Salvianolic Acid B Inhibits Ferroptosis Through ACSL4 Pathway Regulation in sepsis-associated encephalopathy Mice

Salvianolic Acid B Inhibits Ferroptosis Through ACSL4 Pathway Regulation in sepsis-associated encephalopathy Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Salvianolic Acid B Inhibits Ferroptosis Through ACSL4 Pathway Regulation in sepsis-associated encephalopathy Mice Xingyao Li, Zhizhun Mo, Yumei Yang, Chunxia Zhao, Shuwen Zhang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7925353/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Apr, 2026 Read the published version in Molecular Neurobiology → Version 1 posted 12 You are reading this latest preprint version Abstract Sepsis frequently leads to multi-organ injury, with the highly metabolically active nervous system being particularly vulnerable, and ferroptosis has been implicated in driving disease progression. Although Salvianolic acid B (SalB), the most abundant water-soluble active component of Salvia miltiorrhiza Bunge., has demonstrated antioxidant and anti-inflammatory properties, its specific mechanisms in sepsis-associated hippocampal injury remain unclear. To investigate SalB's therapeutic potential against sepsis and its role in mitigating neural damage via ACSL4-mediated ferroptosis, a murine sepsis model was established by cecal ligation and puncture (CLP). SalB's efficacy was evaluated using 7-day survival rates, multi-organ biochemical markers, and critical treatment windows. Inflammatory cytokines were measured by ELISA, and hippocampal morphology was examined histologically. Mechanistic studies included Fe²⁺ staining and lipid peroxidation assays, while protein arrays and Western blotting clarified SalB's interaction with ACSL4, confirming its anti-ferroptotic role. Our results show that SalB significantly improved survival in CLP-induced septic mice, reduced levels of inflammatory factors, alleviated hippocampal neuronal damage, and preserved blood-brain barrier integrity. Data from biochemical assays and Western blot analysis indicated that SalB suppresses ferroptosis by modulating the ACSL4/GPX4 pathway, supporting its therapeutic role in septic hippocampal injury. Additionally, protein array and molecular docking studies provided evidence that SalB likely exerts its pharmacological activity by competitively inhibiting the substrate CoA binding to ACSL4 at amino acid residues LYS-572, LEU-574, and SER-607. In conclusion, SalB protects against sepsis-induced hippocampal injury by targeting ACSL4-mediated ferroptosis, offering a novel herbal-based strategic direction for sepsis treatment. Sepsis sepsis-associated encephalopathy Salvianolic acid B Ferroptosis Neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1 Introduction Sepsis is defined as a life-threatening systemic inflammatory response syndrome caused by a dysregulated host reaction to bacterial, fungal, or viral infections [1], which severely damages the lungs, heart, liver, and other organs, potentially leading to fatal outcomes. The central nervous system (CNS) is recognized as one of the earliest organs affected by sepsis. Clinically, CNS involvement manifests as sepsis-associated encephalopathy (SAE), a diffuse brain dysfunction secondary to systemic infection in sepsis. SAE is characterized by sepsis-related diffuse cerebral impairment detected through clinical or standard laboratory evaluations, in the absence of direct CNS infection, structural abnormalities, or other encephalopathies (e.g. hepatic or renal encephalopathy)[2]. As one of the most severe complications during both acute sepsis and recovery, SAE involves pathological alterations such as neuroinflammation, microcirculatory disturbances, and metabolic failure [3]. Recent studies have linked ferroptosis to glutamate-mediated excitotoxicity and neuronal injury during SAE pathogenesis [4]. Evidence from cecal ligation and puncture (CLP)-induced septic mice reveals reduced GPX4 expression, elevated transferrin and malondialdehyde (MDA) levels, and mitochondrial shrinkage in the brain [5]. Ferroptosis is also observed in the hippocampus, marked by increased reactive oxygen species (ROS) and Fe 2+ content, decreased glutathione (GSH) levels, upregulated long-chain acyl-CoA synthetase 4 (ACSL4) expression, and downregulated glutathione peroxidase 4 (GPX4) protein levels. Clinically, these changes correlate with aggravated cognitive deficits, reduced survival rates, and exacerbated mitochondrial damage in hippocampal neurons [6]. Current clinical management of SAE primarily focuses on treating underlying infections, administering antibiotics, and providing supportive care [7]. Salvianolic acid B (SalB) is the most biologically active water-soluble component of Salvia miltiorrhiza Bunge and exhibits potent anti-inflammatory[8] and anti-lipid peroxidation effects[9]. SalB is a polyphenolic compound formed by the condensation of three danshensu molecules and one caffeic acid. Preclinical and clinical studies indicate that SalB attenuates inflammation, suppresses pro-inflammatory cytokines [10], enhances superoxide dismutase (SOD) activity, inhibits MDA production, and scavenges ROS [11], thereby protecting tissue integrity. However, existing research on SalB in sepsis has predominantly focused on cardiovascular protection, with limited exploration of its regulatory mechanisms and key targets in sepsis-associated neurological dysfunction. This study aims to elucidate the molecular mechanisms underlying SalB’s ameliorative effects on sepsis-induced SAE. First, we evaluated SalB’s impact on survival rates, behavioral phenotypes, histopathological changes, and serum inflammatory responses in septic mice. Next, ferroptosis-related biomarkers and ACSL4/GPX4 pathway proteins were analyzed to validate SalB’s regulatory role in ferroptosis. Finally, molecular docking was employed to predict SalB-ACSL4 interaction sites, further clarifying SalB’s pharmacological mechanism in SAE treatment. 2 Materials and methods 2.1 Experimental materials Salvianolic acid B (SalB, CAS No.: 121521-90-2) was purchased from Target Molecule Corp.(TargetMol). (Product No.: T5725, purity: 99.86%). Ferroptosis inhibitor Ferrostatin-1 (Fer-1, CAS No.: 347174-05-4) was procured from the same supplier (Product No.: T6500, purity: 99.68%). All compounds were freshly prepared as stock solutions immediately prior to experimental use. 2.2 Experimental animals and modeling SPF grade C57BL/6 J mice, 8 weeks old (20-22 g), were used in this study, and the experimental animals were purchased from Beijing Huafukang Biotechnology Company. The mice were housed under controlled environmental conditions: ambient temperature 20-26℃ (daily fluctuation ≤ 4℃), relative humidity 40-70%, and a 12/12-hour light/dark cycle. All animals were provided ad libitum access to autoclaved feed and sterile water. The ethical approval number is BJTCM-M-2023-11-04. Sepsis was induced using the CLP procedure. Briefly, mice were anesthetized with 1% sodium pentobarbital, followed by a 1 cm midline laparotomy to expose the cecum. The proximal third of the cecum was ligated with surgical suture and punctured once through an avascular region using an 18-gauge needle. A small volume of fecal content was gently extruded from the puncture site, after which the cecum was carefully repositioned into the abdominal cavity, and the incision was closed with layered sutures. Postoperative fluid resuscitation was administered via subcutaneous injection of 0.9 % sterile saline (2 mL). Sham-operated controls underwent identical surgical procedures excluding cecal ligation and puncture. 2.3 SalB Treatment Protocol Following a 7-day acclimatization period, mice were administered treatments via intraperitoneal injection (Fig 1). SalB treatment groups received doses of 20 mg/kg (high dose), 10 mg/kg (medium dose), or 5 mg/kg (low dose) in CLP-induced septic mice. The positive control group was treated with Ferrostatin-1 (Fer-1) at a dose of 5 mg/kg in CLP-induced mice. All treatments were administered once daily, with the first dose given 2 hours post-surgery and continued for 3 consecutive days. Survival rates were monitored over a 7-day period. CLP and sham-operated control groups received an equivalent volume of sterile saline. To further investigate the underlying mechanisms, the above grouping and modeling procedures were replicated. At 48 hours post-surgery, mice were euthanized, and experimental samples were collected for subsequent analysis. 2.4 Behavioral Testing In accordance with established literature, two behavioral scoring systems were employed for each group of mice: the Murine Sepsis Score (MSS) [ 12 ] and the modified Neurological Severity Score (mNSS) [ 13 ]. These assessments were conducted to evaluate sepsis severity and neurological functional deficits, respectively. 2.5 Biochemical Analysis The expression levels of Fe 2+ , lipid peroxides (LPO), and reduced GSH in hippocampal tissues were quantified using commercially available assay kits (Elabscience Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer’s instructions. Additionally, the total protein concentration in liver tissues was determined using a BCA protein assay kit (Thermo Fisher Scientific Technology Co., Ltd., Shanghai, China). 2.6 Histological Assessment For histological assessment, whole brain tissues were fixed in 4% paraformaldehyde, followed by standard processing steps including dehydration, paraffin embedding, and sectioning. Tissue sections were stained with hematoxylin and eosin (H&E) and Nissl staining for morphological analysis. Fe 2+ staining was performed using a commercially available Prussian blue staining kit (Solarbio Science & Technology Co., Ltd., Beijing, China; Product No.: G1429) according to the manufacturer’s protocol. Finally, the stained sections were examined and imaged under an optical microscope for detailed histological evaluation. 2.7 Evans Blue Staining A 2% Evans blue dye solution and a heparinized saline solution were prepared for subsequent use. Mice were anesthetized with 1% sodium pentobarbital, and a 2% Evans blue dye solution (50 mg/kg) was injected into the orbital venous plexus. After a 2-hour circulation period, the thoracic cavity was opened, and 0.9% heparinized saline was slowly perfused through the left ventricular apex. The right atrium was incised to allow drainage until the effluent became clear. Brain tissues were carefully dissected, and the hippocampal region was exposed through precise sectioning. Gross photographs of the brain were taken for further analysis. 