SS31 Mitigates Subarachnoid Hemorrhage-Induced Early Brain Injury via Nrf2/Keap1 Pathway Activation

SS31 Mitigates Subarachnoid Hemorrhage-Induced Early Brain Injury via Nrf2/Keap1 Pathway Activation | 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 SS31 Mitigates Subarachnoid Hemorrhage-Induced Early Brain Injury via Nrf2/Keap1 Pathway Activation Ming Liu, Jianchen Shen, Liang Wu, Meiqiu Liu, Cao Zeng, Zhiji Cai, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6797139/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 Background : Subarachnoid hemorrhage (SAH) presents a significant clinical challenge with high mortality and disability rates. Oxidative stress (OS) plays a critical role in early brain injury post-SAH. SS31, an antioxidant agent, has shown promise in mitigating ischemia/reperfusion injury-related damage. This study aims to investigate the role and mechanism of SS31 in SAH, focusing on its impact on early brain injury. Methods : This study systematically investigated the neuroprotective mechanisms of SS31 through both in vivo and in vitro approaches. Initially, we assessed the anti-apoptotic efficacy of SS31 using a subarachnoid hemorrhage (SAH) rat model and an HB-induced SH-SY5Y microglial cell model, concurrently evaluating its impact on neurological functional recovery in SAH animals. Subsequently, we examined the regulatory effects of SS31 on the KEAP1/Nrf2/HO-1 signaling pathway through protein expression analysis in both experimental systems. Furthermore, ultrastructural and functional evaluations were conducted to characterize SS31's protective effects against mitochondrial impairment in SAH pathogenesis. Results: SS31 treatment significantly mitigated early brain injury (EBI) following SAH, including BBB dysfunction, BE, neural cell apoptosis, OS, mitochondrial damage, and neurological deficits. SS31 also inhibits apoptosis, both in vivo and in cell models. At the same time, SS31 inhibited the expression of inflammatory factors in rat brain tissue and serum, which confirmed its anti-inflammatory effect. Furthermore, SS31 administration resulted in a notable reduction in Keap1 expression and a significant increase in Nrf2 and HO-1 expression. Conclusion: This study demonstrated that SS31 enhanced the recovery of nervous function after SAH in rats by mitigating OS-associated neuronal death via the Nrf2/Keap1 axis. SS31 SAH EBI KEAP1 NRF2 HO-1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Subarachnoid hemorrhage (SAH), a severe neurological condition precipitated by the rupture of intracranial aneurysms or vascular malformations, leads to significant morbidity and mortality despite advances in medical and surgical interventions. [1]. The prognosis for SAH patients remains challenging, underscoring the need for novel therapeutic strategies. Early brain injury (EBI), emerging in the aftermath of SAH, is a critical determinant of patient outcomes, marked by complex pathophysiological mechanisms including neuroinflammatory responses, oxidative stress (OS), apoptosis, blood-brain barrier dysfunction, and microvascular impairment. [2–4]. Oxidative stress, in particular, characterized by an imbalance between the production of reactive oxygen species (ROS) and antioxidant defenses, has been identified as a key contributor to neuronal damage and poor outcomes post-SAH[5, 6]. SS31, a mitochondria-targeted antioxidant, offers a promising avenue for mitigating OS-induced damage in SAH. [7–9]. Capable of crossing the blood-brain barrier, SS31 has demonstrated potential in reducing OS and preserving neuronal integrity in various neurodegenerative disease models, suggesting its utility in SAH treatment might be equally significant. [10–12] [13]. Nrf2 is a crucial transcription factor that modulates the antioxidant response [14]. Furthermore, the activation of the Nrf2/Keap1 antioxidant pathway by SS31 highlights a potentially novel mechanism through which it exerts neuroprotective effects in the context of SAH.[15, 16]. This investigation was designed to delineate the dose-responsive neuroprotective capacity of SS31 through systematic evaluation of redox homeostasis, apoptosis markers, and ferroptosis indicators in both in vivo and in vitro SAH models, ultimately establishing its potential as a novel therapeutic intervention targeting oxidative cascades. Materials and methods Animal s Adult female Sprague Dawley (SD) rats weighing 300–350g were obtained from the Hubei Animal Center for Disease Control and Prevention. Rats were maintained on a 12-h light-dark cycle, with unlimited access to water and food. All experimental procedures were approved by the Animal Care and Use Committee of Ningde Municipal Hospital (China) and conducted in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) for the Care and Use of Laboratory Animals. The experimental SAH model was established following previously described methods [17, 18]. Rats were anesthetized with 10% chloral hydrate (400 mg/kg body weight) and positioned in a stereotaxic head frame. Subsequently, fresh arterial blood (0.3 ml), taken from the same rat, was carefully and gradually injected into the prechiasmatic cistern for three minutes using sterile methods. Once the rats regained consciousness after the anesthesia, they were placed in their cages with unlimited access to water and food. The rats were euthanized with a lethal dose of pentobarbital (200 mg/kg) 24h after SAH (Zhou, J., et al., 2023; Shen, R., et al., 2020). Drug a dministration A total of 210 rats were divided into five groups, with 42 rats per group: sham (SG), SAH, SAH+vehicle (SAH+V), SAH+SS31 (10 mg/kg, low dose), and SAH+SS31 (30 mg/kg, high dose). SS31 (catalog number GC34569; GLPBIO, CA, USA) was administered intraperitoneally at doses of 10 or 30 mg/kg at two and 12 hours post-SAH. Dosages were chosen based on previous studies. Rats in the SAH+V group received an equivalent volume of the vehicle via intraperitoneal injection after SAH induction. Brain water content Brain water content was assessed at 24h following SAH establishment. The cerebrum was weighed immediately after being removed from the skull (wet weight) and subsequently weighed again after being dried at 100 °C for 72 h (dry weight). Brain edema was calculated as follows: [(wet weight − dry weight)/wet weight] × 100%. BBB permeability Evans blue (EB) extravasation was conducted to determine BBB permeability in rats at 24h following SAH. In brief, rats were injected intravenously with 2% EB at 24h following SAH. Then the rats were euthanized with a lethal dose of pentobarbital as described above. Subsequently, each rat was perfused transcardially with saline three hour after injection. Afterward, the brains were harvested and homogenized in trichloroacetic acid. Finally, supernatants' absorbance was measured at 610 nm using a spectrophotometer. Neurological score According to previous studies [15, 16], a six-point system was employed to assess the neurological function of rats at 72 h following SAH. Appetite (0-2), Activity (0-2), and Deficits (0-2) were assessed using the scoring system described in Table 1. and the sum of the three parameters represented the final neurological score, with lower scores indicating better neurological function. Measurement of MDA, CAT, SOD, and GSH activities Rats were transcardially perfused using 4◦C saline after euthanasia at 24h after SAH. The brains were harvested and homogenized in phosphate-buffered saline (PBS) after being removed from the skull. The activities of MDA, CAT, SOD, and GSH were determined using the corresponding assay kits (Nanjing Jiancheng Corp., China). TUNEL staining A TUNEL kit (Roche, CA, USA) was utilized to evaluate cell apoptosis in the brain cortex at 24h following SAH. In brief, each brain section was exposed to the TUNEL reaction mixture for 60 minutes at 37◦C, followed by DAPI staining. Finally, the stained sections were examined using a fluorescence microscope (Olympus, Japan). The number of TUNEL-positive cells in each section was calculated using ImageJ software (ImageJ, NIH) (Zhang et al., 2015). Western blot ting Western blotting was conducted as described in our previous publications [19, 20]. In brief, appropriate amounts of protein were separated using 10% SDS-PAGE and transferred onto polyvinylidenedifluoride (PVDF) membranes. After blocking nonspecific binding with 5% skimmed milk for two hours, the membranes were exposed to primary antibodies against Keap-1 (1：1000，10503-2-AP; Proteintech), HO-1 (1:1000，10701-1-AP; Proteintech), Nrf2 (1:1000 16396-1-AP; Proteintech), Bcl-2 (1:1000 26593-1-AP; Proteintech) and Bax (1:1000 50599-2-Ig; Proteintech) overnight at 4°C. The membranes were exposed to the relevant HRP-labelled secondary antibodies (1:5,000 in TBST) for two hours. The protein bands on the membrane were captured on X-ray film. UN-Scan-It 6.1 was used to estimate changes in protein expression by calculating the average pixel density of the bands after normalization to GAPDH. Immunohistochemical s taining Immunohistochemical staining was carried out based on our previous studies [15, 16]. Brain sections were exposed to primary antibodies against Keap-1(1:400), HO-1(1:200), Nrf2(1:300), Bcl-2(1:300), and Bax(1:300) overnight at 4◦C. After rinsing three times in PBS), the sections were exposed to HRP-labelled goat anti-rabbit IgG (1:300; Santa Cruz Biotechnology, USA) for 60 minutes at 37°C. 3,3′- Diaminobenzidine (DAB) was employed to visualize Keap-1, Nrf2, and HO-1. Transmission e lectron m icroscopy (TEM) As described in a previous study (Liu et al., 2014), the temporal lobe tissues of the rats were fixed in glutaraldehyde (2.5%) for four h, followed by post-fixation with osmium tetroxide (1%) for two h and dehydration in an ascending ethanol series. Next, the specimens were embedded in Epon-812. Subsequently, 60 nm semi-thin sections were stained with 2% lead citrate and 2% uranyl acetate. The sections were examined using a TEM Tecnai G2 20 TWIN (FEI), which is a 200kV field emission gun (FEG) high-resolution and analytical TEM/STEM. SH-SY5Y Cell Culture and Drug Treatment SH-SY5Y cells were commercially obtained from Wuhan Procell Biotechnology Service Co., Ltd. (Wuhan, China) and maintained in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) under standard culture conditions (37°C, 5% CO₂ humidified atmosphere). For experimental procedures, cells were seeded in 24-well plates at a density of 1×10⁵ cells/mL. To establish an oxidative stress model, cells were pretreated with 100μM hemoglobin (Hb) for 12 h, while control groups received equivalent volumes of normal saline. Following Hb exposure, the treatment groups were subjected to medium replacement with phosphate-buffered saline (PBS) containing SS31 peptide, maintaining equivalent volumes for 24 h. All cell cultures were subsequently incubated for an additional 12 h prior to sample collection. To ensure methodological rigor, all in vitro experiments were conducted in triplicate with independent biological replicates. JC-1 Staining Mitochondrial membrane potential was assessed using the JC-1 staining assay (Thermo, Waltham, MA, USA). Cells were seeded in a six-well plate at a density of 1×10⁵ cells per well. Following incubation, cells were washed twice with JC-1 staining buffer and resuspended in fresh culture medium or PBS. Then cells were washed twice with phosphate-buffer. A fluorescence microscopy was used with excitation/emission wavelengths of 488/530 nm for the monomeric form (green fluorescence) and 488/590 nm for the aggregated form (red fluorescence). The ratio of red to green fluorescence intensity was calculated to quantify mitochondrial. Data were analyzed using ImageJ 1.52a. All experiments were performed in triplicate, and results were expressed as mean ± standard deviation (SD). Enzyme-Linked Immunosorbent Assay (ELISA) Quantification of inflammatory cytokines was performed using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Proteintech, Rosemont, USA) according to manufacturer protocols for IL-1β, IL-6 and TNF-α. Sample preparation involved collection of 100 μL aliquots from rats serum, which were subsequently loaded into antibody-coated 96-well plates. Quantitative analysis was conducted by measuring absorbance at 450 nm using a Multiskan SkyHigh microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) with wavelength correction at 570 nm. Standard curve generation employed recombinant protein calibrators spanning 0-1000 pg/mL, with analyte concentrations calculated through four-parameter logistic (4PL) regression modeling. Reverse Transcription Quantitative PCR (RT-qPCR) Total RNA was extracted from the brain tissues using an RNA extraction kit (Sangon Biotech, China) following the manufacturer’s instructions. The RNA concentration and purity were assessed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA). The extracted RNA was diluted with RNase-free dH 2 O to a final concentration of 500 ng/ml. Reverse transcription was performed using the PrimeScript RT reagent kit (TaKaRa, China). RT-qPCR was conducted using SYBR Green technology (TaKaRa, China). PCR amplification was performed using a standard two-step protocol on a thermal cycler (StepOnePlus, Thermo). The primer design is provided in Table S1. Relative gene expression was calculated using the ΔΔCt method, with GAPDH as the internal reference. The calculation formula is as follows: ΔΔCt =ΔCt (experimental group)-ΔCt (control group) The fold change in expression level is then calculated as: Fold Change=2−ΔΔCt Each reaction was performed in triplicate, and no-template controls were included to confirm the absence of contamination. Statistical analysis All results were reported as means ± SEM. Statistical tests were conducted with SPSS 26.0, with one-way ANOVA applied to all data，differences between groups were analyzed by LSD analysis. A p-value of less than .05 was considered significant. Results SS31 treatment protects BBB disruption, attenuates BE, and improves neurobehavior Brain water content was significantly higher in the SAH group than in SG ( P < .05). Treatment with SS31 noticeably decreased the water content compared with the SAH + V group ( P < .05; Fig. 1 A). Compared to the SG rats, the SAH-treated rats exhibited a notable rise ( P < .05) in BBB permeability to EB. Similarly, SS31 administration effectively suppressed EB extravasation ( P < .05; Fig. 1 B). Rats in the SAH groups presented remarkably higher neurological scores at 72 h following SAH ( P < .05). Treatment with 10 or 30 mg/kg SS31 reduced the neurological scores at 72 h following SAH ( P .05; Fig. 1 A-C). Effect of SS31 on OS in rats following SAH As shown in Fig. 2 (A, E), compared with the Sham group, the SAH group exhibited significantly elevated ROS levels, which were reduced in a dose-dependent manner following SS31 treatment (P < .05). What’s more, Fig. 2 (B-D, F) shows a marked reduction in the levels of GSH, SOD, and CAT compared to the SG, while increased MDA was seen in the SAH group ( P < .05). SS31 treatment resulted in a remarkable increase in GSH, SOD, and CAT activities, and decreased MDA levels in rat brain tissue compared to the SAH group ( P < .05). Moreover, a dose-related effect was observed ( P < .05). SS31 treatment alleviates neuronal apoptosis in rats following SAH