Oligo-peptide Pena-4 activates SIRT1 and exerts neuroprotective benefits against cerebral ischemia-reperfusion injury in mice

Oligo-peptide Pena-4 activates SIRT1 and exerts neuroprotective benefits against cerebral ischemia-reperfusion injury in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Oligo-peptide Pena-4 activates SIRT1 and exerts neuroprotective benefits against cerebral ischemia-reperfusion injury in mice Guoling Yang, Zhiqi Hou, JinJing Zhao, Anna Zhang, Yuchang Shen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8774215/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose In ischemic stroke, the therapeutic options for mitigating ischemia/reperfusion (I/R) impairments in brain remain critically limited, underscoring an urgent need for novel neuroprotective agents. This study investigated the protective effects and underlying mechanisms of Pena-4, an oligopeptide originally identified from proteolytic digestion of Penaeus japonicus, in a model of transient middle cerebral artery occlusion/reperfusion (MCAO/R) . Methods Neurological function, infarct volume, and histopathological changes were evaluated after Pena-4 treatment. Network pharmacology and molecular docking were used to predict potential targets and pathways. Key proteins related to silent information regulator sirtuin 1 (SIRT1) signaling, oxidative stress, and pyroptosis were analyzed by western blotting, and malondialdehyde (MDA) levels were measured. Results Pena-4 notably improved neurological performance and decreased infarct volume. Histological analysis showed attenuated neuronal damage in the cortex and hippocampus. Network pharmacology approach identified 55 overlapping targets and highlighted silent information regulator sirtuin 1 (SIRT1) as a key hub target, which showed stable binding with Pena-4 in docking analysis. Pena-4 restored I/R-induced SIRT1 downregulation, increased SOD2 levels, reduced MDA content, and suppressed NLRP3-dependent pyroptosis, as evidenced by decreased NLRP3, cleaved caspase-1, and cleaved GSDMD. Conclusion Pena-4 thus confers neuroprotection against I/R impairments in brain through mechanisms that involve SIRT1 activation, enhanced antioxidant defense, and inhibition of NLRP3-dependent pyroptosis. SIRT1 antioxidant defense pyroptosis ischemic stroke network pharmacology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. INTRODUCTION Ischemic stroke (IS) constitutes a significant global public health challenge, ranking among the foremost causes of death and persistent disability worldwide [ 1 , 2 ]. A critical contributor to ischemic brain damage is cerebral I/R injury, a pathological process that paradoxically exacerbates neuronal loss despite the restoration of blood flow [ 3 ]. The complex pathophysiology of I/R injury in brain involves a cascade of molecular events, including pronounced oxidative damage, excitotoxicity, inflammatory activation, and various forms of cell death, which collectively drive the expansion of infarcted tissue [ 4 ]. Despite advances in acute revascularization therapies, effective neuroprotective strategies to mitigate this secondary injury remain scarce, highlighting a significant unmet clinical need [ 5 ]. Pena-4 is an oligopeptide (amino acid sequence: Phe-Ile-Lys-Lys, FIKK; Fig. 1 A) originally identified in proteolytic digests of Penaeus japonicus and Salmo salar L. [ 6 , 7 ], and has previously been reported to exhibit potent antioxidant activity. During preliminary screening of a series of bioactive peptides derived from these sources, Pena-4 emerged as the most promising candidate, demonstrating a superior capacity to reduce infarct volume in mice subjected to MCAO/R (unpublished data). Based on these findings, our team utilized network pharmacology combined with molecular docking to elucidate its mechanism of action, which identified Sirtuin 1 (SIRT1), as a key hub target in the current work. As an NAD⁺-dependent deacetylase, SIRT1 is believed to offer neuroprotection in IS [ 8 – 10 ] via multiple mechanisms. Accordingly, current work was conducted to comprehensively assess the neuroprotective benefits of Pena-4 against I/R injury in brain and investigate whether its mechanisms involve the modulation of SIRT1-related pathways. 2. Methods and materials 2.1. Mice and reagents C57BL/6J mice (at 7–8 weeks postnatal age, male) were sourced from Sipeifu Biotechnology (Suzhou, Jiangsu, China). In accordance with institutional guidelines made by the National Institutes of Health, standard husbandry conditions were applied: temperature 22–25°C, humidity 50 ± 5%, 12-h light/dark cycle, and unrestricted availability of food and water. Pena-4 (sequence ID: FIKK, Shanghai Top-peptide, China) was synthesized with a purity of 98.37%. Antibodies included anti-Caspase-1 antibody (Abcam, Cambridge, UK; ab138483), anti-GSDMD antibody (HUABIO, Hangzhou, China; HA721144), anti-SIRT1 antibody (Abcam, ab189494) and anti-NLRP3 antibody (HUABIO, HA750236), anti-SOD2 antibody (Aladdin, Shanghai, China; Ab169025), and β-Actin antibody (Servicebio, Wuhan, China; ZB15001). Lipid peroxidation levels were quantified using a malondialdehyde (MDA) assay kit (Cat. No. A003-1, Njjcbio, Nanjing, China). 2.2. MCAO/R As described previously [ 11 ], external carotid artery (ECA) was exposed and ligated under anesthesia, and a silicone-coated nylon filament (Beijing CinonTech Co., Ltd.; Cat. No. A5-122050) was introduced via the ECA stump. The filament was then advanced past the left common carotid artery (CCA) bifurcation approximately 10 ± 1 mm into the intracranial segment of the internal carotid artery for blocking the origin of the middle cerebral artery. Following a 2-hour occlusion period, the filament was carefully removed for restoring blood flow. The model's success defined as over 85% reduction during ischemia and over 80% flow recovery during reperfusion by using laser Doppler flowmetry. A thermostatically controlled heating pad was used throughout the procedure for keeping murine core body temperature stable within the range of 37.0 ± 0.5°C. In this experiment, a total of 17 mice were used in the sham-operated group, and 51 mice were assigned to the model group, among which 6 died during surgery and 3 failed to develop the model successfully. The overall surgical mortality rate in MCAO/R was 11.8% (6/51). Animals were assigned to four groups randomly: a sham-operated group (exposure of the left CCA only, without occlusion), an MCAO model group, and two treatment groups that received Pena-4 intravenously at dose of either 1 mg/kg or 5 mg/kg immediately after reperfusion (Fig. 1 A). Behavioral scoring, infarct quantification, and histological analyses were performed blinded 24 hrs after reperfusion. 2.3. Neurobehavioral assessment Neurological function was assessed 24 hrs after reperfusion using the Garcia scale system [ 12 , 13 ]. The Garcia scale is divided into 6 sections, with a total score of 18 points. The higher scores indicating less severe neurological impairment and a score of 18 representing normal function. 2.4. 2,3,5-triphenyltetrazolium chloride (TTC) staining Brain was cut into five slices [ 14 ], which were then immersed in a 1.5% TTC solution (Sangon Biotech, Cat. No. A610558) and incubated for half an hour at 37°C, with gentle flipping at 5-minute intervals for uniform staining. ImageJ software (NIH, USA) was used to quantify the infarct volume. 