2.8 Western blotting (WB) Hippocampal tissues were homogenized, lysed, and centrifuged to extract total protein. Protein concentrations were quantified using a BCA protein assay kit (Thermo Fisher Scientific Technology Co., Ltd., Shanghai, China). Equal amounts of protein samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were blocked with rapid blocking solution (Beyotime, P0252-500 mL, Shanghai, China) at room temperature for 20 minutes, followed by overnight incubation at 4℃ with the following primary antibodies: ACSL4 (1:10,000, ab155282, Abcam, Cambridge, United Kingdom), LPCAT3 (1:3,000, 67882-1-Ig, Proteintech, Wuhan, China), ALOX15 (1:1,000, ab244205, Abcam, Cambridge, United Kingdom), GPX4 (1:5,000, ab125066, Abcam, Cambridge, United Kingdom), SLC7A11 (1:1,000, ab307601, Abcam, Cambridge, United Kingdom), β-actin (1:50,000, 66009-1-Ig, Proteintech, Wuhan, China), and GAPDH (1:80,000, 81640-5-RR, Proteintech, Wuhan, China). After washing with 1×TBST, membranes were incubated with secondary antibodies at room temperature for 1 hour: goat anti-rabbit IgG (1:10,000, B900210, Proteintech, Wuhan, China) and rabbit anti-mouse IgG (1:10,000, B900120, Proteintech, Wuhan, China). Membranes were washed again with 1×TBST, and protein bands were visualized using the Tanon-5200 Multi Gel Imaging System. Band intensities were quantified using ImageJ software (version 1.8.0). 2.9 Enzyme-linked immunoassay (ELISA) Serum levels of IL-1β, IL-4, IL-10, and TNF-α were measured using commercially available mouse cytokine ELISA kits (Meimian Industrial Co., Ltd., Jiangsu, China) according to the manufacturer’s instructions. Reactions were monitored within 15 minutes, and the optical density (OD) of each well was measured at a wavelength of 450 nm using a microplate reader. Data were recorded, and standard curves were generated for quantitative analysis. 2.10 Molecular Docking The small molecule SalB and coenzyme A (CoA) were docked with the pathway protein ACSL4. The three-dimensional (3D) structures of the compounds were retrieved from the PubChem database. The crystal structure of the protein receptor (ACSL4) was obtained from the UniProt protein database (https://www.uniprot.org/). Molecular docking was performed using AutoDock (version 4.2.6), and the results were visualized and analyzed using PyMOL for comparative assessment. 2.11 Statistical analysis Data are expressed as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 8 (GraphPad, La Jolla, USA). One-way analysis of variance (ANOVA) was employed to assess intergroup differences, with statistical significance denoted by an asterisk (*) for P < 0.05. 3 Results 3.1 Time-Dependent Changes of ferroptosis Biomarkers Induced by the CLP Model To investigate the development of ferroptosis in various organs of septic mice over different time periods, we conducted a CLP model. Observations were made at 12, 24, 48, and 72 hours post-surgery (Fig 2 B), during which we assessed the abdominal state of the mice and collected samples from the liver, kidneys, and hippocampus to measure iron ions, GSH, and LPO levels. We aimed to observe the relative expression trends of ferroptosis-related indicators across different time points in comparison to the control group. The results indicated that inflammation and swelling in the cecum of the CLP-operated mice began at 12 hours post-surgery, with purulent changes emerging at 24 hours, and progressively worsening adhesions in a time-dependent manner (Fig 2A and B). Furthermore, the measurements revealed that, compared to the control group, levels of total iron ions and LPO in the liver, kidneys, and hippocampus showed a peak elevation at 12 hours, followed by a slight decrease at 24 hours and a subsequent rise at 48 hours (Fig 2 C and E). In contrast, the GSH levels exhibited a significant downward trend (Fig 2 D). These findings suggest that the CLP model induces ferroptosis, which presents a time-dependent variation, with 48 hours potentially representing a critical time point for ferroptosis occurrence. 3.2 SalB Exhibits Regulatory Effects on Ferroptosis During the Critical Time Window at 48 Hours Based on the previous experimental results, to further investigate the protective effect of SalB during the critical time window of ferroptosis, we administered the drug to mice following the CLP sepsis model. Tissue samples were collected at 48 and 72 hours to conduct further analyses on ferroptosis-related indicators (Fe 2+ , LPO, GSH) in the liver, kidneys, and hippocampus, observing the relative expression trends of these indicators across different time points in comparison to the control group. The results demonstrated that SalB significantly downregulated the expression trends of Fe 2+ and LPO in the liver, kidneys, and hippocampus at both 48 (Fig 3 A and C)and 72 (Fig 3 D and F) hours, while upregulating GSH levels (Fig 3 B and E). Notably, the regulatory effects of SalB on Fe 2+ , LPO, and GSH were most pronounced in the hippocampus at 48 hours, with all three indicators showing significant differences compared to the CLP group(Fig 3 A-C). 3.3 SalB Effectively Improves Behavioral Phenotypes in Mice and Reduces Inflammatory Factor Expression Previous experiments have demonstrated that 48 hours post-CLP modeling is a critical window for ferroptosis, with SalB showing the most significant therapeutic effects on the hippocampus at this time point. To further investigate the specific protective mechanisms of SalB on hippocampal tissue, we administered different concentrations of SalB to mice and recorded mortality rates over a 7-day period. Behavioral phenotypes were assessed at 48 hours, along with measurements of inflammatory factor expression levels. The results indicated that at 10 mg/kg, the mortality rate of mice treated with SalB was significantly reduced compared to the CLP group(Fig 4 B). Behavioral observations revealed improvements in the mental state of the treated mice, with fur appearing glossier than that of the CLP group(Fig 4 A). Additionally, both the MSS and the modified mNSS for the SalB group were significantly lower than those of the CLP group(Fig 4 C-D). Expression levels of inflammatory factors showed a significant decrease, indicating that SalB can notably improve the behavioral phenotypes of mice while controlling inflammation levels(Fig 4 E-H). Interestingly, all four inflammatory factor indicators in the CLP group were significantly elevated, suggesting the occurrence of a systemic inflammatory storm in septic mice following CLP modeling. 3.4 SalB Reduces Pathological Changes in Hippocampal Tissue and Improves Blood-Brain Barrier (BBB) Permeability To further validate the therapeutic effects of SalB on the hippocampus, histological sections and Evans Blue staining were performed on the hippocampal region of the mice. The results showed that in the CLP group, there was a significant reduction in the number of neurons in the CA1 region, with noticeable vacuolization of neuronal cells(Fig 5 A). The CA3 region exhibited abnormal neuronal morphology, characterized by extensive neuronal shrinkage and necrosis, increased intercellular spaces, and a reduced or absent number of Nissl bodies, leading to pale staining(Fig 5 A). Additionally, there was a significant increase in the number of degenerative neurons as indicated by the FJC staining(Fig 5 D). In contrast, the Sham and SalB groups displayed well-organized hippocampal neurons in the CA1 region with clear outlines and distinct Nissl body staining(Fig 5 B). The CA3 region showed relatively regular neuronal arrangement, abundant Nissl bodies, and a reduction in degenerative neurons(Fig 5 B). SalB treatment significantly improved the pathological changes in the hippocampus compared to the model group. Furthermore, the results from Evans Blue staining provided additional evidence that the CLP group exhibited extensive blue staining throughout the brain, with particularly significant staining in the hippocampal structures in coronal sections(Fig 5 C). In the SalB group, however, there was no significant blue staining, with the results closely resembling those of the Sham group. This indicates that SalB can significantly improve the disruption of BBB stability caused by sepsis. It is noteworthy that the degree of pathological damage to hippocampal neurons in the Fer-1 group was not significantly different from that in the CLP group, and a certain degree of blue staining was observed in Evans Blue staining. This suggests that while Fer-1 may improve the expression of ferroptosis-related indicators, its therapeutic effects on neuronal pathological damage and increased BBB permeability are not as pronounced as those of SalB. 3.5 SalB Regulates the Expression of Ferroptosis-Related Factors in the Hippocampus Fe 2+ staining and measurements of ferroptosis-related factors (Fe 2+ , GSH, LPO) were performed on the CA3 region of the hippocampus in mice(Fig 6 A). The results indicated that CLP significantly upregulated the expression level of Fe 2+ in the hippocampal tissue, increased the content of LPO within the tissue, and downregulated GSH levels(Fig 6 B-D). In contrast, SalB treatment significantly decreased the expression levels of Fe 2+ and LPO while simultaneously upregulating GSH levels in the tissue. These findings suggest that SalB can effectively regulate the expression of ferroptosis-related indicators in the hippocampal tissue, thereby improving the accumulation of Fe 2+ within the tissue. 