Therapeutic effect and mechanism of glutathione on brain injury in sepsis model

Therapeutic effect and mechanism of glutathione on brain injury in sepsis model | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 9 March 2025 V1 Latest version Share on Therapeutic effect and mechanism of glutathione on brain injury in sepsis model Authors : hui lu , cailin wang 0009-0000-0350-5117 [email protected] , yong liu , xue xu , and xiangru wen Authors Info & Affiliations https://doi.org/10.22541/au.174151462.26804627/v1 283 views 126 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction caused by sepsis, with an unclear pathogenesis and limited treatment options. This study aimed to investigate the therapeutic effects of glutathione (GSH) in a lipopolysaccharide (LPS)-induced sepsis model and to elucidate its underlying mechanism of action. Adult C57BL/6 mice were intraperitoneally injected with LPS (25 mg/kg) to induce sepsis. Prior to LPS administration, GSH (100 mg/kg) was administered intraperitoneally for four consecutive days. Remarkably, GSH treatment significantly reduced mortality in the sepsis model. Behavioral experiments showed notable improvements in locomotor activity in GSH-treated mice, including increased total distance covered and more frequent crossings in the light/dark field test. Additionally, GSH treatment enhanced exploration, as indicated by an increased number of entries into the open arms of the elevated plus maze. In the tail suspension test, GSH treatment reduced resting time and instances of immobility, suggesting potential antidepressant effects. Histological analyses, including Nissl, HE, and immunofluorescence staining, revealed enhanced neuronal survival in the hippocampal CA1 and DG regions following GSH treatment. To explore the molecular mechanisms, Western blot analysis demonstrated that GSH reduced the release of inflammatory markers (IL-1β, IL-6, IL-10) and apoptosis-related proteins (Caspase-3 and cleaved Caspase-3). Furthermore, GSH downregulated PKA, phosphorylated PKA, and NF-κB, suggesting modulation of the PKA and NF-κB signaling pathways. These findings suggest that GSH mitigates neurological damage in sepsis by reducing inflammation and apoptosis, while also improving depressive behavior and cognitive function. GSH may represent a promising therapeutic approach for reducing mortality in SAE. Therapeutic effect and mechanism of glutathione on brain injury in sepsis model not-yet-known not-yet-known not-yet-known unknown Running title: Glutathione Alleviates Sepsis-Induced Brain Injury Cailin Wang1, Meifen Li3, Yong Liu4, Xue Xu1, Xiangru Wen5#, Hui Lu2# 1 Medical Research Center, People’s Hospital of Suzhou New District, Suzhou, Jiangsu, China 2 Department of Pain, Suzhou Hospital of Integrated Traditional Chinese and Western Medicine, Suzhou, Jiangsu, China 3 Department of Medical Microbiology, People’s Hospital of Suzhou New District, Suzhou, Jiangsu, China 4 Department of Orthopedics, People’s Hospital of Suzhou New District, Suzhou, Jiangsu, China 5 Research Center for Neurobiology and Department of Neurobiology, Xuzhou Medical College, Xuzhou, Jiangsu，China # Corresponding author. Hui Lu：Address: No. 39 Xiashatang, Wuzhong District, Suzhou, Jiangsu，215101, PR China. Email: [email protected] Xiangru Wen：Address:No. 209 Tongshan Road, Xuzhou, Jiangsu, 221004, PR China. Email: [email protected] Absrtact Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction caused by sepsis, with an unclear pathogenesis and limited treatment options. This study aimed to investigate the therapeutic effects of glutathione (GSH) in a lipopolysaccharide (LPS)-induced sepsis model and to elucidate its underlying mechanism of action. Adult C57BL/6 mice were intraperitoneally injected with LPS (25 mg/kg) to induce sepsis. Prior to LPS administration, GSH (100 mg/kg) was administered intraperitoneally for four consecutive days. Remarkably, GSH treatment significantly reduced mortality in the sepsis model. Behavioral experiments showed notable improvements in locomotor activity in GSH-treated mice, including increased total distance covered and more frequent crossings in the light/dark field test. Additionally, GSH treatment enhanced exploration, as indicated by an increased number of entries into the open arms of the elevated plus maze. In the tail suspension test, GSH treatment reduced resting time and instances of immobility, suggesting potential antidepressant effects. Histological analyses, including Nissl, HE, and immunofluorescence staining, revealed enhanced neuronal survival in the