in vitro and vivo Numerous TUNEL-positive cells were observed in the temporal lobe at 24h following SAH. Treatment with SS31 decreased the number of TUNEL-positive cells in the brain tissue of SAH rats compared to untreated animals ( P < .05; Fig. 3 A, C). Flow cytometry analysis of cell apoptosis revealed that the SAH group exhibited a significant increase in apoptotic cells, whereas SS31 treatment reduced apoptosis (P < .05; Fig. 3 B, D, E). Furthermore, SAH increased BAX expression in temporal lobe neurons and decreased BCL-2 expression at 24 hours post-SAH. Compared with the SAH + V group, the SS31 group showed elevated BCL-2 expression and reduced BAX expression ( P < .05; Fig. 4 D-F). Notably, high-dose SS31 treatment further enhanced this trend ( P < .05; Fig. 4 A-C). The in vitro SH-SY5Y cell model corroborated these findings, collectively demonstrating that SS31 inhibits neuronal apoptosis and exerts neuroprotective effects in rats. SS31 attenuated neuroinflammation in SAH rats. To investigate the anti-inflammatory effects of SS31, we measured the levels of IL-1β, IL-6, and TNF-α in rat brain tissues and serum. The results showed that the expression of these inflammatory cytokines was significantly elevated in the SAH group. However, after SS31 treatment, the levels of IL-1β, IL-6, and TNF-α in both brain tissues and serum were markedly reduced. These findings confirm the anti-inflammatory properties of SS31 (P < .05; Fig. 4 G-L). Effect of SS31 on the Nrf2/Keap1 pathway Immunoblotting was used to evaluate the effect of SS31 on the Keap1/HO-1/Nrf2 pathway in EBI following SAH. Western blotting revealed the upregulation of Nrf2 and HO-1 and the downregulation of Keap1 in the SAH + V group compared to the SG. Treatment with SS31 led to a striking increase in Nrf2 and HO-1, while Keap1 decreased compared with the SAH + V group ( P < .05; Fig. 5 A-D). Moreover, the trend was accelerated with the high-dose SS31 treatment ( P < .05; Fig. 5 A-D). Immunohistochemical analysis revealed that neural cells demonstrated positive HO-1, Nrf2, and Keap1 expression after SAH. Furthermore, compared to the SAH + V group, more positive HO-1 and Nrf2 immunostained cells and fewer Keap1-immunostained cells were observed in the SAH + SS31 groups after SAH (Fig. 5 E-H). Moreover, in vitro experiments further confirmed our hypothesis. SS31-treated cells exhibited upregulated expression of Nrf2 and HO-1 alongside downregulated Keap1 levels, with these trends following a dose-dependent manner ( P < .05; Fig. 5 I-L). Mitochondria morphology We used JC-1 staining to observe the early apoptotic signal of neurons. Mitochondrial membrane potential, as evidenced by a diminished red/green fluorescence intensity ratio in SAH groups, consistent with early apoptotic progression. Notably, SS31 administration dose-dependently restored this ratio, demonstrating therapeutic efficacy in maintaining mitochondrial integrity and cellular viability (Fig. 6 A, B). Mitochondria in the SG displayed a typical shape, normal cristae, intact membranes, and a compact matrix space. However, in the SAH + V group, some mitochondria were swollen and presented fractured and indistinct membranes and cristae (Fig. 6 ). Mitochondrial injuries, including swelling and fracture, were reduced by SS31 administration when compared with the SAH + V group (Fig. 6 C). Discussion The key findings of this investigation can be synthesized as follows: Initially, SS31 effectively ameliorated a range of detrimental outcomes, including brain edema, blood-brain barrier permeability, oxidative stress, cellular apoptosis, and motor impairments in rats subjected to subarachnoid hemorrhage (SAH). Additionally, SS31 was observed to upregulate the Nrf2/Keap1 pathway, which exhibited activation in response to SAH in the experimental rats. These results collectively suggest that SS31 exerts a neuroprotective effect by mitigating oxidative stress following SAH in rats. Furthermore, it is hypothesized that the antioxidative mechanism of SS31 may be linked to the activation of the Keap1/HO-1/Nrf2 pathway during the early stages of experimental SAH-induced brain injury. The determinants contributing to an unfavorable prognosis subsequent to SAH are intricate and multifaceted [21]. OS emerges as a pivotal pathological mechanism in early brain injury following SAH. Consequently, pharmacological agents targeting mitochondria, the primary origin of reactive oxygen species, represent promising therapeutic avenues for SAH. Mitochondria-targeted antioxidants, such as Mitoquinone (MitoQ) and SS31, hold potential for mitigating OS-associated damage, cellular demise, and providing neuroprotection against neurodegeneration, ischemia, and reperfusion injury [11, 12, 22–25]. Zhang and colleagues have demonstrated the ability of MitoQ to ameliorate mitochondrial OS-related neuronal demise and neurological deficits in animal models of SAH[16]. Likewise, Shen et al substantiated that SS31 attenuates EBI following SAH owing to its antioxidant attributes[13]. Despite some investigations evaluating the neuroprotective efficacy of SS31 in SAH, its underlying mechanisms remain incompletely elucidated[26]. Further inquiries are imperative to elucidate the mechanisms through which SS31 exerts its actions in SAH before clinical trials can be contemplated. As acknowledged, Nrf2 represents a redox-sensitive transcription factor pivotal in regulating the antioxidant signaling cascade. Typically, Nrf2 forms a complex with Keap1 in the cytoplasm, where it undergoes degradation under basal physiological conditions. Upon encountering acute stressors, Nrf2 dissociates from Keap1 and translocates to the nucleus, orchestrating the transcriptional activities of antioxidant response elements (ARE) and ARE-associated gene products, such as HO-1 [27]. Multiple studies have highlighted the potential benefits of the Nrf2/Keap1 axis in mitigating EBI following SAH [27–30]. This mechanism may operate through preserving mitochondrial functionality and suppressing oxidative stress. Wang et al. demonstrated SS31's ability to modulate oxidative stress status in aged brains via the Nrf2/Keap1 signaling pathway and confer protective effects against aging in vivo[31]. Numerous researchers have provided evidence that SS31 can enhance Nrf2 expression and safeguard liver and cardiac functions in animal models [32, 33]. After SAH in this investigation, levels of MDA were elevated, while the activities of GSH and endogenous antioxidant enzymes (SOD and CAT) were diminished, consistent with previous research findings. Additionally, two different doses of SS31 were administered to evaluate its impact on SAH. The results indicated that SS31 treatment attenuated lipid peroxidation reactions observed after SAH. The higher SS31 dose exhibited a more pronounced antioxidant effect, albeit without yielding additional improvements in neurological function compared to the lower dose. Furthermore, activation of the Nrf2/Keap1 pathway was observed following SAH, with reduced Keap1 expression and increased expression of Nrf2 and HO-1 noted in SAH rats after SS31 administration. These findings suggest that the Nrf2/Keap1 axis may contribute to modulating SS31's antioxidant properties. Complementary experiments in SH-SY5Y cells demonstrated that SS31 treatment attenuated Hb-induced oxidative stress damage and suppressed cellular apoptosis. Mechanistically, SS31 concomitantly upregulated the expression of Nrf2 and HO-1 while downregulating Keap1, suggesting activation of ferroptosis-related pathways. These coordinated molecular changes further confirmed the neuroprotective properties of SS31. Furthermore, it