3.6 SalB Exhibits Pharmacological Activity Through Competitive Binding with the Substrate of ACSL4 Using protein chip technology combined with Autodock molecular docking, we explored the pharmacological activity mechanisms of SalB. The results from the protein chip indicated that SalB can bind specifically and significantly to the ACSL4 protein (Fig 7 C-E), with clear signals and minimal background interference. Further, we conducted molecular docking of SalB and the ACSL4 enzyme substrate CoA with the ACSL4 protein using PyMOL(Fig 7 F-G). The results showed that the binding energy of SalB to ACSL4 is -7.03 kcal/mol(Fig 7 F), while the binding energy of CoA to ACSL4 is -9.24 kcal/mol(Fig 7 G). Both compounds can stably bind to ACSL4; however, it's noteworthy that their binding pockets overlap at the amino acid residues Lys-572, Leu-574, and Ser-607, and both interactions involve hydrogen bonds. By systematically analyzing the hydrogen bond network fosrmed by amino acid residues in overlapping and non-overlapping regions during docking, we further revealed the interaction mechanisms between both compounds and the ACSL4 protein (Table 1). In deeper analysis, we found that at the Lys-572 residue, SalB forms two effective hydrogen bonds, whereas CoA forms only one. Additionally, SalB is closer in terms of H-A and D-A distances, making it more stable in comparison. At the Leu-574 residue, the two hydrogen bonds formed by CoA with the protein had both H-A and D-A distances that were further away than the ones formed by SalB, falling outside the effective range and negatively affecting the binding capability. At the Ser-607 residue, SalB forms three effective hydrogen bonds, while CoA forms two. SalB's maximum donor angle of 163.72° is closer to 180° compared to CoA's donor angle, resulting in higher binding efficiency. Moreover, the hydrogen bonds formed by CoA with the protein show longer H-A and D-A distances compared to those formed by SalB, further impacting the binding effect. In summary, although the overall binding energy of SalB is slightly greater than that of CoA, it appears that SalB has a superior binding effect with ACSL4 at the overlapping amino acid residues Lys-572, Leu-574, and Ser-607 when compared to CoA. Table 1 Analysis of Hydrogen Bonds in Molecular Docking Interactions Residue AA Distance H-A Distance D-A Donor Atom SalB 572A LYS 2.17 Å 2.67 Å 138.63 572A LYS 2.56 Å 2.96 Å 124.94 574A LEU 2.65 Å 3.06 Å 125.95 606A LYS 2.80 Å 3.39 Å 117.2 607A SER 2.27 Å 3.26 Å 163.72 607A SER 2.26 Å 2.79 Å 145.08 607A SER 2.36 Å 2.79 Å 127.98 CoA 504A GLU 2.01 Å 2.6 Å 160.21 504A GLU 2.39 Å 2.7 Å 114.45 572A LYS 2.65 Å 3.26 Å 175.93 574A LEU 3.8 Å 4.03 Å 107.26 574A LEU 2.93 Å 3.5 Å 153.27 576A LYS 2.87 Å 3.86 Å 164.1 576A LYS 3.36 Å 3.94 Å 117.15 582A TYR 2.58 Å 2.77 Å 101.58 607A SER 2.48 Å 2.88 Å 124.73 607A SER 2.46 Å 3.15 Å 130.03 3.7 SalB Modulates the ACSL4/GPX4 Protein Pathway in Hippocampal Tissue To further validate the molecular mechanisms by which SalB regulates ferroptosis, WB analysis was conducted. The results showed that, compared to the Sham group, the expression levels of ACSL4, ALOX15, and LPCAT3, which are proteins in the ACSL4 pathway, were significantly upregulated in the CLP group (Fig 8 A-D). Conversely, the expression levels of GPX4 and SLC7A11, proteins in the GPX4 pathway, were significantly downregulated in the CLP group (Fig 8 E-G). This indicates that the ACSL4 pathway can be activated while the GPX4 pathway is inhibited during the septic process, thereby contributing to ferroptosis and resulting in damage to the hippocampus. In contrast, following administration of SalB, there was a significant downregulation of the ACSL4 pathway proteins ACSL4, ALOX15, and LPCAT3, along with an upregulation of GPX4 and SLC7A11 expression levels compared to the CLP group. These findings demonstrate that SalB can inhibit the occurrence of ferroptosis by regulating the expression of protein markers in both the ACSL4 and GPX4 pathways, thereby providing protective effects against neuronal damage in the hippocampus. 4 Discussion Ferroptosis, an iron-dependent, lipid peroxidation-driven form of cell death, has recently been implicated in sepsis-related organ damage [14, 15]. During sepsis, dysregulated iron metabolism-characterized by aberrant iron transport, increased uptake, and reduced export-contributes to pathological iron overload. This study systematically investigated the temporal dynamics of ferroptosis in septic mice. Key ferroptotic markers (Fe 2+ , GSH, and LPO) were analyzed, revealing a time-dependent progression: lipid peroxidation and iron accumulation were detectable as early as 12 hours post-CLP surgery, accompanied by significant GSH depletion. By 48 hours post-surgery, iron and LPO levels peaked with statistical significance, coinciding with the highest mortality rate in CLP mice. This temporal alignment suggests that ferroptosis reaches its zenith at 48 hours, correlating with the critical window of systemic metabolic derangement. These findings are consistent with endotoxin fluctuation patterns reported in Sepsis Pathophysiology and Therapeutics[16]. SalB, the most bioactive water-soluble component of Salvia miltiorrhiza Bunge, exhibits potent anti-inflammatory and anti-lipid peroxidation effects. However, its role in sepsis-induced neural injury remains underexplored. This study demonstrates that SalB exerts neu-roprotective effects during the acute phase of sepsis (48-72 hours), with peak efficacy observed at 48 hours - the critical window of ferroptosis progression. SalB significantly attenuated hippocampal ferroptosis, suggesting its therapeutic potential via modulation of iron metabolism and lipid peroxidation pathways. These findings provide a novel theoretical foundation for natural product-based interventions in sepsis management. Sepsis-induced multi-organ failure, driven by uncontrolled cytokine storms [17, 18] is a leading cause of clinical mortality. The hippocampus, a brain region critical for memory and learning, is highly vulnerable to inflammatory insults. In this study, CLP mice exhibited elevated behavioral scores, indicative of neurocognitive impairment, which were markedly reduced by SalB treatment. SAE is characterized by neuroinflammatory hyperactivation, particularly microglial polarization imbalance. M1 microglia, which secrete pro-inflammatory cytokines (e.g., IL-1β, TNF-α), exacerbate neuronal damage, while M2 microglia promote neuroprotection and repair via anti-inflammatory mediators (e.g., IL-10) [19]. Paradoxically, excessive IL-10 in sepsis correlates with septic shock and multi-organ dysfunction [20]. Our data revealed elevated serum levels of IL-1β, TNF-α, IL-4, and IL-10 in CLP mice, consistent with the cytokine profiles of septic patients [21]. SalB treatment significantly suppressed pro-inflammatory IL-1β and TNF-α to sham-operated levels, while modulating anti-inflammatory IL-4 and IL-10 within a neuroprotective range. Notably, IL-10 levels in SalB-treated mice remained higher than in sham controls, suggesting a balanced immunoregulatory role of SalB in curbing inflammatory cascades. In the activity of the nervous system, the hippocampus receives neural incoming signals from numerous regions, including the limbic system, cortex, and subcortical areas, through the entorhinal cortex and the subiculum. The main pathway of neural activity entering the hippocampus begins at the entorhinal cortex, projecting via the perforant pathway to the granule cells of the dentate gyrus, which then send collateral branches to the pyramidal cells in the CA1 and CA3 regions. The mossy fibers emitted by the granule cells can activate the CA3 pyramidal cells as well as interneurons in the spatial region. CA3 pyramidal cells, through local and associative excitatory collaterals, not only excite other CA3 pyramidal cells and those in the lateral nucleus but also activate CA1 pyramidal cells [22]. The unique connections and functional divisions between CA3 and CA1 support the hippocampus's functions in memory encoding, integration, and retrieval. The projection pathway between the CA3 and CA1 regions is vital for synaptic plasticity as well as the convergence and projection of information [23, 24]. The collaboration and imbalance of these areas not only affect memory functions but also constitute a significant pathological basis for neuropsychological disorders. Although the hippocampus is not the only brain region critical for memory, damage or dysfunction in this structure can lead to deficiencies in the formation and retention of various types of memory [25]. In this study, pathological section staining and FJC fluorescence staining revealed neuron damage and necrosis in the CA1 and CA3 regions of hippocampal tissue from mice in the CLP-induced sepsis model, characterized by increased intercellular spacing, loss of Nissl bodies, and an increase in degenerative neurons. This suggests that systemic inflammatory storms induced by abdominal sepsis may initiate neuroinflammatory responses, thereby affecting neuronal metabolism and promoting hippocampal neuronal injury and degenerative pathological changes, leading to cognitive dysfunction. Notably, the administration of SalB effectively improved neuronal damage in the CA1 and CA3 regions of the hippocampus, restoring the abundance of Nissl bodies and reducing the number of degenerative neurons. Evans Blue staining further confirmed extensive damage to the BBB induced by CLP, while SalB exhibited significant protective effects on the BBB. Importantly, neurons in the ferroptosis inhibitor group showed milder degeneration of Nissl bodies, which may be related to the inhibition of ferroptosis, while the significant and widespread improvement seen in the SalB group in terms of tissue pathology may stem from its dual action in regulating both inflammation and oxidative stress. Iron-induced oxidative stress represents a crucial pathological feature of neurodegenerative changes. Theoretically, the brain, with its inherently higher lipid content, greater membrane composition, and fewer antioxidant enzymes, shows weaker tolerance to lipid peroxidation compared to other organs and is more susceptible to the effects of iron accumulation, increasing the likelihood of oxidative stress and damage [26]. Evidence suggests a negative correlation between iron ion concentrations in the hippocampal region and memory scores [27]. Furthermore, studies have found that iron accumulation in the hippocampus leads to cognitive impairments in most neurodegenerative diseases, while the use of ferroptosis inhibitors can reduce oxidative stress damage and improve cognitive dysfunction [28]. In this study, observation of Fe 2+ staining in the CA3 region of the hippocampus revealed that CLP significantly induced notable enrichment of Fe 2+ in this region, while the intervention of SalB and the ferroptosis inhibitor Fer-1 effectively reduced Fe 2+ accumulation, indicating their regulatory effect on the excessive production of ferrous ions. To further validate the regulatory effects on ferroptosis, this study measured the expression levels of ferroptosis-related indicators, Fe 2+ , LPO, and GSH. In the pathway of ferroptosis, Fe 2+ promotes the generation of LPO through the Fenton reaction, which triggers membrane damage due to LPO