hippocampal CA1 and DG regions following GSH treatment. To explore the molecular mechanisms, Western blot analysis demonstrated that GSH reduced the release of inflammatory markers (IL-1β, IL-6, IL-10) and apoptosis-related proteins (Caspase-3 and cleaved Caspase-3). Furthermore, GSH downregulated PKA, phosphorylated PKA, and NF-κB, suggesting modulation of the PKA and NF-κB signaling pathways. These findings suggest that GSH mitigates neurological damage in sepsis by reducing inflammation and apoptosis, while also improving depressive behavior and cognitive function. GSH may represent a promising therapeutic approach for reducing mortality in SAE. Key words： Sepsis; SAE; GSH; PKA; NF-κB 1 Introduction Sepsis-associated encephalopathy (SAE) is a critical complication affecting the central nervous system (CNS) in patients with sepsis, characterized by diffuse or multifocal brain dysfunction resulting from systemic infection (Pan et al. 2022). Notably, SAE can occur even in the absence of direct brain infection, making its pathophysiology more complex (de Souza Stork et al. 2022). SAE is common among critically ill patients in the Intensive Care Unit (ICU), affecting up to 70% of individuals with severe systemic infections (Chavan et al. 2017). Its clinical manifestations range from mild neurological disturbances, such as epileptic episodes, to profound coma, with patients often experiencing behavioral, cognitive, arousal, and consciousness disturbances (Zheng et al. 2025). Moreover, many of those affected suffer from long-term cognitive impairments (Zhang et al. 2025). Despite its high prevalence in ICU settings, the exact pathogenesis of SAE remains poorly understood, and there are currently no effective treatments available (Forman et al. 2009). As a result, SAE represents a significant clinical challenge, garnering attention from the medical community worldwide. In addition to cognitive dysfunction, depression is one of the most debilitating complications of sepsis-induced brain dysfunction, contributing to increased morbidity and mortality (Algahtani et al. 2023). Depression in this context remains a major therapeutic challenge, as current treatments can only manage the symptoms rather than address the underlying causes. This highlights an urgent need to better understand the regulatory mechanisms behind sepsis-induced depression and to identify potential treatments that can effectively target these behavioral disturbances. Addressing both the pathogenesis and treatment of sepsis-induced depression has thus become a critical area of research (Montague-Cardoso 2021). Inflammatory mediators, including cytokines and the complement system, as well as disruptions in cellular signaling pathways, are believed to play a key role in the onset and progression of SAE (Lawrence 2009). Among the key players in this process, NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) serves as a central transcription factor involved in the immune and inflammatory responses. It regulates the expression of several pro-inflammatory genes, such as TNF, IL-1α, IL-1β, IL-6, cyclooxygenase-2 (COX-2), and iNOS, thereby contributing to the inflammatory cascade (Yang et al. 2025). Another important pathway, the PKA (protein kinase A) signaling pathway, plays a crucial role in modulating cellular responses to inflammation. Through the regulation of cyclic AMP (cAMP) levels, PKA influences the activation of various signaling molecules that can suppress or enhance inflammatory responses, depending on its activation status (Serezani et al. 2008). While activation of the PKA pathway has been shown to suppress inflammation, inhibition of PKA can exacerbate the inflammatory response, suggesting its potential as a therapeutic target (Lee et al. 2019). Glutathione (GSH), a naturally occurring tripeptide, plays a crucial role in maintaining immune function, regulating oxidative stress, apoptosis, and modulating several intracellular signaling pathways (Wu et al. 2004). Previous clinical trials have shown that GSH can improve the therapeutic outcomes of sepsis, particularly for organ systems such as the lungs, liver, and kidneys (Eve et al. 2020). However, its specific effects on the brain during sepsis, especially in relation to neuronal damage and neuroinflammation, remain underexplored. Based on these considerations, we hypothesize that GSH may alleviate neuroinflammation and neuronal damage in the hippocampus by enhancing the cAMP/PKA signaling pathway and inhibiting NF-κB activation. This, in turn, could help mitigate depressive behavior associated with SAE. This study aims to test this hypothesis by investigating the therapeutic effects of GSH on sepsis-induced brain injury, providing new insights into its potential as a treatment for SAE. not-yet-known not-yet-known not-yet-known unknown 2 METHODS not-yet-known not-yet-known not-yet-known unknown 2. 