetized using an oxygen anesthesia machine to induce a pain-free state. After anesthesia, the rats were placed in a supine position, and the neck hair was shaved and disinfected. The rats were then positioned on a surgical table, maintaining a body temperature of approximately 37°C. The head was fixed in a holder, and a midline cervical skin incision was made. Layers were dissected to expose the left carotid artery and vagus nerve along the trachea. Upon identifying the ”Y”-shaped vascular structure, the external carotid artery and the distal end of the internal carotid artery were ligated using surgical sutures. A 0.15 mm diameter filament was inserted to induce ischemic occlusion. After 30 minutes of occlusion, the filament was removed, and the incision was sealed using an electric coagulator and sutured layer by layer. The rats were transferred to a warming chamber for gradual recovery, with necessary medical interventions and monitoring, including body temperature, heart rate, respiratory rate, and neurological function. Sham Surgery Group:A total of 8 SD rats were used as the sham surgery group. Only a skin incision was made without inducing ischemic injury, and the rats were administered an equivalent volume of saline via oral gavage. They were normally housed for 7 days. All rats were housed individually in transparent cages with a 12-hour light/dark cycle (08:00–20:00). The rats had free access to water and food, and the room temperature was maintained at approximately 25°C. Before the experiment, the rats were fasted for 6 hours but allowed free access to water. FABP5 Over expression and Inhibition Groups:The FABP5-PCDH-CMV-EF1A-EGFP-T2A-PURO overexpression plasmid and the pLKO.1-EGFP-puro-FABP5shRNA knockdown plasmid were constructed, followed by lentiviral packaging. The viral supernatant was collected, filtered through a 0.45 μm filter, and the lentiviral titer was measured. The corresponding lentiviral particles were then injected into the two groups of rats, labeled as the overexpression group (OVER) and the inhibition group (SH), respectively. Biosignaling Pathway Enrichment Analysis in MCAO Rats The GSE166162 dataset was downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). Weighted Gene Co-expression Network Analysis (WGCNA) was performed using R language (version 3.6.2) to explore potential biosignaling pathway enrichment in MCAO rats. A total of 12 samples from the Affymetrix Rat 230 2.0 array were divided into Sham and MCAO groups, with 6 samples in each group (n=6). The GEOquery package was used to obtain the expression matrix, and gene annotation was performed using the GPL1355 platform. An ”unsigned” co-expression network was constructed using WGCNA. The Pearson method was employed to calculate the correlation between sorted module eigengenes (MEs_col) and trait data (traitData). Genes associated with MCAO expression were screened based on gene significance (GS) > 0.6 and module membership (MM) > 0.6. The STRING database (https://string-db.org/) was used to construct a protein-protein interaction (PPI) network and perform pathway enrichment analysis. A total of 56 genes, including 55 genes screened by WGCNA and 4 genes of interest from previous studies (FABP4, FABP5, NSE, BDNF), were included in the network. In the STRING database, the species was set to Rattus norvegicus, and the MCL algorithm was used for cluster analysis. Pathway enrichment analysis of the screened genes was performed using the GO and KEGG databases. The results were visualized using the ggplot2 package in R. Molecular Docking Analysis of RSV and FABP The active structure of the target protein was obtained from the PDB database (https://www.rcsb.org/) in PDB file format. The SDF file of the target small molecule, resveratrol (RSV), was retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) using the search term ”Resveratrol.” Semi-flexible docking was performed using AutoDock 4.2.6. The results showed a low binding energy between the ligand (RSV) and the receptor (FABP), indicating strong binding affinity. 