accumulation, further releasing more Fe 2+ and ROS, thereby creating a vicious cycle. In this process, GPX4 uses GSH as a cofactor to resist lipid peroxidation, thus protecting membrane integrity. The results of this study indicate that CLP-induced sepsis leads to significant increases in Fe 2+ and LPO levels while downregulating GSH levels, thereby promoting the occurrence of ferroptosis. Conversely, SalB can downregulate Fe 2+ and LPO levels and promote GSH secretion, demonstrating its capacity to exert neuroprotective effects on septic mice by inhibiting oxidative stress damage induced by iron ions. Through protein chip experiments, it has been demonstrated that SalB can significantly and specifically bind to the ACSL4 protein. The ACSL4 pathway, as a key protein pathway for lipid peroxidation, plays an important role in the process of ferroptosis, as it is responsible for CoA esterification to free fatty acids in an ATP-dependent manner [29]. The formation of acyl-CoA activates the corresponding fatty acids for fatty acid oxidation or lipid biosynthesis, thereby further initiating the lipid peroxidation process. It, along with the GPX4 pathway, constitutes an important cyclic mechanism for the generation and degradation of lipid peroxides during physiological activities. Specifically, the ACSL4 pathway promotes the generation of lipid peroxides, while GPX4 is responsible for the degradation of these harmful peroxidized lipids. The two pathways complement each other, maintaining the balance of lipid metabolism within cells. This balance is crucial for preventing cellular damage caused by the accumulation of lipid peroxides, particularly evident in the occurrence and development of ferroptosis. Through this cyclic mechanism, the body can effectively regulate lipid peroxidation levels, thus influencing the process of ferroptosis. Therefore, we further performed protein-small molecule docking between SalB and ACSL4 using molecular docking technology, and we also docked CoA with ACSL4, comparing and analyzing the results of these two docking studies. The results showed that SalB shares the same binding sites with CoA at the amino acid residues Lys-572, Leu-574, and Ser-607 in the ACSL4 protein, and its binding effect is superior to that of CoA. Based on the experimental results, it is inferred that SalB may exert its pharmacological activity through competitive binding. Through structural analysis of the ACSL4 protein, we identified that the amino acid residues Lys-572, Leu-574, and Ser-607 are located within the C-terminal catalytic domain (approximately residues 500-711) and the Eukaryotic long-chain fatty acid CoA synthetase (LC-FACS) domain (residues 80-655), both of which constitute the core region governing substrate binding and enzymatic activity. Among these, the regions harboring Lys-572 and Leu-574 may guide drug molecules into the protein interior via hydrogen bonding, thereby altering local or global conformational states of the protein. Such structural changes could modify the geometry and properties of the substrate-binding pocket, ultimately disrupting ACSL4-mediated fatty acid activation. Notably, Ser-607, which also serves as a phosphorylation site, is implicated in this process. Given that phosphorylation of ACSL4 critically amplifies lipid peroxidation linked to ferroptosis [30], the formation of hydrogen bonds between SalB and Ser-607 may either obstruct phosphorylation or perturb post-phosphorylation signaling. This dual mechanism would regulate ACSL4-driven lipid metabolic pathways, thereby influencing the progression of ferroptosis. Finally, we returned to in vivo studies, measuring the expression levels of ACSL4 and GPX4 pathway proteins. The results indicated that in the CLP septic model mice, the protein level of the ACSL4 pathway was significantly elevated compared to the sham surgery group, while the expression of GPX4 pathway proteins was suppressed, suggesting that excessive lipid peroxidation had occurred in the septic mice, promoting ferroptosis. After administration of SalB, the protein level of the ACSL4 pathway was significantly downregulated, while the expression of GPX4 pathway proteins was significantly increased, indicating that SalB can effectively inhibit the occurrence of lipid peroxidation, regulate the generation of lipid peroxides, and inhibit ferroptosis (Fig 9). Notably, the ferroptosis inhibitor Fer-1 was less effective on the ACSL4 pathway protein compared to SalB, but it significantly regulated the expression of GPX4 pathway proteins, demonstrating its specific biological activity on the GPX4 pathway. 5 Conclusion In summary, our study demonstrates that SalB significantly improves the survival rates of mice subjected to CLP-induced sepsis, reduces inflammatory responses, and alleviates neuronal damage in the hippocampus. Furthermore, SalB mediates its therapeutic effects by regulating ferroptosis processes through the ACSL4 pathway in the context of hippocampal injury caused by CLP-induced sepsis. Additionally, our protein arrays and molecular docking analysis further supports the hypothesis that SalB exerts its pharmacological activity via competitive inhibition with the substrate CoA of the ACSL4 protein. Our findings provide new insights into the use of SalB for treating sepsis. Abbreviations ACSL4, Long-chain acyl-CoA synthetase 4; BBB, Blood-brain barrier; CLP, Cecal ligation and puncture; CNS, Central nervous system; CoA, coenzyme A; Fer-1, Ferrostatin-1; GSH, glutathione; GPX4, Glutathione peroxidase 4; H&E, hematoxylin and eosin; IL, Interleukin; LPO, lipid peroxides; MDA, Malondialdehyde; MSS, Murine Sepsis Score; mNSS, modified Neurological Severity Score; OD, optical density; PVDF, polyvinylidene difluoride; ROS, Reactive oxygen species; SalB, Salvianolic acid B; SAE, Sepsis-associated encephalopathy; SNR, Signal-to-Noise Ratio; SOD, superoxide dismutase ; WB, Western blot. Declarations Acknowledgments The graphical abstract figures were generated by FigDraw (www.figdraw.com). Animal Experiment Ethics Approval Number: BJTCM-M-2023-11-04. Clinical trial number: not applicable. Funding This work was supported by National Key Research and Development Program of China (2022YFD1801102), National Natural Science Foundation of China (82474428, 82174348), Sanming Project of Medicine in Shenzhen (No.SZZYSM202411012). Author Contribution The study was conceived and designed by KF, XXL, and QQL. The initial draft of the manuscript was prepared by XYL, ZZM, and YMY, while the main experimental data were collected by XYL, CXZ, and SWZ. YL. further revised and refined the manuscript. YMY and ZZM were responsible for the analysis, interpretation, and visualization of the data. YXL. and LW. directly accessed and validated the data presented in the article. All authors reviewed the manuscript. Data availability Data will be made available on request. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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16:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7925353/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7925353/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12035-026-05876-y","type":"published","date":"2026-04-22T15:56:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":96250491,"identity":"66d5cf96-551b-42dd-9993-18f96061e18e","added_by":"auto","created_at":"2025-11-19 07:38:27","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1354694,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the experimental timeline and dosing schedule\u003c/p\u003e","description":"","filename":"figure1.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7925353/v1/9b29eb1e26e09d2c590a5998.jpg"},{"id":96250289,"identity":"5adff6fd-29bd-4c2d-927b-0801d7076bc5","added_by":"auto","created_at":"2025-11-19 07:37:58","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1604930,"visible":true,"origin":"","legend":"\u003cp\u003eThe changes in biomarkers of ferroptosis exhibit a time effect within different time windows post-CLP model. A. The condition of the cecum in mice at 12, 24, 48, and 72 hours after CLP; B. Clinical manifestations of the cecum in C57BL/6 mice after CLP modeling; C-E. Expression levels of ferroptosis-related biomarkers at different time points. * indicates comparison with the control group, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7925353/v1/9c84e7153b46cd34c257125c.jpg"},{"id":96200887,"identity":"f9e88faa-b8d1-4637-9374-ad07b3e35616","added_by":"auto","created_at":"2025-11-18 16:28:18","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":824263,"visible":true,"origin":"","legend":"\u003cp\u003eRegulation of ferroptosis-related indicators in the liver, kidneys, and hippocampus by SalB at 48 and 72 hours post-CLP modeling. A-C. Expression levels of Fe\u003csup\u003e2+\u003c/sup\u003e, GSH, and LPO in the liver, kidneys, and hippocampus of mice at 48 hours after administration of SalB; D-F. Expression levels of Fe\u003csup\u003e2+\u003c/sup\u003e, GSH, and LPO in the liver, kidneys, and hippocampus of mice at 72 hours after administration of SalB. Data are presented as mean ± SD, and differences between means were assessed using one-way ANOVA. (* indicates comparison with the control group, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7925353/v1/d967d986c615f86cf9cec18b.jpg"},{"id":96200888,"identity":"e9e4b0a7-6815-4b63-9d40-4e606221a349","added_by":"auto","created_at":"2025-11-18 16:28:18","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":796119,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of Mouse Mortality Rates, Behavioral Phenotypes, and Inflammatory Factor Expression. A. General appearance of mice following SalB administration; B. Mortality rates of mice 72 hours after administration of different concentrations of SalB (5 mg/kg, 10 mg/kg, 20 mg/kg); C-D. Behavioral scoring of mice at 48 hours (C: MSS score; D: mNSS score); E-H. Expression levels of inflammatory factor indicators (IL-1β, IL-4, TNF-α, IL-10) in mice. Data are presented as mean ± SD, and differences between means were assessed using one-way ANOVA. (* indicates comparison with the Sham group, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001，# indicates comparison with the CLP group, #P \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7925353/v1/c749952e05f6f5ba97efcc54.jpg"},{"id":96200890,"identity":"7d3982b3-7c6c-4905-b3f8-d88dee3880b6","added_by":"auto","created_at":"2025-11-18 