is pertinent to note that this investigation has delineated the protective impacts of SS31 on mitochondrial function and apoptosis mitigation. These findings hold significance in light of the established involvement of the Nrf2/Keap1 axis in modulating mitochondria-linked apoptosis, as evidenced in prior research. [34]. Several limitations of this study warrant consideration. Firstly, although SS31 was found to upregulate Nrf2/Keap1 expression and modulate the transcription of antioxidant enzymes, we did not assess the impact of SS31 on subarachnoid hemorrhage (SAH) in the absence of Nrf2. Secondly, we did not explore the potential involvement of other signaling pathways that might have contributed to the beneficial effects of SS31 on EBI following SAH. Lastly, further investigation is needed to elucidate the precise mechanism by which SS31 modulates Nrf2/Keap1 activation. Therefore, future research endeavors will prioritize addressing these crucial mechanistic inquiries. Conclusion In summary, our study revealed that SS31 facilitated the recovery of neurological function post-SAH in rats and inhibition of Hb-induced apoptosis, via attenuating oxidative stress-related neuronal demise through the Nrf2/Keap1 axis. Despite the need for additional investigation, this study underscored the potential efficacy of SS31 as a therapeutic candidate for SAH. Declarations Funding: This project is supported by the Youth Scientific Research Guiding Project of Fujian Provincial Health Commission (No.2018-ZQN-80). Conflict of Interest The authors declare that they have no competing interests regarding the publication of this paper. CRediT Authorship Contribution Statement Ming Liu: Writing – original draft. Jianchen Shen: Writing–Formal analysis. Liang Wu: Writing–review & editing. Meiqiu Liu: Validation. Cao Zeng and Zhiji Cai: Investigation. Guanghui Wu: Writing–review & editing. Jian Yin: Conceptualization. Data Availability Statement The datasets for this study are available on request to the corresponding authors. References Alsbrook, D.L., et al., 2023. Neuroinflammation in Acute Ischemic and Hemorrhagic Stroke. Curr Neurol Neurosci Rep. Armstrong, J.S., 2008. Mitochondria-Directed Therapeutics. Antioxidants & Redox Signaling. 10 , 575-578. Bhatti, J.S., et al., 2021. 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A Triple-Targeted Rutin-Based Self-Assembled Delivery Vector for Treating Ischemic Stroke by Vascular Normalization and Anti-Inflammation via ACE2/Ang1-7 Signaling. ACS Cent Sci. 9 , 1180-1199. Zhou, J., et al., 2023. SS31 Confers Cerebral Protection by Reversing Mitochondrial Dysfunction in Early Brain Injury Following Subarachnoid Hemorrhage, via the Nrf2- and PGC-1alpha-Dependent Pathways. Neurochem Res. 48 , 1580-1595. Zhu, Y., et al., 2022. SS-31, a Mitochondria-Targeting Peptide, Ameliorates Kidney Disease. Oxid Med Cell Longev. 2022 , 1295509. Zolnourian, A., Galea, I., Bulters, D., 2019. Neuroprotective Role of the Nrf2 Pathway in Subarachnoid Haemorrhage and Its Therapeutic Potential. Oxidative Medicine and Cellular Longevity. 2019 , 1-21. Additional Declarations No competing interests reported. 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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-6797139","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":466722852,"identity":"0e83db75-ac87-47a5-a772-b644388eb7bc","order_by":0,"name":"Ming Liu","email":"","orcid":"","institution":"Ningde Clinical Medical College of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Liu","suffix":""},{"id":466722853,"identity":"b12e2945-87c9-4fc9-94a5-39922d35a0cc","order_by":1,"name":"Jianchen Shen","email":"","orcid":"","institution":"Ningde Clinical Medical College of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jianchen","middleName":"","lastName":"Shen","suffix":""},{"id":466722855,"identity":"4698815b-2b74-481b-8ae2-39c7d97319aa","order_by":2,"name":"Liang Wu","email":"","orcid":"","institution":"Ningde Clinical Medical College of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Wu","suffix":""},{"id":466722856,"identity":"0bd4641c-fc9b-4ada-9a13-1cab49312980","order_by":3,"name":"Meiqiu Liu","email":"","orcid":"","institution":"Ningde Clinical Medical College of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Meiqiu","middleName":"","lastName":"Liu","suffix":""},{"id":466722857,"identity":"f685ea1f-17e6-4845-ab68-04f3a0e6258b","order_by":4,"name":"Cao Zeng","email":"","orcid":"","institution":"Ningde Clinical Medical College of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Cao","middleName":"","lastName":"Zeng","suffix":""},{"id":466722858,"identity":"c0f582f8-5222-4fe3-92d5-14639dfe5798","order_by":5,"name":"Zhiji Cai","email":"","orcid":"","institution":"Ningde Clinical Medical College of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhiji","middleName":"","lastName":"Cai","suffix":""},{"id":466722859,"identity":"886a2935-d1b6-4be4-a792-16aaa05641c0","order_by":6,"name":"Jian Yin","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Yin","suffix":""},{"id":466722860,"identity":"d4b0940c-f063-421e-ad0a-1e54ecca46e9","order_by":7,"name":"Guanghui Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYDACCSBOqLCR42dmPvyAeC0PzqQZS7azpRkQrYXxYduhxA3neRQkiNIhH91jJpHYdiBx82EeBgOGGptogloM75wxk0g4d8d422HeAw8YjqXlNhDUMiN3m0RC2TPZbYf5EgwYGw4Tq4XtMOPmZh4DCaK0yEuAtLQdVtzATKwWA4n8zxYJwECWOAwM5ARi/CI/Iy3x5g9QVPYfPvzgQ40NEbYcQOYlEFIOtoWgoaNgFIyCUTAKAFmVQ4gBZmYkAAAAAElFTkSuQmCC","orcid":"","institution":"Ningde Clinical Medical College of Fujian Medical University","correspondingAuthor":true,"prefix":"","firstName":"Guanghui","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2025-06-01 18:08:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6797139/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6797139/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84088993,"identity":"e26f5dd3-fd85-4147-aa01-3d3e728e64cb","added_by":"auto","created_at":"2025-06-06 15:42:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":726919,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6797139/v1/7558188ba7f3127fd43d9ff2.png"},{"id":84088994,"identity":"a64dc026-729b-4a63-be46-a03a719c3e03","added_by":"auto","created_at":"2025-06-06 15:42:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":317316,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6797139/v1/b388c3826c6f9d06d84144b4.png"},{"id":84089321,"identity":"c81a5f78-b696-4fc6-bacb-7e7793babd45","added_by":"auto","created_at":"2025-06-06 15:50:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":706203,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6797139/v1/c01c536c9945510d419c027c.png"},{"id":84088998,"identity":"9dc4c495-6a49-4e2d-b04e-e305b6143d92","added_by":"auto","created_at":"2025-06-06 15:42:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":613102,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6797139/v1/37e811a95b8610fe84ad3976.png"},{"id":84089000,"identity":"d2b38e64-d7d7-492d-9f69-77b37ef4bd63","added_by":"auto","created_at":"2025-06-06 15:42:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1689539,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6797139/v1/1d642d1bb48a3e6c1b1de60a.png"},{"id":84089012,"identity":"98984c99-d532-4c37-9b72-8984d970acc5","added_by":"auto","created_at":"2025-06-06 15:42:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1198639,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6797139/v1/aa8342e26e634c65e7481a12.png"},{"id":84714307,"identity":"4d15d0dd-caa6-4751-be9b-f4e032d2c514","added_by":"auto","created_at":"2025-06-16 14:02:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6148769,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6797139/v1/10762ceb-e0e7-4624-bfdd-f650cf044707.pdf"},{"id":84088995,"identity":"b8e64493-2470-4715-8286-5715f5eb3c8f","added_by":"auto","created_at":"2025-06-06 15:42:08","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15057,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6797139/v1/b914b14a493f7bc98a1e3adc.