4. Effects of RSV on Neuronal Morphology and Function in MCAO Rats 4.1 Grouping and Drug Administration in MCAO Rats After the MCAO model was established, the rats in the first part of the experiment were numbered 1–32, and those in the second part were numbered 33–56. Random numbers were generated using the ”RandBetween(1,24)” function in Excel, and the rats were grouped according to the random sequence.First Part of the Experiment:Rats 1–8 were assigned to the MCAO control group.Rats 9–16 were assigned to the RSV5 group.Rats 17–24 were assigned to the RSV10 group.Rats 25–32 were assigned to the RSV20 group.Second Part of the Experiment:Rats 33–40 were assigned to the MCAO control group.Rats 41–48 were assigned to the FABP5 overexpression group.Rats 49–56 were assigned to the FABP5 inhibition group.Drug Administration:First Part of the Experiment:The RSV5, RSV10, and RSV20 groups were administered RSV at doses of 5 mg/kg, 10 mg/kg, and 20 mg/kg, respectively, via oral gavage once daily for 7 consecutive days.The MCAO control group was administered an equivalent volume of saline via oral gavage.Second Part of the Experiment:Both the FABP5 overexpression group and the FABP5 inhibition group were administered RSV at a dose of 10 mg/kg via oral gavage once daily for 7 consecutive days.The MCAO control group was administered an equivalent volume of saline via oral gavage.The rats were observed for 7 days. Behavioral Monitoring of MCAO Rats in Each Group Longa Neurological Score:The neurological deficits were scored as follows:0 points: No neurological deficits.1 point: Incomplete extension of the left forepaw.2 points: Circling to the left while walking.3 points: Falling to the left (paralyzed side) while walking.4 points: Inability to walk spontaneously, with loss of consciousness.Neurological scores were assessed on day 0 (before the experiment) and on days 1, 3, 5, and 7 of the experiment. Open Field Test:The open field apparatus consisted of a black plastic box with dimensions of 90 cm (length) × 90 cm (width) × 45 cm (height). A camera was mounted above the box to capture the entire field of view. Before each test, the floor and walls of the box were wiped with alcohol to eliminate residual odors or excretions from previous animals. Testing began on day 7 of the experiment, with one rat tested at a time for 5 minutes per session. A minimum interval of 24 hours was maintained between tests.The rat was placed at the starting point of the open field, and the recording device was activated to track the animal’s movement trajectory. After data collection, the field was divided into a 3×3 grid using analysis software. The total distance traveled (Total distance) and the duration of immobility (Time of stop moving) were recorded and analyzed. Data analysis was performed using ImageJ. Comparative Analysis of Cerebral Infarct Area, Neuronal Morphology, and Related Factor Expression in MCAO Rats from Each Group Brain Tissue Sampling and Western Blot (WB) Analysis:The scalp of the fetal rats was removed, and the skull was cut open to extract the brain tissue. The brain tissue was rinsed with 0.9% saline and placed on an ice platform. The left and right cerebral hemispheres were separated. The right cerebral hemisphere was homogenized. Specifically, the right brain tissue was placed in a centrifuge tube and stored in a -80°C freezer. Whole-cell lysis buffer was added, followed by ultrasonic homogenization. The sample was kept on ice for 15 minutes and then centrifuged (12,000 rpm, 4°C, 15 minutes). The supernatant was transferred to a new centrifuge tube. Protein concentrations of BDNF, NSE, FABP3, FABP4, FABP5, and AMPK were measured using corresponding assay kits. TTC Staining for Infarct Area Assessment:2,3,5-Triphenyltetrazolium chloride (TTC), a lipid-soluble and light-sensitive compound, was used to detect ischemic cerebral infarction in mammalian brain tissue. In this study, 2% TTC staining solution was applied to brain tissues on the 7th day post-surgery to evaluate ischemic injury. The procedure was as follows: Fresh brain tissue or slices were completely immersed in 2% TTC staining solution and incubated at 37°C for 30 minutes. Color changes were observed during incubation. After staining, the tissues were gently rinsed with PBS buffer, and the staining results were photographed. ImageJ software was used for image recognition and analysis to calculate the percentage of TTC infarct area. The formula used was:Infarct volume ratio = (Sum of white infarct areas in all slices) / (Sum of total brain slice areas) × 100%. Morphological Changes in Stained Brain Tissue:Paraffin-embedded brain tissue sections were deparaffinized in xylene for 10 minutes. The sections were then rehydrated by sequentially immersing them in 95%, 85%, and 70% ethanol gradients for 5 minutes each. After rehydration, the samples were washed three times in PBS, 5 minutes each. Hematoxylin staining was performed for 10 minutes, followed by differentiation using 1% hydrochloric acid ethanol. Eosin staining was then applied for 3 minutes. The tissue sections were dehydrated through an ethanol gradient, mounted with neutral resin, and air-dried before being photographed. Flow Cytometry Analysis of Neuronal Apoptosis Neuronal apoptosis was detected using the Annexin V-FITC/PI Apoptosis Kit (E-CK-A211, Elabscience Biotechnology, China). The