16:28:18","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4141820,"visible":true,"origin":"","legend":"\u003cp\u003eSalB (10 mg/kg) Ameliorates Neuronal Damage in the Hippocampus of Septic Mice and Protects the BBB. A. HE staining results of the CA1 and CA3 regions of the hippocampus in each group of mice, scale bar = 50 μm; B. Nissl staining results of the CA1 and CA3 regions of the hippocampus in each group of mice, scale bar = 50 μm; C. Evans Blue staining photographs of the whole brain in mice; D. FJC staining results of the CA3 region of the hippocampus in each group of mice. Data are presented as mean ± SD, and differences between means were assessed using one-way ANOVA. (* indicates comparison with the control group, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7925353/v1/ea88179659a2edf7289b69bc.jpg"},{"id":96200891,"identity":"9e62f490-5a26-4a68-97c0-34f03cfa09d6","added_by":"auto","created_at":"2025-11-18 16:28:18","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":739348,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of Fe\u003csup\u003e2+\u003c/sup\u003e Staining and Expression of Ferroptosis-Related Indicators in the Hippocampus of Mice. (where the dosage of SalB was 10 mg/kg) A. Fe\u003csup\u003e2+\u003c/sup\u003e staining in the CA3 region of the hippocampus in mice at 48 hours after administration of SalB; B-D. Expression levels of ferroptosis-related indicators in hippocampal tissue (B: Expression of Fe\u003csup\u003e2+\u003c/sup\u003e in the hippocampus; C: Expression of GSH in the hippocampus; D: Expression of LPO in the hippocampus), n = 6 per group. Data are presented as mean ± SD, and differences between means were assessed using one-way ANOVA. (* indicates comparison with the control group, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7925353/v1/ce30f23030a55575aded89fe.jpg"},{"id":96200896,"identity":"9a2a157b-74a7-45cf-9eef-e75cfac3ca79","added_by":"auto","created_at":"2025-11-18 16:28:18","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1113888,"visible":true,"origin":"","legend":"\u003cp\u003eResults from the protein chip experiments for SalB and molecular docking of SalB and CoA with the ACSL4 protein. A. SalB binds to biotin through chemical modification, with the dotted box indicating biotin; B. Schematic representation of the functioning principle of the protein chip; C-E. Protein chip results (C: Protein chip results for Bio-SalB, D: Protein chip results for D-Biotin as a control, E: Signal-to-Noise Ratio for Bio-SalB compared to D-Biotin in the ACSL4 protein); F-G: Molecular docking of SalB and CoA with the ACSL4 protein (F: Binding site of SalB with ACSL4, binding energy -7.03 kcal/mol; G: Binding site of CoA with ACSL4, binding energy -9.24 kcal/mol)\u003c/p\u003e","description":"","filename":"figure7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7925353/v1/14929ec749883e1e84007997.jpg"},{"id":96200895,"identity":"1741ae65-bccf-49a4-91e0-fe0ae11560cf","added_by":"auto","created_at":"2025-11-18 16:28:18","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":257540,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of ACSL4 and GPX4 Pathway Protein Expression in Mice (where the dosage of SalB was 10 mg/kg). A. WB analysis used to evaluate the expression levels of ACSL4, LPCAT3, and ALOX15 in hippocampal tissue, The uncropped blots were presented in Supplementary Fig.S1; B-D. Statistical analysis of protein expression levels for ACSL4, LPCAT3, and ALOX15 ; E. WB analysis used to evaluate the expression levels of GPX4 and SLC7A11 in hippocampal tissue, The uncropped blots were presented in Supplementary Fig.S2; F-G. Statistical analysis of protein expression levels for GPX4 and SLC7A11. Data are presented as mean ± SD, and differences between means were assessed using one-way ANOVA. (* indicates comparison with the control group, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"figure8.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7925353/v1/be3c32cac341aa07a7be998d.jpg"},{"id":96200897,"identity":"524efd9a-66ce-40e9-a403-be688ef1cf7a","added_by":"auto","created_at":"2025-11-18 16:28:18","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":169124,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism of SalB in the Treatment of SAE\u003c/p\u003e","description":"","filename":"figure9.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7925353/v1/1880e54d0d27f057d2150967.jpg"},{"id":107927664,"identity":"2f715710-302d-4355-85e5-908e7b17a335","added_by":"auto","created_at":"2026-04-27 16:00:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11298614,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7925353/v1/d4806e50-4169-44e9-a3dd-e7c64f7740c7.pdf"},{"id":96200894,"identity":"2fce1e34-884c-4fa0-aa8c-12f502041aaf","added_by":"auto","created_at":"2025-11-18 16:28:18","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1184330,"visible":true,"origin":"","legend":"","description":"","filename":"uncroppedGelsandBlotsimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7925353/v1/7ce940394d92b3bba0aac1a9.pdf"},{"id":96250249,"identity":"ad6a4304-9594-40af-a706-c6c6341b0dfb","added_by":"auto","created_at":"2025-11-19 07:37:49","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1571000,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"Graphicalabstract.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7925353/v1/09f50c7ad3e24c9215113f67.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"Salvianolic Acid B Inhibits Ferroptosis Through ACSL4 Pathway Regulation in sepsis-associated encephalopathy Mice","fulltext":[{"header":"1 Introduction ","content":"\u003cp\u003eSepsis is defined as a life-threatening systemic inflammatory response syndrome caused by a dysregulated host reaction to bacterial, fungal, or viral infections [1], which severely damages the lungs, heart, liver, and other organs, potentially leading to fatal outcomes. The central nervous system (CNS) is recognized as one of the earliest organs affected by sepsis. Clinically, CNS involvement manifests as sepsis-associated encephalopathy (SAE), a diffuse brain dysfunction secondary to systemic infection in sepsis. SAE is characterized by sepsis-related diffuse cerebral impairment detected through clinical or standard laboratory evaluations, in the absence of direct CNS infection, structural abnormalities, or other encephalopathies (e.g. hepatic or renal encephalopathy)[2]. As one of the most severe complications during both acute sepsis and recovery, SAE involves pathological alterations such as neuroinflammation, microcirculatory disturbances, and metabolic failure [3]. Recent studies have linked ferroptosis to glutamate-mediated excitotoxicity and neuronal injury during SAE pathogenesis [4]. Evidence from cecal ligation and puncture (CLP)-induced septic mice reveals reduced GPX4 expression, elevated transferrin and malondialdehyde (MDA) levels, and mitochondrial shrinkage in the brain [5]. Ferroptosis is also observed in the hippocampus, marked by increased reactive oxygen species (ROS) and Fe\u003csup\u003e2+\u003c/sup\u003econtent, decreased glutathione (GSH) levels, upregulated long-chain acyl-CoA synthetase 4 (ACSL4) expression, and downregulated glutathione peroxidase 4 (GPX4) protein levels. Clinically, these changes correlate with aggravated cognitive deficits, reduced survival rates, and exacerbated mitochondrial damage in hippocampal neurons [6].\u003c/p\u003e\n\u003cp\u003eCurrent clinical management of SAE primarily focuses on treating underlying infections, administering antibiotics, and providing supportive care [7]. Salvianolic acid B (SalB) is the most biologically active water-soluble component of Salvia miltiorrhiza Bunge and exhibits potent anti-inflammatory[8] and anti-lipid peroxidation effects[9]. SalB is a polyphenolic compound formed by the condensation of three danshensu molecules and one caffeic acid. Preclinical and clinical studies indicate that SalB attenuates inflammation, suppresses pro-inflammatory cytokines [10], enhances superoxide dismutase (SOD) activity, inhibits MDA production, and scavenges ROS [11], thereby protecting tissue integrity. However, existing research on SalB in sepsis has predominantly focused on cardiovascular protection, with limited exploration of its regulatory mechanisms and key targets in sepsis-associated neurological dysfunction.\u003c/p\u003e\n\u003cp\u003eThis study aims to elucidate the molecular mechanisms underlying SalB\u0026rsquo;s ameliorative effects on sepsis-induced SAE. First, we evaluated SalB\u0026rsquo;s impact on survival rates, behavioral phenotypes, histopathological changes, and serum inflammatory responses in septic mice. Next, ferroptosis-related biomarkers and ACSL4/GPX4 pathway proteins were analyzed to validate SalB\u0026rsquo;s regulatory role in ferroptosis. Finally, molecular docking was employed to predict SalB-ACSL4 interaction sites, further clarifying SalB\u0026rsquo;s pharmacological mechanism in SAE treatment.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Experimental materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSalvianolic acid B (SalB, CAS No.: 121521-90-2) was purchased from Target Molecule Corp.(TargetMol). (Product No.: T5725, purity: 99.86%). Ferroptosis inhibitor Ferrostatin-1 (Fer-1, CAS No.: 347174-05-4) was procured from the same supplier (Product No.: T6500, purity: 99.68%). All compounds were freshly prepared as stock solutions immediately prior to experimental use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Experimental animals and modeling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPF grade C57BL/6 J mice, 8 weeks old (20-22 g), were used in this study, and the experimental animals were purchased from Beijing Huafukang Biotechnology Company. The mice were housed under controlled environmental conditions: ambient temperature 20-26℃\u0026nbsp;(daily fluctuation\u0026nbsp;\u0026le;\u0026nbsp;4℃), relative humidity 40-70%, and a 12/12-hour light/dark cycle. All animals were provided ad libitum access to autoclaved feed and sterile water. The ethical approval number is BJTCM-M-2023-11-04.