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"SS31 Mitigates Subarachnoid Hemorrhage-Induced Early Brain Injury via Nrf2/Keap1 Pathway Activation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSubarachnoid hemorrhage (SAH), a severe neurological condition precipitated by the rupture of intracranial aneurysms or vascular malformations, leads to significant morbidity and mortality despite advances in medical and surgical interventions. [1]. The prognosis for SAH patients remains challenging, underscoring the need for novel therapeutic strategies. Early brain injury (EBI), emerging in the aftermath of SAH, is a critical determinant of patient outcomes, marked by complex pathophysiological mechanisms including neuroinflammatory responses, oxidative stress (OS), apoptosis, blood-brain barrier dysfunction, and microvascular impairment. [2\u0026ndash;4]. Oxidative stress, in particular, characterized by an imbalance between the production of reactive oxygen species (ROS) and antioxidant defenses, has been identified as a key contributor to neuronal damage and poor outcomes post-SAH[5, 6].\u003c/p\u003e \u003cp\u003eSS31, a mitochondria-targeted antioxidant, offers a promising avenue for mitigating OS-induced damage in SAH. [7\u0026ndash;9]. Capable of crossing the blood-brain barrier, SS31 has demonstrated potential in reducing OS and preserving neuronal integrity in various neurodegenerative disease models, suggesting its utility in SAH treatment might be equally significant. [10\u0026ndash;12] [13]. Nrf2 is a crucial transcription factor that modulates the antioxidant response [14]. Furthermore, the activation of the Nrf2/Keap1 antioxidant pathway by SS31 highlights a potentially novel mechanism through which it exerts neuroprotective effects in the context of SAH.[15, 16]. This investigation was designed to delineate the dose-responsive neuroprotective capacity of SS31 through systematic evaluation of redox homeostasis, apoptosis markers, and ferroptosis indicators in both in vivo and in vitro SAH models, ultimately establishing its potential as a novel therapeutic intervention targeting oxidative cascades.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdult female Sprague Dawley (SD) rats weighing 300–350g were obtained from the Hubei Animal Center for Disease Control and Prevention. Rats were maintained on a 12-h light-dark cycle, with unlimited access to water and food. All experimental procedures were approved by the Animal Care and Use Committee of Ningde Municipal Hospital (China) and conducted in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) for the Care and Use of Laboratory Animals.\u003c/p\u003e\n\u003cp\u003eThe experimental SAH model was established following previously described methods [17, 18]. Rats were anesthetized with 10% chloral hydrate (400 mg/kg body weight) and positioned in a stereotaxic head frame. Subsequently, fresh arterial blood (0.3 ml), taken from the same rat, was carefully and gradually injected into the prechiasmatic cistern for three minutes using sterile methods. Once the rats regained consciousness after the anesthesia, they were placed in their cages with unlimited access to water and food. The rats were euthanized with a lethal dose of pentobarbital (200 mg/kg) 24h after SAH (Zhou, J., et al., 2023; Shen, R., et al., 2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDrug \u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003edministration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 210 rats were divided into five groups, with 42 rats per group: sham (SG), SAH, SAH+vehicle (SAH+V), SAH+SS31 (10 mg/kg, low dose), and SAH+SS31 (30 mg/kg, high dose). SS31 (catalog number GC34569; GLPBIO, CA, USA) was administered intraperitoneally at doses of 10 or 30 mg/kg at two and 12 hours post-SAH. Dosages were chosen based on previous studies. Rats in the SAH+V group received an equivalent volume of the vehicle via intraperitoneal injection after SAH induction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBrain water content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrain water content was assessed at 24h following SAH establishment. The cerebrum was weighed immediately after being removed from the skull (wet weight) and subsequently weighed again after being dried at 100 °C for 72 h (dry weight). Brain edema was calculated as follows: [(wet weight − dry weight)/wet weight] × 100%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBBB\u003c/strong\u003e\u003cstrong\u003e permeability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEvans blue (EB) extravasation was conducted to determine BBB permeability in rats at 24h following SAH. In brief, rats were injected intravenously with 2% EB at 24h following SAH. Then the rats were euthanized with a lethal dose of pentobarbital as described above. Subsequently, each rat was perfused transcardially with saline three hour after injection. Afterward, the brains were harvested and homogenized in trichloroacetic acid. Finally, supernatants' absorbance was measured at 610 nm using a spectrophotometer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeurological score\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to previous studies [15, 16], a six-point system was employed to assess the neurological function of rats at 72 h following SAH. Appetite (0-2), Activity (0-2), and Deficits (0-2) were assessed using the scoring system described in Table 1. and the sum of the three parameters represented the final neurological score, with lower scores indicating better neurological function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of MDA, CAT, SOD, and GSH activities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats were transcardially perfused using 4◦C saline after euthanasia at 24h after SAH. The brains were harvested and homogenized in phosphate-buffered saline (PBS) after being removed from the skull. The activities of MDA, CAT, SOD, and GSH were determined using the corresponding assay kits (Nanjing Jiancheng Corp., China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTUNEL staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA TUNEL kit (Roche, CA, USA) was utilized to evaluate cell apoptosis in the brain cortex at 24h following SAH. In brief, each brain section was exposed to the TUNEL reaction mixture for 60 minutes at 37◦C, followed by DAPI staining. Finally, the stained sections were examined using a fluorescence microscope (Olympus, Japan). The number of TUNEL-positive cells in each section was calculated using ImageJ software (ImageJ, NIH) (Zhang et al., 2015).