1 Reagents Lipopolysaccharides(LPS) and glutathione (GSH) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The following primary antibodies were used: anti-PKA, polyclonal anti-p-PKA (Thr198), anti-IL-1β, anti-IL-6, anti-NF-κB, mouse monoclonal anti-Caspase-3 (CASP3), and goat monoclonal anti-IL-10, all obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A rabbit polyclonal anti-cleaved-Caspase-3 (cleaved-CASP3) antibody was purchased from Cell Signaling Technology (Beverly, MA, USA). The secondary antibodies used in this study were also purchased from Sigma-Aldrich. 2. 2 Animal model Male C57BL/6 mice (2-3 months, 20 ± 5 g) were obtained from the Experimental Animal Center of Xuzhou Medical College. They were housed under standard conditions with free access to food and water at 21°C on a 12-hour light/dark cycle. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Xuzhou Medical University. Sepsis Model: Sepsis was induced by a single intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) (25 mg/kg) dissolved in normal saline. Mice in the control (CON) group received an equivalent volume of saline. Glutathione Treatment: Mice in the GSH treatment group received daily i.p. injections of GSH (100 mg/kg) for four days before LPS administration.(See Figure S1 for details). 2.3 Pathological behavioral scoring Pathological behavioral scoring was conducted to assess the effects of LPS injection on mice, focusing on piloerection, ptosis, and activity level. Piloerection was scored as 0 (no piloerection), 1 (moderate piloerection), or 2 (extensive piloerection). Ptosis was scored as 0 (normal or >50% open), 1 (slight ptosis, 1/3–1/2 closed), or 2 (severe ptosis, <1/3 open). Activity was scored as 0 (active behavior), 1 (inactivity), or 2 (lethargy or curled up). This scoring was used to evaluate the impact of LPS and treatments, minimizing external disturbances during assessment（Table S1). not-yet-known not-yet-known not-yet-known unknown 2.4 Open-field and closed-field test Depression-like behavior was assessed using the Open-Field Test (OFT) and Closed-Field Test (CFT) based on the method of Prut & Belzung (2003). Open-Field Test (OFT): Mice were placed at the center of an open-field apparatus (50*50*30 cm) and allowed to acclimate for 3 minutes. Their free-moving behavior was then recorded for 5 minutes using an open-field tracking system. Activity was quantified in terms of total movement time, total distance traveled, distance in the central area, and time spent in the central area. Closed-Field Test (CFT): Following the OFT, mice were placed in a dark, closed-box of the same size, and behavior was recorded as in the OFT.The experiment was conducted in a quiet environment to minimize disturbances. 2. 5 Elevated plus maze test The EPMT, used to assess depression-like behavior, consists of a cross-shaped elevated platform (64*64 cm) with two open arms and two closed arms. Mice were acclimated to the test room for 1 hour before being placed at the center of the maze, facing the open arm. They were allowed to explore for 5 minutes while their behavior was recorded. The system tracked entries into the open and closed arms and time spent in the open arms. Parameters calculated included the percentage of time spent in the open arms and total entries into both arms. After each trial, the maze was cleaned with 75% ethanol and allowed to dry before the next test. The experiment was conducted in a quiet environment to minimize disturbances. 2.6 Tail suspension test In the tail suspension test, the tip of the mouse’s tail (1.0–1.5 cm) was affixed to a metallic surface using medical adhesive tape, and the mouse was suspended 30 cm above the surface for 6 minutes under 100 lux illumination. During the test, two parameters were recorded: immobility, defined as the total time the mouse remained immobile for more than 2 seconds, and latency, the time taken for the mouse to first exhibit immobility after being suspended. 2.7 Nissl Staining and HE-staining Mice were transcardially perfused with 200 mL ice-cold phosphate-buffered saline, followed by 300 mL of 10% formalin. After 48 hours of fixation, brains were dehydrated, embedded in paraffin, and sectioned into 10-µm slices. Hippocampal sections were stained with Nissl stain (Solarbio, China) following the manufacturer’s instructions. Surviving pyramidal neurons in the CA1–CA3 and DG regions were counted at ×400 magnification. For HE staining, 5-µm brain sections were prepared to assess hippocampal cell damage. 