\u003c/p\u003e\n\u003cp\u003eSepsis was induced using the CLP procedure. Briefly, mice were anesthetized with 1% sodium pentobarbital, followed by a 1 cm midline laparotomy to expose the cecum. The proximal third of the cecum was ligated with surgical suture and punctured once through an avascular region using an 18-gauge needle. A small volume of fecal content was gently extruded from the puncture site, after which the cecum was carefully repositioned into the abdominal cavity, and the incision was closed with layered sutures. Postoperative fluid resuscitation was administered via subcutaneous injection of 0.9 % sterile saline (2 mL). Sham-operated controls underwent identical surgical procedures excluding cecal ligation and puncture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 SalB Treatment Protocol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing a 7-day acclimatization period, mice were administered treatments via intraperitoneal injection (Fig 1). SalB treatment groups received doses of 20 mg/kg (high dose), 10 mg/kg (medium dose), or 5 mg/kg (low dose) in CLP-induced septic mice. The positive control group was treated with Ferrostatin-1 (Fer-1) at a dose of 5 mg/kg in CLP-induced mice. All treatments were administered once daily, with the first dose given 2 hours post-surgery and continued for 3 consecutive days. Survival rates were monitored over a 7-day period. CLP and sham-operated control groups received an equivalent volume of sterile saline. To further investigate the underlying mechanisms, the above grouping and modeling procedures were replicated. At 48 hours post-surgery, mice were euthanized, and experimental samples were collected for subsequent analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Behavioral Testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn accordance with established literature, two behavioral scoring systems were employed for each group of mice: the Murine Sepsis Score (MSS) [\u003ca href=\"#_ENREF_12\" title=\"Shrum, 2014 #534\"\u003e12\u003c/a\u003e]\u0026nbsp;and the modified Neurological Severity Score (mNSS)\u0026nbsp;[\u003ca href=\"#_ENREF_13\" title=\"Wang, 2024 #535\"\u003e13\u003c/a\u003e]. These assessments were conducted to evaluate sepsis severity and neurological functional deficits, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Biochemical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression levels of Fe\u003csup\u003e2+\u003c/sup\u003e, lipid peroxides (LPO), and reduced GSH in hippocampal tissues were quantified using commercially available assay kits (Elabscience Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer\u0026rsquo;s instructions. Additionally, the total protein concentration in liver tissues was determined using a BCA protein assay kit (Thermo Fisher Scientific Technology Co., Ltd., Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Histological Assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor histological assessment, whole brain tissues were fixed in 4% paraformaldehyde, followed by standard processing steps including dehydration, paraffin embedding, and sectioning. Tissue sections were stained with hematoxylin and eosin (H\u0026amp;E) and Nissl staining for morphological analysis. Fe\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003estaining was performed using a commercially available Prussian blue staining kit (Solarbio Science \u0026amp; Technology Co., Ltd., Beijing, China; Product No.: G1429) according to the manufacturer\u0026rsquo;s protocol. Finally, the stained sections were examined and imaged under an optical microscope for detailed histological evaluation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Evans Blue Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 2% Evans blue dye solution and a heparinized saline solution were prepared for subsequent use. Mice were anesthetized with 1% sodium pentobarbital, and a 2% Evans blue dye solution (50 mg/kg) was injected into the orbital venous plexus. After a 2-hour circulation period, the thoracic cavity was opened, and 0.9% heparinized saline was slowly perfused through the left ventricular apex. The right atrium was incised to allow drainage until the effluent became clear. Brain tissues were carefully dissected, and the hippocampal region was exposed through precise sectioning. Gross photographs of the brain were taken for further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Western blotting (WB)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHippocampal tissues were homogenized, lysed, and centrifuged to extract total protein. Protein concentrations were quantified using a BCA protein assay kit (Thermo Fisher Scientific Technology Co., Ltd., Shanghai, China). Equal amounts of protein samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were blocked with rapid blocking solution (Beyotime, P0252-500 mL, Shanghai, China) at room temperature for 20 minutes, followed by overnight incubation at 4℃\u0026nbsp;with the following primary antibodies: ACSL4 (1:10,000, ab155282, Abcam, Cambridge, United Kingdom), LPCAT3 (1:3,000, 67882-1-Ig, Proteintech, Wuhan, China), ALOX15 (1:1,000, ab244205, Abcam, Cambridge, United Kingdom), GPX4 (1:5,000, ab125066, Abcam, Cambridge, United Kingdom), SLC7A11 (1:1,000, ab307601, Abcam, Cambridge, United Kingdom),\u0026nbsp;\u0026beta;-actin (1:50,000, 66009-1-Ig, Proteintech, Wuhan, China), and GAPDH (1:80,000, 81640-5-RR, Proteintech, Wuhan, China). After washing with 1\u0026times;TBST, membranes were incubated with secondary antibodies at room temperature for 1 hour: goat anti-rabbit IgG (1:10,000, B900210, Proteintech, Wuhan, China) and rabbit anti-mouse IgG (1:10,000, B900120, Proteintech, Wuhan, China). Membranes were washed again with 1\u0026times;TBST, and protein bands were visualized using the Tanon-5200 Multi Gel Imaging System. Band intensities were quantified using ImageJ software (version 1.8.0).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Enzyme-linked immunoassay (ELISA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum levels of IL-1\u0026beta;, IL-4, IL-10, and TNF-\u0026alpha;\u0026nbsp;were measured using commercially available mouse cytokine ELISA kits (Meimian Industrial Co., Ltd., Jiangsu, China) according to the manufacturer\u0026rsquo;s instructions. Reactions were monitored within 15 minutes, and the optical density (OD) of each well was measured at a wavelength of 450 nm using a microplate reader. Data were recorded, and standard curves were generated for quantitative analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10 Molecular Docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe small molecule SalB and coenzyme A (CoA) were docked with the pathway protein ACSL4. The three-dimensional (3D) structures of the compounds were retrieved from the PubChem database. The crystal structure of the protein receptor (ACSL4) was obtained from the UniProt protein database (https://www.uniprot.org/). Molecular docking was performed using AutoDock (version 4.2.6), and the results were visualized and analyzed using PyMOL for comparative assessment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are expressed as mean\u0026nbsp;\u0026plusmn;\u0026nbsp;standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 8 (GraphPad, La Jolla, USA). One-way analysis of variance (ANOVA) was employed to assess intergroup differences, with statistical significance denoted by an asterisk (*) for\u003cem\u003e\u0026nbsp;P\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"3 Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Time-Dependent Changes of ferroptosis Biomarkers Induced by the CLP Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the development of ferroptosis in various organs of septic mice over different time periods, we conducted a CLP model. Observations were made at 12, 24, 48, and 72 hours post-surgery (Fig 2 B), during which we assessed the abdominal state of the mice and collected samples from the liver, kidneys, and hippocampus to measure iron ions, GSH, and LPO levels. We aimed to observe the relative expression trends of ferroptosis-related indicators across different time points in comparison to the control group. The results indicated that inflammation and swelling in the cecum of the CLP-operated mice began at 12 hours post-surgery, with purulent changes emerging at 24 hours, and progressively worsening adhesions in a time-dependent manner (Fig 2A and B). Furthermore, the measurements revealed that, compared to the control group, levels of total iron ions and LPO in the liver, kidneys, and hippocampus showed a peak elevation at 12 hours, followed by a slight decrease at 24 hours and a subsequent rise at 48 hours (Fig 2 C and E). In contrast, the GSH levels exhibited a significant downward trend (Fig 2 D). These findings suggest that the CLP model induces ferroptosis, which presents a time-dependent variation, with 48 hours potentially representing a critical time point for ferroptosis occurrence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 SalB Exhibits Regulatory Effects on Ferroptosis During the Critical Time Window at 48 Hours\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the previous experimental results, to further investigate the protective effect of SalB during the critical time window of ferroptosis, we administered the drug to mice following the CLP sepsis model. Tissue samples were collected at 48 and 72 hours to conduct further analyses on ferroptosis-related indicators (Fe\u003csup\u003e2+\u003c/sup\u003e, LPO, GSH) in the liver, kidneys, and hippocampus, observing the relative expression trends of these indicators across different time points in comparison to the control group. The results demonstrated that SalB significantly downregulated the expression trends of Fe\u003csup\u003e2+\u003c/sup\u003e and LPO in the liver, kidneys, and hippocampus at both 48 (Fig 3 A and C)and 72 (Fig 3 D and F) hours, while upregulating GSH levels (Fig 3 B and E). Notably, the regulatory effects of SalB on Fe\u003csup\u003e2+\u003c/sup\u003e, LPO, and GSH were most pronounced in the hippocampus at 48 hours, with all three indicators showing significant differences compared to the CLP group(Fig 3 A-C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 SalB Effectively Improves Behavioral Phenotypes in Mice and Reduces Inflammatory Factor Expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious experiments have demonstrated that 48 hours post-CLP modeling is a critical window for ferroptosis, with SalB showing the most significant therapeutic effects on the hippocampus at this time point. To further investigate the specific protective mechanisms of SalB on hippocampal tissue, we administered different concentrations of SalB to mice and recorded mortality rates over a 7-day