procedure was as follows:Apoptosis was induced according to the experimental protocol.Cells were centrifuged at 300 × g for 5 minutes, and the supernatant was discarded.The cell pellet was collected, washed once with PBS, gently resuspended, and counted.A total of 1 × 10^6 resuspended cells were centrifuged at 300 × g for 5 minutes, and the supernatant was discarded.The cells were washed once with PBS, centrifuged again, and the supernatant was discarded.The cell pellet was resuspended in 100 μL of diluted 1× Annexin V binding buffer.Then, 2.5 μL of Annexin V-FITC reagent and 2.5 μL of PI reagent (50 μg/mL) were added.The mixture was gently mixed and incubated at room temperature in the dark for 20 minutes.After incubation, 400 μL of diluted 1× Annexin V binding buffer was added, and the sample was mixed thoroughly.Apoptosis was analyzed using a BD FACSCanto II flow cytometer. Results IS Neuronal Injury and Protection Are Closely Associated with FABP5, a Fatty Acid Metabolism Pathway Protein with Strong Binding Affinity to RSV Cluster analysis, soft threshold screening, module construction, phenotype data correlation analysis, and gene significance analysis of MCAO rat samples revealed a total of 56 genes significantly associated with the ”MCAO” phenotype. Protein-protein interaction (PPI) pathway enrichment analysis showed that genes in Cluster 1 were primarily related to fatty acid metabolism, suggesting that the regulation of FABP5, a fatty acid metabolism pathway protein, is closely linked to IS neuronal injury and protection (Fig.1A-D). Semi-flexible molecular docking experiments demonstrated that RSV has a binding site with FABP5 (Fig.1E), indicating an interaction between RSV and FABP5. RSV Improves Motor and Cognitive Functions in MCAO Rats Open field test and Longa neurological scores were conducted on the rats from the first part of the experiment. The movement trajectories in the open field test showed that the Sham group rats had the longest total distance traveled and the shortest stationary time, and exhibited central grid exploration behavior (Fig. 2A1). In contrast, the MCAO group rats remained stationary in the corners for long periods, had a small range of activity, and moved slowly (Fig. 2A2). After RSV treatment, the motor functions of the rats in each group significantly recovered, with increased total distance traveled and reduced stationary time, particularly notable in the RSV10 and RSV20 groups. The RSV20 group showed more central exploration behavior compared to the RSV10 group (Fig. 2A3-5). Statistical results of stationary time in the open field test showed significant differences between the RSV treatment groups at different doses and the MCAO control group (p < 0.001) (Fig. 2B). Statistical results of total distance traveled in the open field test showed a significant difference between the RSV10 group and the MCAO control group (P = 0.0358) (Fig. 2C). Longa scores of the rats in each group showed that the RSV20 group had a significant difference compared to the MCAO group on day 5 (P = 0.0119). The RSV10 and RSV20 groups showed significant differences compared to the MCAO group on day 7 (P = 0.0407 and P < 0.0001, respectively) (Fig. 2D). The above results indicate that the behavioral and cognitive functions of MCAO rats were significantly impaired, but after RSV treatment, the behavioral abilities of MCAO rats were significantly improved. As the concentration of RSV increased, the improvement in motor and cognitive behavioral abilities of MCAO rats gradually enhanced, indicating that RSV has a significant effect in improving the behavioral aspects of MCAO rats, and the effect is dose-dependent. RSV Ameliorates Neuronal Morphology in MCAO Rats, with the Degree of Improvement Positively Correlated with RSV Concentration TTC staining results revealed that RSV significantly reduced brain tissue damage induced by MCAO. Statistical analysis of cerebral infarction area showed a significant difference between the RSV10 group and the MCAO group (P = 0.0133), and an extremely significant difference between the RSV20 group and the MCAO group (P < 0.001). As the concentration of RSV increased, the area of brain damage caused by ischemia gradually decreased, and the brain tissue damage in the RSV10 and RSV20 groups was significantly alleviated compared to the MCAO group (Fig. 3A, B). HE staining results demonstrated that the neuronal structure in the MCAO group was disrupted, with disordered neuronal morphology. After RSV intervention, neuronal morphology was significantly improved, and the degree of improvement gradually increased with higher RSV doses (Fig. 3C). These results indicate that RSV has a neuroprotective effect