2.8 Immunohistochemistry Frozen mouse brain sections (30 μm) were washed 3-4 times with 0.1 M PBS for 5 minutes each, then incubated in blocking solution containing 5% bovine serum albumin (BSA) for 2 hours at room temperature. After blocking, sections were incubated overnight at 4°C with primary antibodies: anti-GFAP (1:100, Abcam), and either anti-NeuN or anti-BrdU (1:150) in 10% goat serum diluted in PBS. The next day, sections were washed 3-4 times with PBS for 5 minutes each and incubated with appropriate anti-rabbit secondary antibodies for 2 hours at room temperature. After washing, the sections were counterstained with DAPI (1:2000, Sigma) for 3 minutes to label the nuclei. Finally, sections were mounted and observed under a fluorescence microscope. 2.9 Western Blot Analysis Hippocampal tissues from each group of 10 mice were homogenized in RIPA lysis buffer containing 1% PMSF. After adding SDS loading buffer, samples were boiled for 5 minutes and proteins were separated by SDS-PAGE. Following electrophoresis, proteins were transferred to a membrane, which was blocked with skimmed milk to reduce nonspecific binding. The membrane was incubated overnight at 4°C with primary antibodies (PI3K, P-PI3K, Pan-AKT, AKT1, caspase-3, and cleaved caspase-3). After washing with buffer, the membrane was incubated with a secondary antibody (1:200) for 2 hours. Protein bands were scanned and analyzed using Quantity One software (BIO-RAD, USA). not-yet-known not-yet-known not-yet-known unknown 2.10 Statistical Analysis Densitometric analysis of immunofluorescence and Western blot bands was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Data were analyzed with GraphPad Prism 7 (GraphPad Software, CA, USA) and images were processed using Photoshop CS6. Statistical analyses were conducted using SPSS version 13.0 for Windows. Data are presented as mean ± SEM. One-way ANOVA followed by the Newman-Keuls test was used for statistical comparisons, with P < 0.05 considered significant. In behavioral tests, all animals were treated as independent samples. 3 RESULT not-yet-known not-yet-known not-yet-known unknown 3.1 GSH Significantly Improves Mortality and Pathological Behavior in LPS-Induced Sepsis Model To assess the effect of GSH pretreatment on sepsis mortality, mouse deaths within 6 days of LPS injection were recorded (Figure 1A). Survival analysis showed that 24 hours post-LPS injection, the survival rate was 50% in the untreated LPS group, while the GSH-pretreated LPS+GSH group had a survival rate of 60%, with significant differences between the groups (NS vs LPS: p < 0.01; LPS vs LPS+GSH: p < 0.01). Pathological scoring on day 12 revealed functional impairments in both LPS and LPS+GSH groups, but no deficits in Saline and GSH groups. The LPS group had significantly higher scores than the Saline group (p < 0.001), and LPS-treated mice had worse scores than the LPS+GSH group (p < 0.001), indicating the protective effect of GSH(Figure 1B). 3.2 Effect of GSH on depression-like behavior of LPS-induced SAE model To evaluate the protective effect of GSH on LPS-induced depression-like behavior, a series of behavioral tests were conducted, including the Open-Field, Closed-Field, Elevated Plus Maze, and Tail Suspension Test. In the Open-Field and Closed-Field Tests, LPS-treated mice showed reduced movement and fewer crossings compared to controls (total distance: P = 0.008; number of crossings: P < 0.001), while GSH treatment restored spontaneous activity, with a significantly greater distance traveled in the open field (P = 0.009) and a reduced number of crossings compared to the LPS group (P < 0.001)(Figure 2A-C). In the Elevated Plus Maze, the LPS group exhibited less time spent in the open arms (P = 0.001) and fewer entries (P = 0.046), indicative of anxiety-like behavior, but GSH treatment increased both the time spent (P = 0.143) and the number of entries (P < 0.001) into the open arms(Figure 2D-F). The Tail Suspension Test further confirmed GSH’s impact, with LPS-treated mice displaying increased immobility (P < 0.05), a hallmark of depression-like behavior, which was significantly reduced in the GSH-treated group (P < 0.05). These findings demonstrate that GSH effectively improves depression-like and sickness behaviors in the LPS-induced sepsis model (Figure 2G-I). 