2.5. Histopathological Evaluation Histopathological assessment was performed using hematoxylin and eosin (H&E) and Nissl labeling for evaluating neuronal morphology and integrity in both hippocampus and cortex [ 15 , 16 ]. Hematoxylin and eosin (H&E)–stained sections were scored according to a previously established grading system[ 17 ]. Nissl-stained sections were imaged using a light microscope under identical magnification and acquisition parameters. Quantitative analysis was performed using ImageJ software (National Institutes of Health, USA). 2.6. Prediction of potential targets for Pena-4 The two-dimensional chemical structure of Pena-4 was sketched using ChemDraw 19.0, and the PDB file of the compound was obtained. The SMILES notation of the molecule was acquired through the NovoPro Bio-Tools online platform ( https://www.novopro.cn/tools/mol2smiles.html ) for molecular format conversion. Potential targets of Pena-4 were predicted [ 18 ] using the SwissTargetPrediction platform ( http://www.swisstargetprediction.ch/ ). Using the search terms "ischemic stroke (IS)" and "cerebral ischemia," candidate genes were retrieved from four databases, including Therapeutic Target Database (TTD; https://db.idrblab.net/ttd/ ), GeneCards ( https://www.genecards.org/ ), OMIM ( https://omim.org/ ), and PharmGKB ( https://www.pharmgkb.org/ ). The intersection of these genes was defined as the disease targets for IS. Finally, we uploaded the targets associated with Pena-4 and those linked to IS into Venny 2.1.0 platform ( https://bioinfogp.cnb.csic.es/tools/venny/ ) for identifying shared targets. 2.7. Interaction and Pathway analysis We generated a protein–protein interaction (PPI) network based on the overlapping targets using the STRING database (v12.0, species: Homo sapiens), with isolated nodes excluded to retain a connected network. Then, we imported the resulting network into Cytoscape (v3.8.0) for visualization and further analysis [ 19 ], where core genes (hub nodes) were identified using the Centiscape (v2.2) plugin based on topological centrality measures. Our team performed GO and KEGG pathway enrichment analyses on the putative Pena-4 targets relevant to IS on the online Metascape platform ( https://metascape.org/gp/index.html#/main/step1 ), in which statistical significance was set at p < 0.01 [ 20 ]. 2.8. Molecular docking Briefly, the three-dimensional SIRT1 (PDB: 4kxq) structure was preprocessed by eliminating water molecules and co-crystallized ligands, followed by the addition of hydrogen atoms and charge assignment using AutoDock Tools 1.5.6 [ 21 ]. The three-dimensional Pena-4 structure was energy-minimized, and its rotatable bonds were defined. Docking grid boxes were centered on the known active site of each protein. The conformation with the most favorable (lowest) binding free energy was selected for subsequent visualization and analysis using PyMOL [ 22 ]. 2.9. Western blot We extracted proteins from the peri-infarct brain tissue using 1× RIPA buffer, which were then subjected to SDS-PAGE separation and blotted onto PVDF membranes. After a 2-hour blockade at room temperature with non-fat milk (5%), membranes were probed using antibodies as described above. We used ECL for detecting the protein bands and finally quantified them with ImageJ, with levels normalized to β-actin [ 23 ]. 2.10. Statistical analysis When normality or equal variance assumptions were violated, Kruskal–Wallis test followed by Dunn’s post hoc analysis was applied. Otherwise, one-way ANOVA with Tukey’s correction was used. Data are presented as mean ± SD. 3. Results 3.1. Pena-4 attenuates neurological dysfunction and reduces infarct volume At 24 hours post-reperfusion, mice subjected to MCAO/R exhibited significant neurological deficits, accompanied by markedly lower neurological scores relative to the sham-operated group (Fig. 1 B). Consistent with these behavioral impairments, TTC staining revealed the extensive white areas in the MCAO/R mice (Fig. 1 C and D), confirming successful model establishment. Administration of Pena-4 at dose of 5 mg/kg significantly improved neurological performance and reduced infarct volume. To further assess the benefits of Pena-4 on brain tissue integrity following I/R, histopathological changes were examined via H&E staining (Fig. 2 ). Brain sections from the MCAO/R mice showed severe pathological alterations compared with the sham group, including neuronal shrinkage, cell loss, disrupted cortical lamination, and prominent nuclear pyknosis. Comparable damage was observed across hippocampal subfields—especially in CA1, CA3, and the dentate gyrus (DG)—where neurons appeared sparse and morphologically disorganized. In contrast, Pena-4 markedly attenuated these histopathological abnormalities. Cortical and hippocampal neurons exhibited improved structural integrity, reduced degeneration, and better-preserved cellular density. To further investigate the neuroprotective effect of Pena-4 on neuronal survival post-ischemia, Nissl staining was also performed (Fig. 3 ). In sham-operated animals, neurons exhibited normal morphology, abundant Nissl substance, and orderly arrangement. By contrast, the MCAO/R group showed extensive neuronal injury, characterized by a pronounced reduction in Nissl-positive cells, diminished staining intensity, cytoplasmic vacuolization, and widespread neuronal loss. Pena-4 markedly attenuated these pathological alterations, as indicated by the preservation of Nissl body structure and a higher density of neurons in both the hippocampus and cortex. 3.2. Mapping shared targets and building the PPI network for Pena-4 in ischemic stroke To explore the molecular targets of Pena-4, we obtained 100 drug-related targets from the SwissTargetPrediction database. We simultaneously gathered IS-related targets from 4 databases (TTD, PharmGKB, OMIM, and GeneCards) and removed duplicates to obtain a final set of 4,680 unique targets (Fig. 4 A). Intersection analysis identified 55 overlapping genes, suggesting their potential therapeutic relevance. These shared targets were utilized to build a PPI network through the STRING database (confidence score: 0.4). After excluding two disconnected nodes, a final network of 53 interacting proteins was obtained (Fig. 4 B). The dense interconnectivity reflects a high degree of functional association and underscores the multi-target nature of Pena-4 in ischemic stroke treatment. Hub gene analysis was performed using CytoScape plugin Centiscape 2.2, which ranked nodes by topological metrics (Fig. 4 C). Nodes with darker red shading represent higher centrality values, indicating that Pena-4’s pharmacological actions may depend significantly on these hub targets. Enrichment analysis of the 53 overlapping targets was conducted, revealing 61 significantly enriched KEGG pathways and 1309 GO terms. GO analysis included 1092 biological process (BP) entries—such as histone H3 deacetylation, collagen catabolic process, regulation of smooth muscle contraction, collagen metabolic process, and protein processing; 116 cellular component (CC) entries-including membrane microdomain, membrane raft, ficolin-1-rich granule lumen, histone deacetylase complex, and Golgi apparatus subcompartment. Among the enriched molecular functions, terms encompassing NAD-dependent deacetylase activity (Fig. 5 A) converged on the functional profile of Sirtuin proteins. This computational insight led us to prioritize SIRT1, a central regulator with corresponding deacetylase functions, for experimental validation of Pena-4's mechanism of action. KEGG pathway analysis identified 61 significant pathways, such as the renin-angiotensin system, neuroactive ligand-receptor interaction, bladder cancer, neuroactive ligand-receptor signaling, and apoptosis (Fig. 5 B). 