on MCAO rats, and this protective effect is enhanced with increasing doses. RSV Upregulates BDNF Expression and Downregulates FABP5 and NSE Expression in the Brain Tissue of MCAO Rats. Western blot (WB) results showed that, compared to the Sham group, the expression of NSE and FABP5 was significantly upregulated in MCAO rats. After intervention with different doses of RSV, NSE expression was downregulated, with significant differences observed in the RSV5, RSV10, and RSV20 groups compared to the MCAO group (P = 0.0357, P = 0.0012, P < 0.0001, respectively) (Fig. 4A, C). FABP5 expression was also significantly downregulated after RSV intervention, with significant differences in the RSV10 and RSV20 groups compared to the MCAO group (P = 0.0072, P = 0.0008, respectively) (Fig. 4A, D). Additionally, BDNF expression was downregulated in MCAO rats but significantly upregulated after RSV intervention, with significant differences in the RSV10 and RSV20 groups compared to the MCAO group (P = 0.0016) (Fig. 4A, E).qPCR results revealed that NSE and FABP5 expression was significantly upregulated in MCAO rats. After intervention with different doses of RSV, NSE expression was downregulated, with significant differences in the RSV10 and RSV20 groups compared to the MCAO group (P = 0.0217, P = 0.0002, respectively) (Fig. 4F). FABP5 expression was also significantly downregulated after RSV intervention, with significant differences in the RSV10 and RSV20 groups compared to the MCAO group (P = 0.0315, P = 0.0251, respectively) (Fig. 4G). Furthermore, compared to the Sham group, BDNF expression was downregulated in MCAO rats but significantly upregulated after RSV intervention, with significant differences in the RSV5, RSV10, and RSV20 groups compared to the MCAO group (P = 0.0399, P = 0.0179, P = 0.0009, respectively) (Fig. 4H).Immunofluorescence results showed that, compared to the Sham group, FABP5 levels were significantly upregulated in the brain tissue of MCAO rats. After RSV intervention, FABP5 expression gradually decreased with increasing RSV concentrations. However, the expression of FABP3 and FABP4 showed no significant changes after RSV intervention.These results indicate that BDNF expression was downregulated, while NSE and FABP5 expression was upregulated in MCAO rats. After RSV intervention, FABP5 and NSE expression were significantly downregulated, and BDNF expression was significantly upregulated. RSV Protects and Repairs IS Neuronal Cells by Downregulating FABP5 Expression. In the second part of the experiment, the OVER group (FABP5 overexpression group) and the SH group (FABP5 inhibition group) were subjected to FABP5 overexpression and inhibition treatments, respectively (Fig. 5). Subsequently, MCAO modeling was performed, followed by RSV 10 mg/kg intervention.Results from brain tissue ischemia and flow cytometry showed that neuronal damage was significantly more severe in the OVER group, while neuronal damage was markedly improved in the SH group (Fig. 6A, B). After RSV 10 mg/kg intervention, samples were taken from the three groups, and the apoptosis rates of brain neuronal cells were analyzed (Fig. 6C). Compared to the MCAO group, the early neuronal apoptosis rate was significantly increased in the OVER group (P = 0.0012), while it was significantly reduced in the SH group (P = 0.0004). Additionally, the early neuronal apoptosis rate was significantly higher in the OVER group compared to the SH group (P < 0.0001), and the late neuronal apoptosis rate was also significantly increased in the OVER group (P = 0.0442).These results indicate that FABP5 overexpression exacerbates IS neuronal cell damage, while FABP5 inhibition exerts a protective effect on IS neuronal cells. RSV may exert its neuroprotective and reparative effects by regulating FABP5 expression. RSV Downregulates FABP5 to Protect Brain Neurons Possibly Through an AMPK-Mediated Energy Metabolism Regulation Mechanism. Immunofluorescence results showed that FABP5 expression was significantly increased in the FABP5 overexpression group (OVER group), while it was significantly reduced in the FABP5 inhibition group (SH group) (Fig. 7A). Simultaneously, AMPK expression was decreased in the OVER group and increased in the SH group (Fig. 7B). Western Blot band results demonstrated that AMPK expression was significantly reduced in the FABP5 overexpression group (OVER group), while it was partially restored in the FABP5 knockdown group (SH group) (Fig. 7C). RT-qPCR results revealed that, compared to the MCAO group, FABP5 expression was significantly upregulated in the FABP5 overexpression group (OVER group) (P = 0.0002), while it was significantly downregulated in the FABP5 inhibition group (SH group) (P = 0.0002). Additionally, FABP5 expression was significantly higher in the OVER group compared to the SH group (P < 0.0001) (Fig. 7D). Regarding AMPK expression, compared to the MCAO group, AMPK expression was significantly downregulated in the OVER group (P = 0.0002), while it was significantly upregulated in the SH group (P = 0.0024). Furthermore, AMPK expression was significantly higher in the SH group compared to the OVER group (P < 0.0001) (Fig. 7E).These results indicate that FABP5 overexpression leads to a reduction in AMPK expression, while FABP5 knockdown upregulates AMPK expression. RSV may exert its neuroprotective effects by regulating FABP5 and subsequently modulating the AMPK-mediated energy metabolism mechanism. Discussion The pathogenesis of ischemic stroke involves multiple factors, including neuronal apoptosis, increased excitotoxicity, oxidative stress, and inflammatory responses 16, 17 . Recent studies have shown that the physiological processes of stroke are driven by interactions among neurons, glial cells, vascular cells, and extracellular matrix components, all of which play critical roles in brain tissue damage and repair 18 .The loss of neurons due to ischemia and infarction is the most direct cause of neurological functional impairment. Therefore, reducing neuronal apoptosis in the early stages of ischemic stroke (IS) is one of the key focuses of clinical research. Brain-derived neurotrophic factor (BDNF) is an important neuroprotective factor and is often regarded as a critical indicator of neuronal repair following stroke 19, 20 . Neuron-specific enolase (NSE), a marker of neuronal damage, is significantly elevated during stroke and serves as an important indicator for assessing neuronal injury post-stroke 21, 22 .In our study, after administering RSV to MCAO rats, we observed a significant increase in BDNF expression and a significant decrease in NSE expression in brain tissue. These findings suggest that RSV exerts a notable protective effect on neurons following IS. In recent years, the role of fatty acid metabolism and its signaling pathways in neural repair following cerebral infarction has garnered significant attention in the academic community. Studies have shown that certain fatty acids exert neuroprotective and reparative functions by binding to highly specific receptors within different lipid families 23 .Research indicates that fatty acid-binding proteins (FABPs) exacerbate cerebral ischemic injury by accelerating matrix metalloproteinase-9 (MMP-9)-mediated blood-brain barrier (BBB) disruption. Therefore, inhibiting FABPs is considered a potential strategy to improve outcomes in ischemic stroke (IS) 24 . FABPs contribute to ischemic neuronal damage by mediating oxidative stress in neural cells, thereby affecting mitochondrial function 25 . Related studies have demonstrated that five subtypes of FABPs—FABP3, FABP5, FABP7, FABP8, and FABP12—are widely distributed in the cerebral cortex, hippocampal neuronal layers, and retinal interneurons 26, 27 . In ischemic stroke research, FABP3 and FABP5 have been identified as key factors contributing to mitochondrial damage in brain neurons.In this experiment, analysis of fatty acid-related pathway proteins in the brain tissue of MCAO rats revealed that FABP4 and FABP5 are significantly associated with neuronal repair and apoptosis following ischemic stroke. However, after RSV administration, only FABP5 levels showed significant changes, while FABP4 levels remained unaltered. Molecular docking experiments demonstrated that RSV has a strong binding affinity for FABP5, suggesting that RSV may regulate fatty acid metabolism by directly influencing the binding or transport of fatty acids with FABP5.In MCAO rat models with FABP5 overexpression and inhibition, we observed that FABP5 overexpression significantly exacerbated neurological functional impairment, while FABP5 inhibition markedly alleviated such damage. These results confirm that FABP5 can induce central nervous system functional injury. The central nervous system (CNS) is a complex network composed of various cell types. In the human body, lipids rank second only to adipose tissue in terms of mass proportion within the CNS. Lipids primarily serve as energy reserves and utilization sources in lipid droplets, playing a crucial role in neurological diseases such as ischemic stroke 28, 29 . When neuronal cells experience abnormal energy metabolism due to ischemia and hypoxia, this is considered a major cause of neuronal apoptosis .Fatty acid metabolism provides ATP energy to cells through β-oxidation, while also serving as a component of phospholipids, structural units of membranes, and a substrate reservoir