period. Behavioral phenotypes were assessed at 48 hours, along with measurements of inflammatory factor expression levels. The results indicated that at 10 mg/kg, the mortality rate of mice treated with SalB was significantly reduced compared to the CLP group(Fig 4 B). Behavioral observations revealed improvements in the mental state of the treated mice, with fur appearing glossier than that of the CLP group(Fig 4 A). Additionally, both the MSS and the modified mNSS for the SalB group were significantly lower than those of the CLP group(Fig 4 C-D). Expression levels of inflammatory factors showed a significant decrease, indicating that SalB can notably improve the behavioral phenotypes of mice while controlling inflammation levels(Fig 4 E-H). Interestingly, all four inflammatory factor indicators in the CLP group were significantly elevated, suggesting the occurrence of a systemic inflammatory storm in septic mice following CLP modeling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 SalB Reduces Pathological Changes in Hippocampal Tissue and Improves Blood-Brain Barrier (BBB) Permeability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further validate the therapeutic effects of SalB on the hippocampus, histological sections and Evans Blue staining were performed on the hippocampal region of the mice. The results showed that in the CLP group, there was a significant reduction in the number of neurons in the CA1 region, with noticeable vacuolization of neuronal cells(Fig 5 A). The CA3 region exhibited abnormal neuronal morphology, characterized by extensive neuronal shrinkage and necrosis, increased intercellular spaces, and a reduced or absent number of Nissl bodies, leading to pale staining(Fig 5 A). Additionally, there was a significant increase in the number of degenerative neurons as indicated by the FJC staining(Fig 5 D). In contrast, the Sham and SalB groups displayed well-organized hippocampal neurons in the CA1 region with clear outlines and distinct Nissl body staining(Fig 5 B). The CA3 region showed relatively regular neuronal arrangement, abundant Nissl bodies, and a reduction in degenerative neurons(Fig 5 B). SalB treatment significantly improved the pathological changes in the hippocampus compared to the model group. Furthermore, the results from Evans Blue staining provided additional evidence that the CLP group exhibited extensive blue staining throughout the brain, with particularly significant staining in the hippocampal structures in coronal sections(Fig 5 C). In the SalB group, however, there was no significant blue staining, with the results closely resembling those of the Sham group. This indicates that SalB can significantly improve the disruption of BBB stability caused by sepsis. It is noteworthy that the degree of pathological damage to hippocampal neurons in the Fer-1 group was not significantly different from that in the CLP group, and a certain degree of blue staining was observed in Evans Blue staining. This suggests that while Fer-1 may improve the expression of ferroptosis-related indicators, its therapeutic effects on neuronal pathological damage and increased BBB permeability are not as pronounced as those of SalB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 SalB Regulates the Expression of Ferroptosis-Related Factors in the Hippocampus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFe\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003estaining and measurements of ferroptosis-related factors (Fe\u003csup\u003e2+\u003c/sup\u003e, GSH, LPO) were performed on the CA3 region of the hippocampus in mice(Fig 6 A). The results indicated that CLP significantly upregulated the expression level of Fe\u003csup\u003e2+\u003c/sup\u003e in the hippocampal tissue, increased the content of LPO within the tissue, and downregulated GSH levels(Fig 6 B-D). In contrast, SalB treatment significantly decreased the expression levels of Fe\u003csup\u003e2+\u003c/sup\u003e and LPO while simultaneously upregulating GSH levels in the tissue. These findings suggest that SalB can effectively regulate the expression of ferroptosis-related indicators in the hippocampal tissue, thereby improving the accumulation of Fe\u003csup\u003e2+\u003c/sup\u003e within the tissue.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 SalB Exhibits Pharmacological Activity Through Competitive Binding with the Substrate of ACSL4\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing protein chip technology combined with Autodock molecular docking, we explored the pharmacological activity mechanisms of SalB. The results from the protein chip indicated that SalB can bind specifically and significantly to the ACSL4 protein (Fig 7 C-E), with clear signals and minimal background interference. Further, we conducted molecular docking of SalB and the ACSL4 enzyme substrate CoA with the ACSL4 protein using PyMOL(Fig 7 F-G). The results showed that the binding energy of SalB to ACSL4 is -7.03 kcal/mol(Fig 7 F), while the binding energy of CoA to ACSL4 is -9.24 kcal/mol(Fig 7 G). Both compounds can stably bind to ACSL4; however, it\u0026apos;s noteworthy that their binding pockets overlap at the amino acid residues Lys-572, Leu-574, and Ser-607, and both interactions involve hydrogen bonds. By systematically analyzing the hydrogen bond network fosrmed by amino acid residues in overlapping and non-overlapping regions during docking, we further revealed the interaction mechanisms between both compounds and the ACSL4 protein (Table 1). In deeper analysis, we found that at the Lys-572 residue, SalB forms two effective hydrogen bonds, whereas CoA forms only one. Additionally, SalB is closer in terms of H-A and D-A distances, making it more stable in comparison. At the Leu-574 residue, the two hydrogen bonds formed by CoA with the protein had both H-A and D-A distances that were further away than the ones formed by SalB, falling outside the effective range and negatively affecting the binding capability. At the Ser-607 residue, SalB forms three effective hydrogen bonds, while CoA forms two. SalB\u0026apos;s maximum donor angle of 163.72\u0026deg; is closer to 180\u0026deg; compared to CoA\u0026apos;s donor angle, resulting in higher binding efficiency. Moreover, the hydrogen bonds formed by CoA with the protein show longer H-A and D-A distances compared to those formed by SalB, further impacting the binding effect. In summary, although the overall binding energy of SalB is slightly greater than that of CoA, it appears that SalB has a superior binding effect with ACSL4 at the overlapping amino acid residues Lys-572, Leu-574, and Ser-607 when compared to CoA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eAnalysis of Hydrogen Bonds in Molecular Docking Interactions\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"87%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eResidue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eAA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eDistance H-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eDistance D-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003eDonor Atom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"7\" valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003eSalB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e572A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eLYS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.17 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.67 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e138.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e572A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eLYS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.56 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.96 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e124.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e574A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eLEU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.65 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e3.06 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e125.95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e606A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eLYS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.80 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e3.39 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e117.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e607A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eSER\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.27 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e3.26 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e163.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e607A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eSER\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.26 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.79 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e145.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e607A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eSER\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.36 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.79 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e127.98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"10\" valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003eCoA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e504A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eGLU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.01 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.6 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e160.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e504A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eGLU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.39 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.7 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e114.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e572A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eLYS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.65 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e3.26 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e175.93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e574A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eLEU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e3.8 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e4.03 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e107.