3.3. Molecular docking predicted a stable binding interaction between Pena-4 and SIRT1 To validate the potential regulation of SIRT1 by Pena-4, molecular docking was done to evaluate the Pena-4–SIRT1 interaction. Conventional docking criteria define absolute scores > 4.25 as moderate, > 5.0 as good, and > 7.0 as strong binding affinity. The results, visualized using PyMOL (Fig. 6 A), indicated a favorable binding mode. Pena-4 was predicted to occupy the active site of SIRT1 via multiple hydrogen bonds and hydrophobic interactions, suggesting direct modulation of SIRT1 activity. To assess biological relevance, SIRT1 protein expression was measured in peri-infarct brain tissue by Western blot (Fig. 6 B). Relative to the sham mice, SIRT1 expression was markedly lowered in the MCAO/R group and Pena-4 administration notably reversed this downregulation, restoring SIRT1 levels. 3.4. Pena-4 attenuates oxidative stress by restoring SOD2 expression and reducing lipid peroxidation in injured brain To determine whether Pena-4 modulates oxidative stress, we assessed SOD2 expression and lipid peroxidation in peri-infarct tissue. Western blot analysis (Fig. 7 A) revealed a marked decrease in SOD2 levels following MCAO/R compared with sham, indicating impaired antioxidant defense. Pena-4 treatment significantly reversed this decline. Correspondingly, MDA, a key indicator of lipid peroxidation, significantly accumulated in the peri-infarct region (Fig. 7 B), which was partly reversed by Pena-4 administration. 3.5. Pena-4 inhibits pyroptosis in injured brains Western blot was conducted to examine the effect of Pena-4 on NLRP3 inflammasome activation and pyroptosis-related proteins in peri-infarct brain tissue (Fig. 8 A). I/R injury markedly increased proteins expression of NLRP3, Clv-GSDMD and Clv-Caspase-1. Pena-4 administration reduced NLRP3 (Fig. 8 B), Clv-GSDMD (Fig. 8 C), and Clv-Caspase-1 (Fig. 8 D) levels in MCAO/R mice significantly. 4. DISCUSSION These findings collectively revealed that Pena-4 exerted robust neuroprotective benefits in mice exposed to cerebral I/R injury. A key highlight of current work is the immediate administration of Pena-4 upon reperfusion, reflecting a realistic treatment scenario for most stroke patients who receive care after ischemia onset and blood flow restoration. This approach is clinically relevant, unlike many neuroprotective agents that failed in trials due to impractical pre- or peri-ischemic dosing requirements [ 24 , 25 ]. This robust post-ischemic efficacy underscores the translational promise of Pena-4 as a viable adjunctive therapy that could extend the treatment window following recanalization. Mechanistically, the robust neuroprotective effects observed when Pena-4 is administered at the point of reperfusion suggests that Pena-4 treatment actively engages key pathological processes activated during reperfusion. During cerebral I/R, the electron transport chain impairment results in overproduction of reactive oxygen species [ 26 ], a pivotal factor in the progression of reperfusion injury [ 27 ]. Sirt1 protein enhanced cellular tolerance to oxidative damage by modulating various genes and their associated signaling pathways [ 28 ]. Within the Sirt1 signaling network, SOD2 holds a central position as a key antioxidant protein [ 29 ]. Sirt1 promotes the transcriptional activation and functional enhancement of SOD2 [ 30 ], thereby bolstering mitochondrial antioxidant capacity and maintaining redox homeostasis. In our work, Pena-4 upregulated Sirt1 and SOD2 protein expression and reduced MDA formation in injured brains. Therefore, Pena-4 might mitigate oxidative damages during cerebral I/R through its intrinsic antioxidant activity and its modulation of Sirt1/SOD2 pathway. A growing body of reports underscored the involvement of multiple regulated cell death pathways during I/R impairments in brain [ 31 ]. Among these, pyroptosis, driven by caspase-1 activation via inflammasomes like NLRP3 [ 32 ], is an inflammatory type of programmed cell death. Highly expressed in the brain, NLRP3 detects cell damage and triggers inflammation, leading to cell death, and is crucial in I/R impairments [ 33 , 34 ]. Emerging evidence has underscored the involvement of SIRT1 in modulating pyroptotic cell death. Sirt1 inhibited NLRP3 inflammasome against cardiac I/R injury [ 35 ]. Sirt1 knockdown in HK-2 cells aggravate LPS-activated NLRP3-mediated pyroptosis [ 36 ] and Sirt1 knockout led to NLRP3 inflammasome activation in podocytes [ 37 ]. In our study, Pena-4 decreased NLRP3 expression and inhibited cleaved GSDMD and Caspase-1 formation in injured brains. Its ability to suppress pyroptosis in injured brain may stem from modulating the Sirt1/NLRP3 pathway. A major limitation of our investigation is the lack of mechanistic validation for SIRT1's role. While our data from network pharmacology, molecular docking, and Western blot strongly suggest an interaction between Pena-4 and SIRT1, they do not provide definitive evidence that SIRT1 activation is directly responsible for the observed suppression of oxidative stress and pyroptosis. Future studies employing SIRT1-knockdown or knockout models are essential to confirm this causal link and rule out other potential Sirt1-independent pathways. Collectively, our findings suggest that Pena-4 holds promise as a neuroprotective agent in brain I/R injury, potentially through its interaction with Sirt1 and subsequent attenuation of oxidative stress and NLRP3-driven pyroptosis. Declarations Competing Interests The authors declare no competing interests. Data Availability The raw data could be obtained upon reasonable request to the corresponding author. Ethics approval The animal-related experimental interventions received approval from the Shanghai University Ethics Committee (Approval No. ECSHU2024-006). Author contributions Conceptualization: Guoling Yang, Zhiqi Hou, Yuefan Zhang; Formal analysis and investigation: Guoling Yang, Zhiqi Hou, JinJing Zhao,Anna Zhang, Yuchang Shen, Yihan Lin, Keying Chen; Writing - original draft preparation: Guoling Yang, Yongsheng Yu, Yuefan Zhang; Writing - review and editing: Guoling Yang, Yuefan Zhang; Supervision: Yongsheng Yu, Yuefan Zhang. 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World J Pediatr Surg 6(4):e000602. https://doi.org/10.1136/wjps-2023-000602 Jiang M, Zhao M, Bai M, Lei J, Yuan Y, Huang S, Zhang Y, Ding G, Jia Z, Zhang A (2021) SIRT1 alleviates aldosterone-induced podocyte injury by suppressing mitochondrial dysfunction and NLRP3 inflammasome activation. Kidney Dis (Basel) 7(4):293–305. https://doi.org/10.1159/000513884 Additional Declarations No competing interests reported. Supplementary Files thefulluncroppedGelsandBlotsimages.