for the synthesis of second messengers and cytokines. Additionally, fatty acids can induce changes in cell morphology, regulate gene expression, modulate hormone secretion, and mediate various cellular responses 30, 31 .AMP-activated protein kinase (AMPK) can sense cellular energy status and regulate energy metabolism pathways through AMP activation 32 . Fatty acid-binding proteins (FABPs) influence cellular energy supply by transporting fatty acids and mediate downstream lipid oxidative stress responses, thereby modulating neuronal cell function 33 . By monitoring FABPs, which are markers of fatty acid metabolism, the energy metabolic state of corresponding organ tissues and cells can be assessed, allowing for the inference of underlying molecular mechanisms. The natural polyphenol resveratrol (RSV) found in plants has been demonstrated to possess neuroprotective, cognitive-enhancing, and immunomodulatory effects 12, 13 . Additionally, RSV is a potent activator of AMP-activated protein kinase (AMPK) 34 . In vitro and in vivo studies have shown that RSV can improve brain tissue energy metabolism and reduce energy loss by modulating the AMPK signaling pathway 35, 36 . In ischemic stroke (IS) rat models, RSV exerts neuroprotective effects by inhibiting phosphodiesterase (PDE) and regulating the cAMP/AMPK/SIRT1 pathway, thereby reducing ATP energy consumption during ischemia and significantly mitigating the detrimental effects of cerebral ischemic injury 37 .In this experiment, after administering RSV to MCAO rats, improvements in open field test performance and neurobehavioral scores were observed compared to the model group. Furthermore, RSV significantly alleviated cerebral ischemia and neuronal apoptosis in MCAO rats. These results suggest that early RSV intervention in IS is a promising clinical treatment strategy that can effectively prevent neuronal damage.In MCAO rat models with FABP5 overexpression and inhibition, we found that AMPK expression slightly decreased in the FABP5 inhibition group, while it significantly decreased in the FABP5 overexpression group. This indicates that FABP5 exerts an inhibitory effect on energy metabolism in central nervous system cells. After RSV intervention, the neurological function and injury in the FABP5 inhibition and overexpression groups exhibited significant polarization. This suggests that RSV improves brain neurological function in MCAO rats by inhibiting FABP5 and regulating brain energy metabolism pathways. Summary This study demonstrates that RSV exerts neuroprotective and reparative effects in ischemic stroke (IS), with the improvement in neurological function significantly enhanced as the concentration of RSV increases. RSV ameliorates neuronal damage caused by ischemia and hypoxia by inhibiting FABP5 levels in fatty acid metabolism and regulating brain energy metabolism pathways, thereby promoting the recovery of neurological function in IS.However, this study has certain limitations. For instance, whether RSV, through its mediation of FABP5, also regulates other cell types (such as glial cells and vascular endothelial cells) as well as mitochondrial metabolism and antioxidant stress responses remains to be further explored in future research. Conflict of interest The authors have no competing interests to declare that are relevant to the content of this article. Funding This study has been approved by the Guangdong Medical Science and Technology Research Fund, with project number: C2024079. Authors’ contributions Bingfeng Xing and Min Hong designed the study. Zhou xin and WeiHao Lin completed the follow up work and collected study data. Changqin Xiang analyzed the data. Bingfeng Xing and Min Hong wrote the manuscript. Min Hong reviewed the manuscript. The manuscript has been read and approved by all co‑authors. The author(s) read and approved the final manuscript. Acknowledgments We thank for the support of the staffs and all the participants enrolled in the study from the department of acupuncture, The First Affiliated Hospital, Sun Yat-sen University, the department of traditional Chinese medicine of the First Affiliated Hospital/The First Clinical Medicine School of Guangdong Pharmaceutical University, Guangzhou, Guangdong, China. Reference 1.Virani SS, Alonso A, Benjamin EJ, et al. Heart disease and stroke statistics-2020 update: A report from the American Heart Association. Circulation. 2020;141:e139-e596 . doi:10.1161/CIR.0000000000000757. 2.Campbell BCV, De Silva DA, Macleod MR, et al. Ischaemic stroke. Nat Rev Dis Primers. 2019;5:70. doi:10.1038/s41572-019-0118-8. 3.Storch J, Corsico B. 