3.3 GSH improves the damage of neurons in the hippocampus of the LPS-induced brain GSH treatment significantly mitigated neuronal damage in the hippocampus of the LPS-induced sepsis model. Nissl and HE staining revealed substantial neuronal degeneration in the CA1 region following LPS injection, characterized by disorganized neurons with blurred Nissl bodies and irregular cell structures. However, GSH pre-treatment reduced neuronal degeneration, preserving neuron morphology and increasing survival. In contrast to the LPS group, the GSH-treated group showed fewer signs of cellular damage, with more intact pyramidal cells and reduced nuclear shrinkage (Figure 3 and 4). Immunofluorescence staining for NeuN further confirmed these results, showing a notable increase in the number of surviving neurons in the GSH-treated group compared to the LPS group. Although neuronal survival in the GSH group was lower than in the control group, the difference was statistically significant, highlighting GSH’s protective effect on hippocampal neurons against LPS-induced damage (Figure5). 3.4 Protective Effects of GSH Against LPS-Induced Neuronal Damage and Inflammation in Sepsis To investigate the protective effects of GSH in the LPS-induced sepsis model, we focused on its impact on neuronal damage, inflammation, and key cellular signaling pathways. Our findings demonstrated that GSH significantly reduced neuronal apoptosis in the hippocampus, as shown by lower levels of caspase-3 and cleaved caspase-3 expression in both the saline and LPS+GSH groups compared to the LPS group (P < 0.05), suggesting that GSH helps preserve neuronal survival in the context of sepsis(Figure 6A and a). In addition to reducing apoptosis, GSH also alleviated the inflammatory response. We observed that GSH treatment significantly reduced the expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in the hippocampus (P < 0.05), further supporting its anti-inflammatory effects (Figure 6D-G and 6d-g). This effect was likely mediated through the inhibition of the NF-κB pathway. GSH pretreatment lowered the phosphorylation of IKKβ in the cytoplasm and prevented the nuclear translocation of p65, indicating a suppression of NF-κB activation (P < 0.05). Moreover, GSH appeared to modulate the PKA signaling pathway, which is involved in cellular responses to inflammation. Both the saline and LPS+GSH groups showed significantly higher phosphorylation of PKA and CREB compared to the LPS group (P < 0.05), suggesting that GSH may also exert its protective effects through the activation of this pathway(Figure6B and b). Overall, GSH protects against LPS-induced neuronal damage and inflammation, likely through the modulation of multiple signaling pathways, including NF-κB and PKA. These findings highlight GSH’s potential as a therapeutic agent in sepsis-related brain injury. 4 DISCUSSION Sepsis-associated encephalopathy (SAE) represents a major complication of sepsis, contributing to severe cognitive dysfunction and depression in affected patients. Despite its significant impact on patient outcomes, the underlying pathogenesis of SAE remains poorly understood, and no specific treatments are currently available (Lu et al. 2025). Our study investigates the neuroprotective potential of Glutathione (GSH) in a murine model of sepsis, providing valuable insights into its role in mitigating the neuroinflammatory and neurodegenerative consequences of sepsis. Our findings demonstrate that GSH pre-treatment significantly improves survival in a LPS-induced sepsis model, underscoring the therapeutic potential of GSH in alleviating both systemic and neurological effects of sepsis. Notably, GSH treatment resulted in a 10% increase in survival rate compared to untreated LPS mice, suggesting a critical role in modulating the inflammatory response and preventing organ dysfunction (Wang et al. 2024). Furthermore, pathological assessments revealed that GSH treatment substantially improved the pathological behavior and reduced functional impairment in the mice. These results support the hypothesis that GSH may play a pivotal role in protecting against sepsis-induced brain damage, particularly in the hippocampus, a key region involved in cognitive functions (Hong et al. 2023). Behavioral analyses also revealed that GSH significantly alleviated depression-like behaviors in the sepsis model. Mice treated with GSH exhibited increased activity in open-field and closed-field tests, with more frequent crossings and a reduced tendency to remain immobile compared to the LPS group. These improvements were consistent with