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8774215","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":589570429,"identity":"ede02c7c-ff07-45e5-93aa-ca59bb50e550","order_by":0,"name":"Guoling Yang","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Guoling","middleName":"","lastName":"Yang","suffix":""},{"id":589570430,"identity":"6537e984-ee26-4757-89ee-8df4fbc41bae","order_by":1,"name":"Zhiqi Hou","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Zhiqi","middleName":"","lastName":"Hou","suffix":""},{"id":589570431,"identity":"64d37908-2723-414f-b71f-6e7a765db9b4","order_by":2,"name":"JinJing Zhao","email":"","orcid":"","institution":"The 305 Hospital of the People's Liberation Army","correspondingAuthor":false,"prefix":"","firstName":"JinJing","middleName":"","lastName":"Zhao","suffix":""},{"id":589570432,"identity":"1fb49c87-1b49-429e-ba15-16525f9a65ec","order_by":3,"name":"Anna Zhang","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Zhang","suffix":""},{"id":589570433,"identity":"dbff5582-b1a8-477d-aa71-8dfe6fa8a04b","order_by":4,"name":"Yuchang Shen","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Yuchang","middleName":"","lastName":"Shen","suffix":""},{"id":589570434,"identity":"719a947f-b6b8-450b-bdc0-035d852223c8","order_by":5,"name":"Yihan Lin","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Yihan","middleName":"","lastName":"Lin","suffix":""},{"id":589570435,"identity":"91d680c9-576e-41a4-b008-fc43797060b4","order_by":6,"name":"Keying Chen","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Keying","middleName":"","lastName":"Chen","suffix":""},{"id":589570436,"identity":"cfc3f1ec-e155-4d55-8089-b1770114ac05","order_by":7,"name":"Yongsheng Yu","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Yongsheng","middleName":"","lastName":"Yu","suffix":""},{"id":589570437,"identity":"9fd935f5-2574-4cf1-85a8-a625a4cd9138","order_by":8,"name":"Yuefan Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYJCCD0CcAMSMDxgbGKBs/IBxBlQZswHJWtgkiNJicCP5YMPPHXV5/LPbr1X+3HGYgZ89x4Dh5w58WtISG3vPsBVL3DlTdpv3zGEGyZ43Boy9Z/BpyTF/wNvGk9hwIyftNmPbYZCIATNjGz4t+R8b/7ZJJM4Hain8CdRiT1hLDmMzb5tB4oYb6ccYeEG2SBDQInnmmWGzbFtC4sYbOczSvG3pPBJnnhUc7MWjhe948sPGt211ifNupD/8+LPNWo6/PXnjg594tCgcgDN5DMAkiDiAVS0UyDfAmewP8CkcBaNgFIyCEQwAG5VbUTuYALUAAAAASUVORK5CYII=","orcid":"","institution":"Shanghai University","correspondingAuthor":true,"prefix":"","firstName":"Yuefan","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2026-02-03 10:10:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8774215/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8774215/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102963172,"identity":"a3db18a9-c516-44fc-b9ce-f9280be05153","added_by":"auto","created_at":"2026-02-19 04:14:07","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":116402,"visible":true,"origin":"","legend":"\u003cp\u003ePena-4 ameliorates neurological dysfunction and decreases cerebral infarct volume.\u003c/p\u003e\n\u003cp\u003e(A) Overview of the experimental workflow, (B) Neurological scores, (C) TTC-staining representative images, (D) and quantitative results of infarct volume. n=5, *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8774215/v1/5cf80e2c8f800d7d9f43989e.jpeg"},{"id":102963014,"identity":"7432776d-6b6d-4c59-8fdf-14a8a222bb74","added_by":"auto","created_at":"2026-02-19 04:12:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":815244,"visible":true,"origin":"","legend":"\u003cp\u003ePena-4 alleviates histopathological impairment in both cortex and hippocampus of injured brain.\u003c/p\u003e\n\u003cp\u003eRepresentative H\u0026amp;E-stained images (A) and scores of cortex (B), CA1 (C), CA3 (D), and DG (E) were shown. n=3, *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8774215/v1/d57e0365564cb74dbc131c3c.png"},{"id":102827822,"identity":"91d7d32f-4238-414b-a0c0-64d15459b338","added_by":"auto","created_at":"2026-02-17 09:13:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":707156,"visible":true,"origin":"","legend":"\u003cp\u003ePena-4 preserves neuronal integrity in both hippocampus and cortex of injured brain.\u003c/p\u003e\n\u003cp\u003eRepresentative Nissl-stained images (A) and quantification of Nissl-positive neurons in the cortex (B), CA1 (C), CA3 (D), and DG (E) were shown. n=3, *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8774215/v1/4941783c3d5538df9f583a76.png"},{"id":102827826,"identity":"2a12e5b8-03d9-4324-8e85-6ab94156dffb","added_by":"auto","created_at":"2026-02-17 09:13:54","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":160317,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of overlapping targets and PPI network construction for Pena-4 against IS.\u003c/p\u003e\n\u003cp\u003e(A) Venn diagram illustrating 55 overlapping genes between Pena-4 targets and IS-related targets. (B) PPI network of the overlapping targets generated from the STRING database (confidence score = 0.4). Two disconnected nodes were omitted, resulting in 53 interacting proteins. Nodes represent proteins; edges indicate functional associations. (C) Hub gene analysis performed using CentiScape 2.2. Node color intensity corresponds to topological centrality, with darker red indicating higher importance.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8774215/v1/82101413be47ba2487f7e67d.jpeg"},{"id":102963319,"identity":"a88a4e8b-67e2-4c27-9a97-ccf7833964ce","added_by":"auto","created_at":"2026-02-19 04:15:25","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":191108,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional enrichment analysis of overlapping targets of Pena-4 in ischemic stroke.\u003c/p\u003e\n\u003cp\u003e(A) Leading GO Enrichment Results; (B) Leading KEGG pathways based on enrichment significance. The number of genes is visualized via bubble size, and the adjusted \u003cem\u003ep\u003c/em\u003e-value is mapped to the color scale.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8774215/v1/6b61ef1f4b78ac624fd51116.jpeg"},{"id":102962546,"identity":"030677c8-b2fa-40f5-ad6e-4d5c8e26bcb8","added_by":"auto","created_at":"2026-02-19 04:09:46","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":156277,"visible":true,"origin":"","legend":"\u003cp\u003ePena-4 directly interacts with SIRT1 and restores its expression in injured brain.\u003c/p\u003e\n\u003cp\u003e(A) Predicted binding conformation of Pena-4 within the SIRT1 active site, as shown by molecular docking. (B) SIRT1 expression in peri-infarct region was assayed and quantified by Western blot. *p \u0026lt; 0.05. n=3.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8774215/v1/725c993223c807464a7e26df.jpeg"},{"id":102963022,"identity":"600cd713-2857-4b23-b279-8a53158c35e8","added_by":"auto","created_at":"2026-02-19 04:12:53","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":87786,"visible":true,"origin":"","legend":"\u003cp\u003ePena-4 mitigates oxidative stress by upregulating SOD2 and reducing lipid peroxidation in injured brain.