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The MEbrown module, with a negative correlation coefficient of 0.84 (p = 0.001), was selected as the module of interest, with a focus on the MCAO group. B. Gene Distribution and Gene Significance in the Module of Interest (Module Membership and Gene Significance)Genes from the MEbrown module in the MCAO group were extracted. Using the criteria of GS > 0.6 and MM > 0.6, genes highly correlated with the phenotype were screened. These genes, which are also key genes associated with the phenotype, were visualized. A total of 56 genes were significantly associated with the ”MCAO” phenotype.C. Construction of PPI Network and Identification of the FABP5 Core Network A protein-protein interaction (PPI) network was constructed using the 56 genes, and the core network centered on FABP5 was identified.D. Pathway Enrichment Analysis Reveals FABP5’s Association with Metabolism Pathway enrichment analysis demonstrated that FABP5 is primarily involved in metabolic pathways.E. Semi-Flexible Molecular Docking of Resveratrol with FABP5 Semi-flexible molecular docking analysis revealed binding sites between resveratrol and FABP5. Fig. 2 Assessment of Motor Function in Rats.A. Open Field Test Trajectory Diagrams. The yellow central point represents the starting position, and the yellow lines indicate movement trajectories. A1, A2, A3, A4, and A5 correspond to the blank control group, MCAO control group, RSV5 group, RSV10 group, and RSV20 group, respectively.B. Statistical Analysis of Immobility Time in the Open Field Test (Unit: s).C. Statistical Analysis of Total Distance Traveled in the Open Field Test (Unit: mm).D. Longa Scores for Each Group of Rats. Fig. 3 A & B. Analysis and Statistical Results of TTC Staining for Cerebral Infarction Area in Each Group.In panel A, red indicates surviving neurons, while white indicates neuronal death.Panel B shows the statistical analysis of cerebral infarction areas in each group.C. HE Staining of Brain Tissues in Each Group.In the sham operation group, neurons exhibited intact structures, orderly arrangement, abundant neurotransmitters in the cytoplasm, and normal synaptic gaps.In the model group, neuronal nuclei were shrunken, cytoplasmic contents were reduced, and cell alignment was disorganized. Fig. 4 A. Western Blot Analysis of BDNF, NSE, and FABP5 Protein Expression in Brain Tissues of Each Group.B. Immunofluorescence Detection of FABP5 in Brain Tissues of Each Group. Green fluorescence indicates FABP5 protein expression.C-E. Western Blot and Relative Quantification of NSE, FABP5, and BDNF Expression in Each Group.F-H. Statistical Analysis of mRNA Levels of NSE, FABP5, and BDNF in Each Group. Fig. 5 A. Schematic Diagrams of Overexpression and Knockdown Plasmids.B. Transfection Status of Each Plasmid in 293T Cells.C. Validation of Plasmid Sizes by qPCR. Fig. 6 A. TTC Staining of Brain Tissues in Each Group. Overexpression of FABP5 increased brain tissue damage, while knockdown reversed this trend.B. Early and Late Apoptosis Rates of Brain Cells in Each Group.C. Statistical Analysis of Early and Late Apoptosis Rates of Brain Cells. Fig. 7 A. Immunofluorescence Staining of FABP5 in Brain Tissues of the Three Groups. Green fluorescence indicates FABP5 expression, and blue fluorescence indicates DAPI staining.B. Immunofluorescence Staining of AMPK in Brain Tissues of the Three Groups. Green fluorescence indicates AMPK expression, and blue fluorescence indicates DAPI staining.C. Western Blot Bands of Brain Tissues in the Three Groups.D & E. RT-qPCR Results Showing FABP5 and AMPK Expression in Brain Tissues of the Three Groups. Supplementary Material File (fig6.tif) Download 3.77 MB Information & Authors Information Version history V1 Version 1 05 April 2025 Peer review timeline Published Open Life Sciences Version of Record 20 Jan 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords epilepsy herbal medicine neurology other stroke Authors Affiliations Bingfeng Xing The First Affiliated Hospital of Guangdong Pharmaceutical University View all articles by this author Min Hong 0009-0007-0048-5748 [email protected] View all articles by this author Xin Zhou The First Affiliated Hospital of Sun Yat-sen University View all articles by this author Weihao Lin The First Affiliated Hospital of Guangdong Pharmaceutical University View all articles by this author Changqin Xiang The First Affiliated Hospital of Guangdong Pharmaceutical University View all articles by this author Metrics & Citations Metrics Article Usage 342 views 154 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Bingfeng Xing, Min Hong, Xin Zhou, et al. Resveratrol Modulates FABP5 to Reduce Neuronal Apoptosis Following Ischemic Stroke. Authorea . 05 April 2025. 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