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003cstrong\u003eting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blotting was conducted as described in our previous publications [19, 20]. In brief, appropriate amounts of protein were separated using 10% SDS-PAGE and transferred onto polyvinylidenedifluoride (PVDF) membranes. After blocking nonspecific binding with 5% skimmed milk for two hours, the membranes were exposed to primary antibodies against Keap-1 (1：1000，10503-2-AP; Proteintech), HO-1 (1:1000，10701-1-AP; Proteintech), Nrf2 (1:1000 16396-1-AP; Proteintech), Bcl-2 (1:1000 26593-1-AP; Proteintech) and Bax (1:1000 50599-2-Ig; Proteintech) overnight at 4°C. The membranes were exposed to the relevant HRP-labelled secondary antibodies (1:5,000 in TBST) for two hours. The protein bands on the membrane were captured on X-ray film. UN-Scan-It 6.1 was used to estimate changes in protein expression by calculating the average pixel density of the bands after normalization to GAPDH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical \u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003cstrong\u003etaining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemical staining was carried out based on our previous studies [15, 16]. Brain sections were exposed to primary antibodies against Keap-1(1:400), HO-1(1:200), Nrf2(1:300), Bcl-2(1:300), and Bax(1:300) overnight at 4◦C. After rinsing three times in PBS), the sections were exposed to HRP-labelled goat anti-rabbit IgG (1:300; Santa Cruz Biotechnology, USA) for 60 minutes at 37°C. 3,3′-\u003cem\u003eDiaminobenzidine\u003c/em\u003e (DAB) was employed to visualize Keap-1, Nrf2, and HO-1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission \u003c/strong\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003cstrong\u003electron \u003c/strong\u003e\u003cstrong\u003em\u003c/strong\u003e\u003cstrong\u003eicroscopy (TEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs described in a previous study (Liu et al., 2014), the temporal lobe tissues of the rats were fixed in glutaraldehyde (2.5%) for four h, followed by post-fixation with osmium tetroxide (1%) for two h and dehydration in an ascending ethanol series. Next, the specimens were embedded in Epon-812. Subsequently, 60 nm semi-thin sections were stained with 2% lead citrate and 2% uranyl acetate. The sections were examined using a TEM Tecnai G2 20 TWIN (FEI), which is a 200kV field emission gun (FEG) high-resolution and analytical TEM/STEM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSH-SY5Y Cell Culture and Drug Treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSH-SY5Y cells were commercially obtained from Wuhan Procell Biotechnology Service Co., Ltd. (Wuhan, China) and maintained in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) under standard culture conditions (37°C, 5% CO₂ humidified atmosphere). For experimental procedures, cells were seeded in 24-well plates at a density of 1×10⁵ cells/mL. \u003c/p\u003e\n\u003cp\u003eTo establish an oxidative stress model, cells were pretreated with 100μM hemoglobin (Hb) for 12 h, while control groups received equivalent volumes of normal saline. Following Hb exposure, the treatment groups were subjected to medium replacement with phosphate-buffered saline (PBS) containing SS31 peptide, maintaining equivalent volumes for 24 h. All cell cultures were subsequently incubated for an additional 12 h prior to sample collection. To ensure methodological rigor, all in vitro experiments were conducted in triplicate with independent biological replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJC-1 Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitochondrial membrane potential was assessed using the JC-1 staining assay (Thermo, Waltham, MA, USA). Cells were seeded in a six-well plate at a density of 1×10⁵ cells per well. Following incubation, cells were washed twice with JC-1 staining buffer and resuspended in fresh culture medium or PBS. Then cells were washed twice with phosphate-buffer. A fluorescence microscopy was used with excitation/emission wavelengths of 488/530 nm for the monomeric form (green fluorescence) and 488/590 nm for the aggregated form (red fluorescence). The ratio of red to green fluorescence intensity was calculated to quantify mitochondrial. Data were analyzed using ImageJ 1.52a. All experiments were performed in triplicate, and results were expressed as mean ± standard deviation (SD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantification of inflammatory cytokines was performed using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Proteintech, Rosemont, USA) according to manufacturer protocols for IL-1β, IL-6 and TNF-α. Sample preparation involved collection of 100 μL aliquots from rats serum, which were subsequently loaded into antibody-coated 96-well plates. Quantitative analysis was conducted by measuring absorbance at 450 nm using a Multiskan SkyHigh microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) with wavelength correction at 570 nm. Standard curve generation employed recombinant protein calibrators spanning 0-1000 pg/mL, with analyte concentrations calculated through four-parameter logistic (4PL) regression modeling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReverse Transcription Quantitative PCR (RT-qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from the brain tissues using an RNA extraction kit (Sangon Biotech, China) following the manufacturer’s instructions. The RNA concentration and purity were assessed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA). The extracted RNA was diluted with RNase-free dH\u003csub\u003e2\u003c/sub\u003eO to a final concentration of 500 ng/ml. Reverse transcription was performed using the PrimeScript RT reagent kit (TaKaRa, China). RT-qPCR was conducted using SYBR Green technology (TaKaRa, China). PCR amplification was performed using a standard two-step protocol on a thermal cycler (StepOnePlus, Thermo). The primer design is provided in Table S1. Relative gene expression was calculated using the ΔΔCt method, with GAPDH as the internal reference. The calculation formula is as follows:\u003c/p\u003e\n\u003cp\u003eΔΔCt =ΔCt (experimental group)-ΔCt (control group)\u003c/p\u003e\n\u003cp\u003eThe fold change in expression level is then calculated as:\u003c/p\u003e\n\u003cp\u003eFold Change=2−ΔΔCt\u003c/p\u003e\n\u003cp\u003eEach reaction was performed in triplicate, and no-template controls were included to confirm the absence of contamination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll results were reported as means ± SEM. Statistical tests were conducted with SPSS 26.0, with one-way ANOVA applied to all data，differences between groups were analyzed by LSD analysis. A p-value of less than .05 was considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSS31 treatment protects BBB disruption, attenuates BE, and improves neurobehavior\u003c/h2\u003e \u003cp\u003eBrain water content was significantly higher in the SAH group than in SG (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05). Treatment with SS31 noticeably decreased the water content compared with the SAH\u0026thinsp;+\u0026thinsp;V group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Compared to the SG rats, the SAH-treated rats exhibited a notable rise (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05) in BBB permeability to EB. Similarly, SS31 administration effectively suppressed EB extravasation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Rats in the SAH groups presented remarkably higher neurological scores at 72 h following SAH (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;.05). Treatment with 10 or 30 mg/kg SS31 reduced the neurological scores at 72 h following SAH (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;.05). However, there was no discernable difference among the different doses used in the SS31 treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEffect of SS31 on OS in rats following SAH\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (A, E), compared with the Sham group, the SAH group exhibited significantly elevated ROS levels, which were reduced in a dose-dependent manner following SS31 treatment (P\u0026thinsp;\u0026lt;\u0026thinsp;.05). What\u0026rsquo;s more, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (B-D, F) shows a marked reduction in the levels of GSH, SOD, and CAT compared to the SG, while increased MDA was seen in the SAH group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05). SS31 treatment resulted in a remarkable increase in GSH, SOD, and CAT activities, and decreased MDA levels in rat brain tissue compared to the SAH group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05). Moreover, a dose-related effect was observed (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSS31 treatment alleviates neuronal apoptosis in rats following SAH in vitro and vivo\u003c/h2\u003e \u003cp\u003eNumerous TUNEL-positive cells were observed in the temporal lobe at 24h following SAH. Treatment with SS31 decreased the number of TUNEL-positive cells in the brain tissue of SAH rats compared to untreated animals (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, C). Flow cytometry analysis of cell apoptosis revealed that the SAH group exhibited a significant increase in apoptotic cells, whereas SS31 treatment reduced apoptosis (P\u0026thinsp;\u0026lt;\u0026thinsp;.