\u003c/p\u003e\n\u003cp\u003e(A) Western blots of SOD2 expression in peri-infarct brain tissue were conducted, n=3. (B) MDA levels in peri-infarct brain tissue were assayed, n=6. *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8774215/v1/133cb554e73a5fd9d983afc5.jpeg"},{"id":102827830,"identity":"0801ea98-7707-4984-af77-7efc019e4f5d","added_by":"auto","created_at":"2026-02-17 09:13:54","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":115640,"visible":true,"origin":"","legend":"\u003cp\u003ePena-4 suppresses pyroptosis in injured brain.\u003c/p\u003e\n\u003cp\u003e(A) Images of western blots targeting NLRP3, full-length GSDMD (FL-GSDMD), cleaved GSDMD (Clv-GSDMD), pro-Caspase-1, and cleaved Caspase-1 (Clv-Caspase-1) in peri-infarct region. Quantitative analysis of (B) NLRP3, (C) Clv-GSDMD, and (D) Clv-Caspase-1 protein levels. n=3, *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8774215/v1/f4540e7fec6c3f14b0b15f31.jpeg"},{"id":107139668,"identity":"61adb530-c3df-497f-a55b-84cd35ccbb34","added_by":"auto","created_at":"2026-04-17 08:44:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2597700,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8774215/v1/604b7908-f468-429d-9a03-49574ce4d460.pdf"},{"id":102827824,"identity":"aa7f5650-a6de-4095-acbf-874384825a2a","added_by":"auto","created_at":"2026-02-17 09:13:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":437531,"visible":true,"origin":"","legend":"","description":"","filename":"thefulluncroppedGelsandBlotsimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8774215/v1/bfe2148167762cd25198d180.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Oligo-peptide Pena-4 activates SIRT1 and exerts neuroprotective benefits against cerebral ischemia-reperfusion injury in mice","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eIschemic stroke (IS) constitutes a significant global public health challenge, ranking among the foremost causes of death and persistent disability worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. A critical contributor to ischemic brain damage is cerebral I/R injury, a pathological process that paradoxically exacerbates neuronal loss despite the restoration of blood flow [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The complex pathophysiology of I/R injury in brain involves a cascade of molecular events, including pronounced oxidative damage, excitotoxicity, inflammatory activation, and various forms of cell death, which collectively drive the expansion of infarcted tissue [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite advances in acute revascularization therapies, effective neuroprotective strategies to mitigate this secondary injury remain scarce, highlighting a significant unmet clinical need [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePena-4 is an oligopeptide (amino acid sequence: Phe-Ile-Lys-Lys, FIKK; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) originally identified in proteolytic digests of \u003cem\u003ePenaeus japonicus\u003c/em\u003e and \u003cem\u003eSalmo salar\u003c/em\u003e L. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and has previously been reported to exhibit potent antioxidant activity. During preliminary screening of a series of bioactive peptides derived from these sources, Pena-4 emerged as the most promising candidate, demonstrating a superior capacity to reduce infarct volume in mice subjected to MCAO/R (unpublished data). Based on these findings, our team utilized network pharmacology combined with molecular docking to elucidate its mechanism of action, which identified Sirtuin 1 (SIRT1), as a key hub target in the current work. As an NAD⁺-dependent deacetylase, SIRT1 is believed to offer neuroprotection in IS [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] via multiple mechanisms. Accordingly, current work was conducted to comprehensively assess the neuroprotective benefits of Pena-4 against I/R injury in brain and investigate whether its mechanisms involve the modulation of SIRT1-related pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Methods and materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Mice and reagents\u003c/h2\u003e \u003cp\u003eC57BL/6J mice (at 7\u0026ndash;8 weeks postnatal age, male) were sourced from Sipeifu Biotechnology (Suzhou, Jiangsu, China). In accordance with institutional guidelines made by the National Institutes of Health, standard husbandry conditions were applied: temperature 22\u0026ndash;25\u0026deg;C, humidity 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5%, 12-h light/dark cycle, and unrestricted availability of food and water.\u003c/p\u003e \u003cp\u003ePena-4 (sequence ID: FIKK, Shanghai Top-peptide, China) was synthesized with a purity of 98.37%. Antibodies included anti-Caspase-1 antibody (Abcam, Cambridge, UK; ab138483), anti-GSDMD antibody (HUABIO, Hangzhou, China; HA721144), anti-SIRT1 antibody (Abcam, ab189494) and anti-NLRP3 antibody (HUABIO, HA750236), anti-SOD2 antibody (Aladdin, Shanghai, China; Ab169025), and β-Actin antibody (Servicebio, Wuhan, China; ZB15001). Lipid peroxidation levels were quantified using a malondialdehyde (MDA) assay kit (Cat. No. A003-1, Njjcbio, Nanjing, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. MCAO/R\u003c/h2\u003e \u003cp\u003eAs described previously [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], external carotid artery (ECA) was exposed and ligated under anesthesia, and a silicone-coated nylon filament (Beijing CinonTech Co., Ltd.; Cat. No. A5-122050) was introduced via the ECA stump. The filament was then advanced past the left common carotid artery (CCA) bifurcation approximately 10\u0026thinsp;\u0026plusmn;\u0026thinsp;1 mm into the intracranial segment of the internal carotid artery for blocking the origin of the middle cerebral artery. Following a 2-hour occlusion period, the filament was carefully removed for restoring blood flow. The model's success defined as over 85% reduction during ischemia and over 80% flow recovery during reperfusion by using laser Doppler flowmetry. A thermostatically controlled heating pad was used throughout the procedure for keeping murine core body temperature stable within the range of 37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C. In this experiment, a total of 17 mice were used in the sham-operated group, and 51 mice were assigned to the model group, among which 6 died during surgery and 3 failed to develop the model successfully. The overall surgical mortality rate in MCAO/R was 11.8% (6/51).\u003c/p\u003e \u003cp\u003eAnimals were assigned to four groups randomly: a sham-operated group (exposure of the left CCA only, without occlusion), an MCAO model group, and two treatment groups that received Pena-4 intravenously at dose of either 1 mg/kg or 5 mg/kg immediately after reperfusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Behavioral scoring, infarct quantification, and histological analyses were performed blinded 24 hrs after reperfusion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Neurobehavioral assessment\u003c/h2\u003e \u003cp\u003eNeurological function was assessed 24 hrs after reperfusion using the Garcia scale system [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The Garcia scale is divided into 6 sections, with a total score of 18 points. The higher scores indicating less severe neurological impairment and a score of 18 representing normal function.