histological data, which showed significantly reduced neuronal damage in the hippocampal CA1 and DG regions. This suggests that GSH helps preserve hippocampal neurons, likely preventing the cognitive deficits associated with SAE (Krzyzaniak et al. 2023). Further investigation into the inflammatory response revealed that GSH pre-treatment reduced the levels of key inflammatory mediators, including TNF-α, IL-1β, and IL-6, in the hippocampus. These cytokines are critical drivers of neuroinflammation and are known to exacerbate neuronal damage in sepsis (Jiang & Li 2024). By inhibiting the release of these inflammatory mediators, GSH appears to exert a significant anti-inflammatory effect that could play a major role in protecting the brain during sepsis. At the molecular level, our study suggests that GSH exerts its neuroprotective effects primarily through the inhibition of the NF-κB signaling pathway. NF-κB is a central regulator of inflammation and is involved in the transcription of pro-inflammatory genes. Our results show that GSH significantly suppressed the phosphorylation and nuclear translocation of p65, a key subunit of NF-κB, thereby preventing the activation of pro-inflammatory target genes (He et al. 2024). This inhibition of NF-κB signaling may explain GSH’s ability to attenuate neuroinflammation and protect against neuronal death during sepsis. Although our study primarily focused on the anti-inflammatory mechanisms of GSH, it is worth noting that the antioxidant properties of GSH may also contribute to its neuroprotective effects, as GSH is a well-known antioxidant. However, since we did not specifically investigate the antioxidant capacity of GSH in this study, future research will be necessary to elucidate the potential role of oxidative stress in the protective effects observed (Zhang et al. 2016). In conclusion, our study provides strong evidence that GSH pre-treatment offers significant neuroprotection in a LPS-induced sepsis model. By reducing inflammation and preventing neuronal damage, GSH demonstrates considerable therapeutic potential for mitigating the devastating effects of SAE. These findings not only reveal the molecular mechanisms through which GSH exerts its protective effects but also pave the way for future studies that could investigate the clinical application of GSH in treating sepsis patients with SAE. Given its potential to improve survival and reduce long-term cognitive impairment, GSH offers a promising intervention for sepsis-associated encephalopathy. Conflict of interest All authors state that there is no conflict of interest. Acknowledgments The People’s Hospital of SND (SGY2021A01); Suzhou people’s Livelihood Science and technology guidance project under Grant (SKJY2021060); References [1] Algahtani, M. M., S. Alshehri, S. S. Alqarni, et al. 2023. ”Inhibition of ITK Signaling Causes Amelioration in Sepsis-Associated Neuroinflammation and Depression-like State in Mice.” Int J Mol Sci 24(9):. https://doi.org/10.3390/ijms24098101. [2] Chavan, S. S., V. A. Pavlov, and K. J. Tracey. 2017. ”Mechanisms and Therapeutic Relevance of Neuro-immune Communication.” Immunity 46(6): 927-942. https://doi.org/10.1016/j.immuni.2017.06.008. [3] de Souza Stork, S., M. Hubner, E. Biehl, et al. 2022. ”Diabetes Exacerbates Sepsis-Induced Neuroinflammation and Brain Mitochondrial Dysfunction.” Inflammation 45(6): 2352-2367. https://doi.org/10.1007/s10753-022-01697-y. [4] Eve, A. A., X. Liu, Y. Wang, M. J. Miller, E. H. Jeffery, and Z. 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Hu. 2025. ”Molecular Mechanism of METTL3 Regulating Hippocampal Neuronal Injury Induced by Sepsis-Associated Encephalopathy.” Arch Physiol Biochem 1-11. https://doi.org/10.1080/13813455.2025.2465337. [21] Zheng, G., J. Yan, W. Li, and Z. Chen. 2025. ”Frailty as an Independent Risk Factor for Sepsis-Associated Delirium: A Cohort Study of 11,740 Older Adult ICU Patients.” Aging Clin Exp Res 37(1): 52. https://doi.org/10.1007/s40520-025-02956-2. not-yet-known not-yet-known not-yet-known unknown Legends: Fig. 1 Survival curve and Neurological scores (A) Survival curve showing the survival rates of mice from four groups over four days post-LPS injection: saline (blue), LPS (red), LPS + GSH (green), and GSH (yellow).