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, D, E). Furthermore, SAH increased BAX expression in temporal lobe neurons and decreased BCL-2 expression at 24 hours post-SAH. Compared with the SAH\u0026thinsp;+\u0026thinsp;V group, the SS31 group showed elevated BCL-2 expression and reduced BAX expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F). Notably, high-dose SS31 treatment further enhanced this trend (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). The in vitro SH-SY5Y cell model corroborated these findings, collectively demonstrating that SS31 inhibits neuronal apoptosis and exerts neuroprotective effects in rats.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSS31 attenuated neuroinflammation in SAH rats.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the anti-inflammatory effects of SS31, we measured the levels of IL-1β, IL-6, and TNF-α in rat brain tissues and serum. The results showed that the expression of these inflammatory cytokines was significantly elevated in the SAH group. However, after SS31 treatment, the levels of IL-1β, IL-6, and TNF-α in both brain tissues and serum were markedly reduced. These findings confirm the anti-inflammatory properties of SS31 (P\u0026thinsp;\u0026lt;\u0026thinsp;.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-L).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eEffect of SS31 on the Nrf2/Keap1 pathway\u003c/h2\u003e \u003cp\u003eImmunoblotting was used to evaluate the effect of SS31 on the Keap1/HO-1/Nrf2 pathway in EBI following SAH. Western blotting revealed the upregulation of Nrf2 and HO-1 and the downregulation of Keap1 in the SAH\u0026thinsp;+\u0026thinsp;V group compared to the SG. Treatment with SS31 led to a striking increase in Nrf2 and HO-1, while Keap1 decreased compared with the SAH\u0026thinsp;+\u0026thinsp;V group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D). Moreover, the trend was accelerated with the high-dose SS31 treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D). Immunohistochemical analysis revealed that neural cells demonstrated positive HO-1, Nrf2, and Keap1 expression after SAH. Furthermore, compared to the SAH\u0026thinsp;+\u0026thinsp;V group, more positive HO-1 and Nrf2 immunostained cells and fewer Keap1-immunostained cells were observed in the SAH\u0026thinsp;+\u0026thinsp;SS31 groups after SAH (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-H). Moreover, in vitro experiments further confirmed our hypothesis. SS31-treated cells exhibited upregulated expression of Nrf2 and HO-1 alongside downregulated Keap1 levels, with these trends following a dose-dependent manner (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI-L).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMitochondria morphology\u003c/h2\u003e \u003cp\u003eWe used JC-1 staining to observe the early apoptotic signal of neurons. Mitochondrial membrane potential, as evidenced by a diminished red/green fluorescence intensity ratio in SAH groups, consistent with early apoptotic progression. Notably, SS31 administration dose-dependently restored this ratio, demonstrating therapeutic efficacy in maintaining mitochondrial integrity and cellular viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Mitochondria in the SG displayed a typical shape, normal cristae, intact membranes, and a compact matrix space. However, in the SAH\u0026thinsp;+\u0026thinsp;V group, some mitochondria were swollen and presented fractured and indistinct membranes and cristae (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Mitochondrial injuries, including swelling and fracture, were reduced by SS31 administration when compared with the SAH\u0026thinsp;+\u0026thinsp;V group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe key findings of this investigation can be synthesized as follows: Initially, SS31 effectively ameliorated a range of detrimental outcomes, including brain edema, blood-brain barrier permeability, oxidative stress, cellular apoptosis, and motor impairments in rats subjected to subarachnoid hemorrhage (SAH). Additionally, SS31 was observed to upregulate the Nrf2/Keap1 pathway, which exhibited activation in response to SAH in the experimental rats. These results collectively suggest that SS31 exerts a neuroprotective effect by mitigating oxidative stress following SAH in rats. Furthermore, it is hypothesized that the antioxidative mechanism of SS31 may be linked to the activation of the Keap1/HO-1/Nrf2 pathway during the early stages of experimental SAH-induced brain injury.\u003c/p\u003e \u003cp\u003eThe determinants contributing to an unfavorable prognosis subsequent to SAH are intricate and multifaceted [21]. OS emerges as a pivotal pathological mechanism in early brain injury following SAH. Consequently, pharmacological agents targeting mitochondria, the primary origin of reactive oxygen species, represent promising therapeutic avenues for SAH. Mitochondria-targeted antioxidants, such as Mitoquinone (MitoQ) and SS31, hold potential for mitigating OS-associated damage, cellular demise, and providing neuroprotection against neurodegeneration, ischemia, and reperfusion injury [11, 12, 22\u0026ndash;25]. Zhang and colleagues have demonstrated the ability of MitoQ to ameliorate mitochondrial OS-related neuronal demise and neurological deficits in animal models of SAH[16]. Likewise, Shen et al substantiated that SS31 attenuates EBI following SAH owing to its antioxidant attributes[13]. Despite some investigations evaluating the neuroprotective efficacy of SS31 in SAH, its underlying mechanisms remain incompletely elucidated[26]. Further inquiries are imperative to elucidate the mechanisms through which SS31 exerts its actions in SAH before clinical trials can be contemplated.\u003c/p\u003e \u003cp\u003eAs acknowledged, Nrf2 represents a redox-sensitive transcription factor pivotal in regulating the antioxidant signaling cascade. Typically, Nrf2 forms a complex with Keap1 in the cytoplasm, where it undergoes degradation under basal physiological conditions. Upon encountering acute stressors, Nrf2 dissociates from Keap1 and translocates to the nucleus, orchestrating the transcriptional activities of antioxidant response elements (ARE) and ARE-associated gene products, such as HO-1 [27]. Multiple studies have highlighted the potential benefits of the Nrf2/Keap1 axis in mitigating EBI following SAH [27\u0026ndash;30]. This mechanism may operate through preserving mitochondrial functionality and suppressing oxidative stress. Wang et al. demonstrated SS31's ability to modulate oxidative stress status in aged brains via the Nrf2/Keap1 signaling pathway and confer protective effects against aging in vivo[31]. Numerous researchers have provided evidence that SS31 can enhance Nrf2 expression and safeguard liver and cardiac functions in animal models [32, 33].