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. 2,3,5-triphenyltetrazolium chloride (TTC) staining\u003c/h2\u003e \u003cp\u003eBrain was cut into five slices [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], which were then immersed in a 1.5% TTC solution (Sangon Biotech, Cat. No. A610558) and incubated for half an hour at 37\u0026deg;C, with gentle flipping at 5-minute intervals for uniform staining. ImageJ software (NIH, USA) was used to quantify the infarct volume.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Histopathological Evaluation\u003c/h2\u003e \u003cp\u003eHistopathological assessment was performed using hematoxylin and eosin (H\u0026amp;E) and Nissl labeling for evaluating neuronal morphology and integrity in both hippocampus and cortex [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Hematoxylin and eosin (H\u0026amp;E)\u0026ndash;stained sections were scored according to a previously established grading system[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Nissl-stained sections were imaged using a light microscope under identical magnification and acquisition parameters. Quantitative analysis was performed using ImageJ software (National Institutes of Health, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Prediction of potential targets for Pena-4\u003c/h2\u003e \u003cp\u003eThe two-dimensional chemical structure of Pena-4 was sketched using ChemDraw 19.0, and the PDB file of the compound was obtained. The SMILES notation of the molecule was acquired through the NovoPro Bio-Tools online platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.novopro.cn/tools/mol2smiles.html\u003c/span\u003e\u003cspan address=\"https://www.novopro.cn/tools/mol2smiles.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for molecular format conversion. Potential targets of Pena-4 were predicted [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] using the SwissTargetPrediction platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"http://www.swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUsing the search terms \"ischemic stroke (IS)\" and \"cerebral ischemia,\" candidate genes were retrieved from four databases, including Therapeutic Target Database (TTD; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://db.idrblab.net/ttd/\u003c/span\u003e\u003cspan address=\"https://db.idrblab.net/ttd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), GeneCards (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), OMIM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://omim.org/\u003c/span\u003e\u003cspan address=\"https://omim.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and PharmGKB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.pharmgkb.org/\u003c/span\u003e\u003cspan address=\"https://www.pharmgkb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The intersection of these genes was defined as the disease targets for IS. Finally, we uploaded the targets associated with Pena-4 and those linked to IS into Venny 2.1.0 platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinfogp.cnb.csic.es/tools/venny/\u003c/span\u003e\u003cspan address=\"https://bioinfogp.cnb.csic.es/tools/venny/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for identifying shared targets.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Interaction and Pathway analysis\u003c/h2\u003e \u003cp\u003eWe generated a protein\u0026ndash;protein interaction (PPI) network based on the overlapping targets using the STRING database (v12.0, species: Homo sapiens), with isolated nodes excluded to retain a connected network. Then, we imported the resulting network into Cytoscape (v3.8.0) for visualization and further analysis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], where core genes (hub nodes) were identified using the Centiscape (v2.2) plugin based on topological centrality measures.\u003c/p\u003e \u003cp\u003eOur team performed GO and KEGG pathway enrichment analyses on the putative Pena-4 targets relevant to IS on the online Metascape platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metascape.org/gp/index.html#/main/step1\u003c/span\u003e\u003cspan address=\"https://metascape.org/gp/index.html#/main/step1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), in which statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Molecular docking\u003c/h2\u003e \u003cp\u003eBriefly, the three-dimensional SIRT1 (PDB: 4kxq) structure was preprocessed by eliminating water molecules and co-crystallized ligands, followed by the addition of hydrogen atoms and charge assignment using AutoDock Tools 1.5.6 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The three-dimensional Pena-4 structure was energy-minimized, and its rotatable bonds were defined. Docking grid boxes were centered on the known active site of each protein. The conformation with the most favorable (lowest) binding free energy was selected for subsequent visualization and analysis using PyMOL [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Western blot\u003c/h2\u003e \u003cp\u003eWe extracted proteins from the peri-infarct brain tissue using 1\u0026times; RIPA buffer, which were then subjected to SDS-PAGE separation and blotted onto PVDF membranes. After a 2-hour blockade at room temperature with non-fat milk (5%), membranes were probed using antibodies as described above. We used ECL for detecting the protein bands and finally quantified them with ImageJ, with levels normalized to β-actin [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e \u003cp\u003eWhen normality or equal variance assumptions were violated, Kruskal\u0026ndash;Wallis test followed by Dunn\u0026rsquo;s post hoc analysis was applied. Otherwise, one-way ANOVA with Tukey\u0026rsquo;s correction was used. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Pena-4 attenuates neurological dysfunction and reduces infarct volume\u003c/h2\u003e \u003cp\u003eAt 24 hours post-reperfusion, mice subjected to MCAO/R exhibited significant neurological deficits, accompanied by markedly lower neurological scores relative to the sham-operated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Consistent with these behavioral impairments, TTC staining revealed the extensive white areas in the MCAO/R mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and D), confirming successful model establishment. Administration of Pena-4 at dose of 5 mg/kg significantly improved neurological performance and reduced infarct volume.\u003c/p\u003e \u003cp\u003eTo further assess the benefits of Pena-4 on brain tissue integrity following I/R, histopathological changes were examined via H\u0026amp;E staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Brain sections from the MCAO/R mice showed severe pathological alterations compared with the sham group, including neuronal shrinkage, cell loss, disrupted cortical lamination, and prominent nuclear pyknosis. Comparable damage was observed across hippocampal subfields\u0026mdash;especially in CA1, CA3, and the dentate gyrus (DG)\u0026mdash;where neurons appeared sparse and morphologically disorganized. In contrast, Pena-4 markedly attenuated these histopathological abnormalities. Cortical and hippocampal neurons exhibited improved structural integrity, reduced degeneration, and better-preserved cellular density.