(B) Neurological scores over time for the four groups: saline (●, purple), LPS (■, red), LPS + GSH (▲, blue), and GSH (▼, green). Data are presented as mean ± SEM (n = 12). *P < 0.05 vs. saline, #P < 0.05 vs. LPS. Fig. 2 Behavioral test (A) The autonomous trajectory map of mice in the open and closed fields after 14 days of LPS (i.p.) injection. (B) Total distance traveled by mice in open and closed fields. (C) Number of crossings in the open and closed fields. (D) Autonomous trajectory map of mice in the cross maze 24 hours after LPS (i.p.) injection. (E) Percentage of time spent in the open arm. (F) Percentage of time spent in the open arm during the median time. (G) Time spent still. (H) Number of times the mouse remained still. Data are expressed as mean ± SEM (n = 20). *P < 0.05 vs saline group, #P < 0.05 vs LPS group. Fig.3 Nissl Staining of Hippocampal CA1 and DG Regions (A) Representative Nissl-stained images of hippocampal CA1 and DG regions. Panels a, b, c, and d represent saline, LPS, LPS+GSH, and GSH groups, with magnified views (x40).(B) Quantification of apoptotic cells in the CA1 region.(C) Quantification of apoptotic cells in the DG region.Data are expressed as mean ± SEM (n = 6). *P < 0.05 vs saline group, #P < 0.05 vs LPS group. Fig.4 HE Staining of Hippocampal CA1 and DG Regions (A) Representative HE-stained images of hippocampal CA1 and DG regions. Panels a, b, c, and d represent saline, LPS, LPS+GSH, and GSH groups, with magnified views (x40).(B) Quantification of apoptotic cells in the CA1 region.(C) Quantification of apoptotic cells in the DG region.Data are expressed as mean ± SEM (n = 6). *P < 0.05 vs saline group, #P < 0.05 vs LPS group. Fig.5 Immunofluorescence staining (A) Representative photomicrographs of the saline group. Panels A1-A4 show NEUN, BRDU, DAPI, and merged staining of the hippocampal CA1 region (×40). A1-a4 show fluorescent staining of NEUN, BRDU, DAPI, and merge of the hippocampal DG region. (B) Representative photomicrographs of the LPS group. Panels B1-B4 and b1-b4 correspond to the saline group in A. (C) Representative photomicrographs of the LPS+GSH group. Panels C1-C4 and c1-c4 correspond to the saline group in A. (D) Quantitative analysis of the number of BrdU-positive cells per unit area in the hippocampal DG. (E) Quantitative analysis of NEUN and DAPI ratio per unit area of the hippocampal CA1. (F) Quantitative analysis of NEUN and DAPI ratio per unit area of the hippocampal DG. Data are expressed as mean ± SEM (n = 6). *P < 0.05 vs saline group, #P < 0.05 vs LPS group. Fig6. Western blot (A, a) Activation of Caspase 3 and Cleaved Caspase 3 in the hippocampus 14 days after LPS injection was analyzed by immunoblot. (A) Scanning immunoblot bands. (a) The intensity of the bands is expressed as optical density (O.D.).(B, b) Expression changes in P65 in the cytoplasm and phosphorylation levels of IKKβ 14 days after LPS injection, analyzed by immunoblot. (B) Scanning immunoblot bands. (b) The intensity of the bands is expressed as optical density (O.D.).(C, c) Immunoblot analysis of PKA and p-PKA levels in the hippocampus 14 days after LPS injection. (C) Scanning immunoblot bands. (c) The intensity of the bands is expressed as optical density (O.D.).(D, d) Expression of IL-1β, IL-6, IL-10, and TNF-α in the hippocampus 4 days after LPS injection was analyzed by immunoblot. (D) Scanning immunoblot bands. (d) The intensity of the bands is expressed as optical density (O.D.).(Data are expressed as mean ± SEM, n = 6. *P < 0.05 vs saline group, #P < 0.05 vs LPS group.) not-yet-known not-yet-known not-yet-known unknown Fig S1. Experimental flowchart Table S1. Pathological Scoring Supplementary Material File (figure1.tif) Download 830.88 KB File (figure3.tif) Download 10.10 MB File (figure4.tif) Download 13.22 MB File (figure5.tif) Download 2.92 MB File (figure6.tif) Download 17.30 MB File (table s1.docx) Download 11.80 KB Information & Authors Information Version history V1 Version 1 09 March 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords gsh nf-κb pka sae sepsis Authors Affiliations hui lu Suzhou Hospital of Integrated Traditional Chinese and Western Medicine View all articles by this author cailin wang 0009-0000-0350-5117 [email protected] People’s Hospital of Suzhou New District, Suzhou, Jiangsu, China View all articles by this author yong liu People’s Hospital of Suzhou New District View all articles by this author xue xu People’s Hospital of Suzhou New District View all articles by this author xiangru wen Xu Zhou Medical College View all articles by this author Metrics & Citations Metrics Article Usage 283 views 126 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation hui lu, cailin wang, yong liu, et al. Therapeutic effect and mechanism of glutathione on brain injury in sepsis model. Authorea . 09 March 2025. 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