\u003c/p\u003e \u003cp\u003eAfter SAH in this investigation, levels of MDA were elevated, while the activities of GSH and endogenous antioxidant enzymes (SOD and CAT) were diminished, consistent with previous research findings. Additionally, two different doses of SS31 were administered to evaluate its impact on SAH. The results indicated that SS31 treatment attenuated lipid peroxidation reactions observed after SAH. The higher SS31 dose exhibited a more pronounced antioxidant effect, albeit without yielding additional improvements in neurological function compared to the lower dose. Furthermore, activation of the Nrf2/Keap1 pathway was observed following SAH, with reduced Keap1 expression and increased expression of Nrf2 and HO-1 noted in SAH rats after SS31 administration. These findings suggest that the Nrf2/Keap1 axis may contribute to modulating SS31's antioxidant properties.\u003c/p\u003e \u003cp\u003eComplementary experiments in SH-SY5Y cells demonstrated that SS31 treatment attenuated Hb-induced oxidative stress damage and suppressed cellular apoptosis. Mechanistically, SS31 concomitantly upregulated the expression of Nrf2 and HO-1 while downregulating Keap1, suggesting activation of ferroptosis-related pathways. These coordinated molecular changes further confirmed the neuroprotective properties of SS31.\u003c/p\u003e \u003cp\u003eFurthermore, it is pertinent to note that this investigation has delineated the protective impacts of SS31 on mitochondrial function and apoptosis mitigation. These findings hold significance in light of the established involvement of the Nrf2/Keap1 axis in modulating mitochondria-linked apoptosis, as evidenced in prior research. [34].\u003c/p\u003e \u003cp\u003eSeveral limitations of this study warrant consideration. Firstly, although SS31 was found to upregulate Nrf2/Keap1 expression and modulate the transcription of antioxidant enzymes, we did not assess the impact of SS31 on subarachnoid hemorrhage (SAH) in the absence of Nrf2. Secondly, we did not explore the potential involvement of other signaling pathways that might have contributed to the beneficial effects of SS31 on EBI following SAH. Lastly, further investigation is needed to elucidate the precise mechanism by which SS31 modulates Nrf2/Keap1 activation. Therefore, future research endeavors will prioritize addressing these crucial mechanistic inquiries.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our study revealed that SS31 facilitated the recovery of neurological function post-SAH in rats and inhibition of Hb-induced apoptosis, via attenuating oxidative stress-related neuronal demise through the Nrf2/Keap1 axis. Despite the need for additional investigation, this study underscored the potential efficacy of SS31 as a therapeutic candidate for SAH.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis project is supported by the Youth Scientific Research Guiding Project of Fujian Provincial Health Commission (No.2018-ZQN-80).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests regarding the publication of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT Authorship Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMing Liu: Writing – original draft. Jianchen Shen: Writing–Formal analysis. Liang Wu: Writing–review \u0026amp; editing. Meiqiu Liu: Validation. Cao Zeng and Zhiji Cai: Investigation. Guanghui Wu: Writing–review \u0026amp; editing. Jian Yin: Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets for this study are available on request to the corresponding authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlsbrook, D.L., et al., 2023. Neuroinflammation in Acute Ischemic and Hemorrhagic Stroke. Curr Neurol Neurosci Rep.\u003c/li\u003e\n \u003cli\u003eArmstrong, J.S., 2008. Mitochondria-Directed Therapeutics. Antioxidants \u0026amp; Redox Signaling. 10\u003cstrong\u003e,\u003c/strong\u003e 575-578.\u003c/li\u003e\n \u003cli\u003eBhatti, J.S., et al., 2021. Protective effects of a mitochondria-targeted small peptide SS31 against hyperglycemia-induced mitochondrial abnormalities in the liver tissues of diabetic mice, Tallyho/JngJ mice. Mitochondrion. 58\u003cstrong\u003e,\u003c/strong\u003e 49-58.\u003c/li\u003e\n \u003cli\u003eChaturvedi, R.K., Beal, M.F., 2008. 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Neuroprotective Role of the Nrf2 Pathway in Subarachnoid Haemorrhage and Its Therapeutic Potential. Oxidative Medicine and Cellular Longevity. 2019\u003cstrong\u003e,\u003c/strong\u003e 1-21.\u003c/li\u003e\n\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":"SS31, SAH, EBI, KEAP1, NRF2, HO-1","lastPublishedDoi":"10.21203/rs.3.rs-6797139/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6797139/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Subarachnoid hemorrhage (SAH) presents a significant clinical challenge with high mortality and disability rates. Oxidative stress (OS) plays a critical role in early brain injury post-SAH. SS31, an antioxidant agent, has shown promise in mitigating ischemia/reperfusion injury-related damage. This study aims to investigate the role and mechanism of SS31 in SAH, focusing on its impact on early brain injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: This study systematically investigated the neuroprotective mechanisms of SS31 through both in vivo and in vitro approaches. Initially, we assessed the anti-apoptotic efficacy of SS31 using a subarachnoid hemorrhage (SAH) rat model and an HB-induced SH-SY5Y microglial cell model, concurrently evaluating its impact on neurological functional recovery in SAH animals. Subsequently, we examined the regulatory effects of SS31 on the KEAP1/Nrf2/HO-1 signaling pathway through protein expression analysis in both experimental systems. Furthermore, ultrastructural and functional evaluations were conducted to characterize SS31's protective effects against mitochondrial impairment in SAH pathogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eSS31 treatment significantly mitigated early brain injury (EBI) following SAH, including BBB dysfunction, BE, neural cell apoptosis, OS, mitochondrial damage, and neurological deficits. SS31 also inhibits apoptosis, both in vivo and in cell models. At the same time, SS31 inhibited the expression of inflammatory factors in rat brain tissue and serum, which confirmed its anti-inflammatory effect. Furthermore, SS31 administration resulted in a notable reduction in Keap1 expression and a significant increase in Nrf2 and HO-1 expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e This study demonstrated that SS31 enhanced the recovery of nervous function after SAH in rats by mitigating OS-associated neuronal death via the Nrf2/Keap1 axis.\u003c/p\u003e","manuscriptTitle":"SS31 Mitigates Subarachnoid Hemorrhage-Induced Early Brain Injury via Nrf2/Keap1 Pathway Activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-06 15:42:03","doi":"10.21203/rs.3.rs-6797139/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":"3c4d8cde-e7cb-4347-a648-a0fcd4789895","owner":[],"postedDate":"June 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-16T13:53:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-06 15:42:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6797139","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6797139","identity":"rs-6797139","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}