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the neuroprotective effect of Pena-4 on neuronal survival post-ischemia, Nissl staining was also performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In sham-operated animals, neurons exhibited normal morphology, abundant Nissl substance, and orderly arrangement. By contrast, the MCAO/R group showed extensive neuronal injury, characterized by a pronounced reduction in Nissl-positive cells, diminished staining intensity, cytoplasmic vacuolization, and widespread neuronal loss. Pena-4 markedly attenuated these pathological alterations, as indicated by the preservation of Nissl body structure and a higher density of neurons in both the hippocampus and cortex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Mapping shared targets and building the PPI network for Pena-4 in ischemic stroke\u003c/h2\u003e \u003cp\u003eTo explore the molecular targets of Pena-4, we obtained 100 drug-related targets from the SwissTargetPrediction database. We simultaneously gathered IS-related targets from 4 databases (TTD, PharmGKB, OMIM, and GeneCards) and removed duplicates to obtain a final set of 4,680 unique targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIntersection analysis identified 55 overlapping genes, suggesting their potential therapeutic relevance. These shared targets were utilized to build a PPI network through the STRING database (confidence score: 0.4). After excluding two disconnected nodes, a final network of 53 interacting proteins was obtained (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The dense interconnectivity reflects a high degree of functional association and underscores the multi-target nature of Pena-4 in ischemic stroke treatment.\u003c/p\u003e \u003cp\u003eHub gene analysis was performed using CytoScape plugin Centiscape 2.2, which ranked nodes by topological metrics (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Nodes with darker red shading represent higher centrality values, indicating that Pena-4\u0026rsquo;s pharmacological actions may depend significantly on these hub targets.\u003c/p\u003e \u003cp\u003eEnrichment analysis of the 53 overlapping targets was conducted, revealing 61 significantly enriched KEGG pathways and 1309 GO terms. GO analysis included 1092 biological process (BP) entries\u0026mdash;such as histone H3 deacetylation, collagen catabolic process, regulation of smooth muscle contraction, collagen metabolic process, and protein processing; 116 cellular component (CC) entries-including membrane microdomain, membrane raft, ficolin-1-rich granule lumen, histone deacetylase complex, and Golgi apparatus subcompartment. Among the enriched molecular functions, terms encompassing NAD-dependent deacetylase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) converged on the functional profile of Sirtuin proteins. This computational insight led us to prioritize SIRT1, a central regulator with corresponding deacetylase functions, for experimental validation of Pena-4's mechanism of action.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKEGG pathway analysis identified 61 significant pathways, such as the renin-angiotensin system, neuroactive ligand-receptor interaction, bladder cancer, neuroactive ligand-receptor signaling, and apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Molecular docking predicted a stable binding interaction between Pena-4 and SIRT1\u003c/h2\u003e \u003cp\u003eTo validate the potential regulation of SIRT1 by Pena-4, molecular docking was done to evaluate the Pena-4\u0026ndash;SIRT1 interaction. Conventional docking criteria define absolute scores\u0026thinsp;\u0026gt;\u0026thinsp;4.25 as moderate, \u0026gt;\u0026thinsp;5.0 as good, and \u0026gt;\u0026thinsp;7.0 as strong binding affinity. The results, visualized using PyMOL (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), indicated a favorable binding mode. Pena-4 was predicted to occupy the active site of SIRT1 via multiple hydrogen bonds and hydrophobic interactions, suggesting direct modulation of SIRT1 activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess biological relevance, SIRT1 protein expression was measured in peri-infarct brain tissue by Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Relative to the sham mice, SIRT1 expression was markedly lowered in the MCAO/R group and Pena-4 administration notably reversed this downregulation, restoring SIRT1 levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Pena-4 attenuates oxidative stress by restoring SOD2 expression and reducing lipid peroxidation in injured brain\u003c/h2\u003e \u003cp\u003eTo determine whether Pena-4 modulates oxidative stress, we assessed SOD2 expression and lipid peroxidation in peri-infarct tissue. Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) revealed a marked decrease in SOD2 levels following MCAO/R compared with sham, indicating impaired antioxidant defense. Pena-4 treatment significantly reversed this decline.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCorrespondingly, MDA, a key indicator of lipid peroxidation, significantly accumulated in the peri-infarct region (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), which was partly reversed by Pena-4 administration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Pena-4 inhibits pyroptosis in injured brains\u003c/h2\u003e \u003cp\u003eWestern blot was conducted to examine the effect of Pena-4 on NLRP3 inflammasome activation and pyroptosis-related proteins in peri-infarct brain tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). I/R injury markedly increased proteins expression of NLRP3, Clv-GSDMD and Clv-Caspase-1. Pena-4 administration reduced NLRP3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), Clv-GSDMD (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC), and Clv-Caspase-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD) levels in MCAO/R mice significantly.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThese findings collectively revealed that Pena-4 exerted robust neuroprotective benefits in mice exposed to cerebral I/R injury. A key highlight of current work is the immediate administration of Pena-4 upon reperfusion, reflecting a realistic treatment scenario for most stroke patients who receive care after ischemia onset and blood flow restoration. This approach is clinically relevant, unlike many neuroprotective agents that failed in trials due to impractical pre- or peri-ischemic dosing requirements [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This robust post-ischemic efficacy underscores the translational promise of Pena-4 as a viable adjunctive therapy that could extend the treatment window following recanalization.\u003c/p\u003e \u003cp\u003eMechanistically, the robust neuroprotective effects observed when Pena-4 is administered at the point of reperfusion suggests that Pena-4 treatment actively engages key pathological processes activated during reperfusion. During cerebral I/R, the electron transport chain impairment results in overproduction of reactive oxygen species [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], a pivotal factor in the progression of reperfusion injury [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Sirt1 protein enhanced cellular tolerance to oxidative damage by modulating various genes and their associated signaling pathways [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Within the Sirt1 signaling network, SOD2 holds a central position as a key antioxidant protein [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Sirt1 promotes the transcriptional activation and functional enhancement of SOD2 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], thereby bolstering mitochondrial antioxidant capacity and maintaining redox homeostasis. In our work, Pena-4 upregulated Sirt1 and SOD2 protein expression and reduced MDA formation in injured brains. Therefore, Pena-4 might mitigate oxidative damages during cerebral I/R through its intrinsic antioxidant activity and its modulation of Sirt1/SOD2 pathway.\u003c/p\u003e \u003cp\u003eA growing body of reports underscored the involvement of multiple regulated cell death pathways during I/R impairments in brain [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Among these, pyroptosis, driven by caspase-1 activation via inflammasomes like NLRP3 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], is an inflammatory type of programmed cell death. Highly expressed in the brain, NLRP3 detects cell damage and triggers inflammation, leading to cell death, and is crucial in I/R impairments [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Emerging evidence has underscored the involvement of SIRT1 in modulating pyroptotic cell death. Sirt1 inhibited NLRP3 inflammasome against cardiac I/R injury [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Sirt1 knockdown in HK-2 cells aggravate LPS-activated NLRP3-mediated pyroptosis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and Sirt1 knockout led to NLRP3 inflammasome activation in podocytes [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In our study, Pena-4 decreased NLRP3 expression and inhibited cleaved GSDMD and Caspase-1 formation in injured brains. Its ability to suppress pyroptosis in injured brain may stem from modulating the Sirt1/NLRP3 pathway.\u003c/p\u003e \u003cp\u003eA major limitation of our investigation is the lack of mechanistic validation for SIRT1's role. While our data from network pharmacology, molecular docking, and Western blot strongly suggest an interaction between Pena-4 and SIRT1, they do not provide definitive evidence that SIRT1 activation is directly responsible for the observed suppression of oxidative stress and pyroptosis. Future studies employing SIRT1-knockdown or knockout models are essential to confirm this causal link and rule out other potential Sirt1-independent pathways.\u003c/p\u003e \u003cp\u003eCollectively, our findings suggest that Pena-4 holds promise as a neuroprotective agent in brain I/R injury, potentially through its interaction with Sirt1 and subsequent attenuation of oxidative stress and NLRP3-driven pyroptosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data could be obtained upon reasonable request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal-related experimental interventions received approval from the Shanghai University Ethics Committee (Approval No. ECSHU2024-006).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Guoling Yang, Zhiqi Hou, Yuefan Zhang;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFormal analysis and investigation: Guoling Yang, Zhiqi Hou, JinJing Zhao,Anna Zhang, Yuchang Shen, Yihan Lin, Keying Chen;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWriting - original draft preparation: Guoling Yang, Yongsheng Yu, Yuefan Zhang;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWriting - review and editing: Guoling Yang, Yuefan Zhang;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSupervision: Yongsheng Yu, Yuefan Zhang.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSaini V, Guada L, Yavagal DR (2021) Global epidemiology of stroke and access to acute ischemic stroke interventions. 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Kidney Dis (Basel) 7(4):293\u0026ndash;305. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1159/000513884\u003c/span\u003e\u003cspan address=\"10.1159/000513884\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SIRT1, antioxidant defense, pyroptosis, ischemic stroke, network pharmacology","lastPublishedDoi":"10.21203/rs.3.rs-8774215/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8774215/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eIn ischemic stroke, the therapeutic options for mitigating ischemia/reperfusion (I/R) impairments in brain remain critically limited, underscoring an urgent need for novel neuroprotective agents. This study investigated the protective effects and underlying mechanisms of Pena-4, an oligopeptide originally identified from proteolytic digestion of Penaeus japonicus, in a model of transient middle cerebral artery occlusion/reperfusion (MCAO/R) .\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eNeurological function, infarct volume, and histopathological changes were evaluated after Pena-4 treatment. Network pharmacology and molecular docking were used to predict potential targets and pathways. Key proteins related to silent information regulator sirtuin 1 (SIRT1) signaling, oxidative stress, and pyroptosis were analyzed by western blotting, and malondialdehyde (MDA) levels were measured.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePena-4 notably improved neurological performance and decreased infarct volume. Histological analysis showed attenuated neuronal damage in the cortex and hippocampus. Network pharmacology approach identified 55 overlapping targets and highlighted silent information regulator sirtuin 1 (SIRT1) as a key hub target, which showed stable binding with Pena-4 in docking analysis. Pena-4 restored I/R-induced SIRT1 downregulation, increased SOD2 levels, reduced MDA content, and suppressed NLRP3-dependent pyroptosis, as evidenced by decreased NLRP3, cleaved caspase-1, and cleaved GSDMD.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003ePena-4 thus confers neuroprotection against I/R impairments in brain through mechanisms that involve SIRT1 activation, enhanced antioxidant defense, and inhibition of NLRP3-dependent pyroptosis.\u003c/p\u003e","manuscriptTitle":"Oligo-peptide Pena-4 activates SIRT1 and exerts neuroprotective benefits against cerebral ischemia-reperfusion injury in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 09:13:47","doi":"10.21203/rs.3.rs-8774215/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"82e32385-167e-4a2c-9fc2-b68badbea1ec","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-17T08:42:40+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-17 09:13:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8774215","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8774215","identity":"rs-8774215","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}