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e574A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eLEU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.93 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e3.5 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e153.27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e576A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eLYS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.87 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e3.86 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e164.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e576A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eLYS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e3.36 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e3.94 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e117.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e582A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eTYR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.58 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.77 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e101.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e607A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eSER\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.48 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.88 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e124.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e607A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eSER\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.46 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e3.15 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e130.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 SalB Modulates the ACSL4/GPX4 Protein Pathway in Hippocampal Tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further validate the molecular mechanisms by which SalB regulates ferroptosis, WB analysis was conducted. The results showed that, compared to the Sham group, the expression levels of ACSL4, ALOX15, and LPCAT3, which are proteins in the ACSL4 pathway, were significantly upregulated in the CLP group (Fig 8 A-D). Conversely, the expression levels of GPX4 and SLC7A11, proteins in the GPX4 pathway, were significantly downregulated in the CLP group (Fig 8 E-G). This indicates that the ACSL4 pathway can be activated while the GPX4 pathway is inhibited during the septic process, thereby contributing to ferroptosis and resulting in damage to the hippocampus. In contrast, following administration of SalB, there was a significant downregulation of the ACSL4 pathway proteins ACSL4, ALOX15, and LPCAT3, along with an upregulation of GPX4 and SLC7A11 expression levels compared to the CLP group. These findings demonstrate that SalB can inhibit the occurrence of ferroptosis by regulating the expression of protein markers in both the ACSL4 and GPX4 pathways, thereby providing protective effects against neuronal damage in the hippocampus.\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eFerroptosis, an iron-dependent, lipid peroxidation-driven form of cell death, has recently been implicated in sepsis-related organ damage [14, 15]. During sepsis, dysregulated iron metabolism-characterized by aberrant iron transport, increased uptake, and reduced export-contributes to pathological iron overload. This study systematically investigated the temporal dynamics of ferroptosis in septic mice. Key ferroptotic markers (Fe\u003csup\u003e2+\u003c/sup\u003e, GSH, and LPO) were analyzed, revealing a time-dependent progression: lipid peroxidation and iron accumulation were detectable as early as 12 hours post-CLP surgery, accompanied by significant GSH depletion. By 48 hours post-surgery, iron and LPO levels peaked with statistical significance, coinciding with the highest mortality rate in CLP mice. This temporal alignment suggests that ferroptosis reaches its zenith at 48 hours, correlating with the critical window of systemic metabolic derangement. These findings are consistent with endotoxin fluctuation patterns reported in Sepsis Pathophysiology and Therapeutics[16].\u003c/p\u003e\n\u003cp\u003eSalB, the most bioactive water-soluble component of Salvia miltiorrhiza Bunge, exhibits potent anti-inflammatory and anti-lipid peroxidation effects. However, its role in sepsis-induced neural injury remains underexplored. This study demonstrates that SalB exerts neu-roprotective effects during the acute phase of sepsis (48-72 hours), with peak efficacy observed at 48 hours - the critical window of ferroptosis progression. SalB significantly attenuated hippocampal ferroptosis, suggesting its therapeutic potential via modulation of iron metabolism and lipid peroxidation pathways. These findings provide a novel theoretical foundation for natural product-based interventions in sepsis management.\u003c/p\u003e\n\u003cp\u003eSepsis-induced multi-organ failure, driven by uncontrolled cytokine storms [17, 18] is a leading cause of clinical mortality. The hippocampus, a brain region critical for memory and learning, is highly vulnerable to inflammatory insults. In this study, CLP mice exhibited elevated behavioral scores, indicative of neurocognitive impairment, which were markedly reduced by SalB treatment. SAE is characterized by neuroinflammatory hyperactivation, particularly microglial polarization imbalance. M1 microglia, which secrete pro-inflammatory cytokines (e.g., IL-1\u0026beta;, TNF-\u0026alpha;), exacerbate neuronal damage, while M2 microglia promote neuroprotection and repair via anti-inflammatory mediators (e.g., IL-10) [19]. Paradoxically, excessive IL-10 in sepsis correlates with septic shock and multi-organ dysfunction [20]. Our data revealed elevated serum levels of IL-1\u0026beta;, TNF-\u0026alpha;, IL-4, and IL-10 in CLP mice, consistent with the cytokine profiles of septic patients [21]. SalB treatment significantly suppressed pro-inflammatory IL-1\u0026beta; and TNF-\u0026alpha; to sham-operated levels, while modulating anti-inflammatory IL-4 and IL-10 within a neuroprotective range. Notably, IL-10 levels in SalB-treated mice remained higher than in sham controls, suggesting a balanced immunoregulatory role of SalB in curbing inflammatory cascades.\u003c/p\u003e\n\u003cp\u003eIn the activity of the nervous system, the hippocampus receives neural incoming signals from numerous regions, including the limbic system, cortex, and subcortical areas, through the entorhinal cortex and the subiculum. The main pathway of neural activity entering the hippocampus begins at the entorhinal cortex, projecting via the perforant pathway to the granule cells of the dentate gyrus, which then send collateral branches to the pyramidal cells in the CA1 and CA3 regions. The mossy fibers emitted by the granule cells can activate the CA3 pyramidal cells as well as interneurons in the spatial region. CA3 pyramidal cells, through local and associative excitatory collaterals, not only excite other CA3 pyramidal cells and those in the lateral nucleus but also activate CA1 pyramidal cells [22]. The unique connections and functional divisions between CA3 and CA1 support the hippocampus\u0026apos;s functions in memory encoding, integration, and retrieval. The projection pathway between the CA3 and CA1 regions is vital for synaptic plasticity as well as the convergence and projection of information [23, 24]. The collaboration and imbalance of these areas not only affect memory functions but also constitute a significant pathological basis for neuropsychological disorders. Although the hippocampus is not the only brain region critical for memory, damage or dysfunction in this structure can lead to deficiencies in the formation and retention of various types of memory [25]. In this study, pathological section staining and FJC fluorescence staining revealed neuron damage and necrosis in the CA1 and CA3 regions of hippocampal tissue from mice in the CLP-induced sepsis model, characterized by increased intercellular spacing, loss of Nissl bodies, and an increase in degenerative neurons. This suggests that systemic inflammatory storms induced by abdominal sepsis may initiate neuroinflammatory responses, thereby affecting neuronal metabolism and promoting hippocampal neuronal injury and degenerative pathological changes, leading to cognitive dysfunction. Notably, the administration of SalB effectively improved neuronal damage in the CA1 and CA3 regions of the hippocampus, restoring the abundance of Nissl bodies and reducing the number of degenerative neurons. Evans Blue staining further confirmed extensive damage to the BBB induced by CLP, while SalB exhibited significant protective effects on the BBB. Importantly, neurons in the ferroptosis inhibitor group showed milder degeneration of Nissl bodies, which may be related to the inhibition of ferroptosis, while the significant and widespread improvement seen in the SalB group in terms of tissue pathology may stem from its dual action in regulating both inflammation and oxidative stress.\u003c/p\u003e\n\u003cp\u003eIron-induced oxidative stress represents a crucial pathological feature of neurodegenerative changes. Theoretically, the brain, with its inherently higher lipid content, greater membrane composition, and fewer antioxidant enzymes, shows weaker tolerance to lipid peroxidation compared to other organs and is more susceptible to the effects of iron accumulation, increasing the likelihood of oxidative stress and damage [26]. Evidence suggests a negative correlation between iron ion concentrations in the hippocampal region and memory scores [27]. Furthermore, studies have found that iron accumulation in the hippocampus leads to cognitive impairments in most neurodegenerative diseases, while the use of ferroptosis inhibitors can reduce oxidative stress damage and improve cognitive dysfunction [28]. In this study, observation of Fe\u003csup\u003e2+\u003c/sup\u003e staining in the CA3 region of the hippocampus revealed that CLP significantly induced notable enrichment of Fe\u003csup\u003e2+\u003c/sup\u003e in this region, while the intervention of SalB and the ferroptosis inhibitor Fer-1 effectively reduced Fe\u003csup\u003e2+\u003c/sup\u003e accumulation, indicating their regulatory effect on the excessive production of ferrous ions. To further validate the regulatory effects on ferroptosis, this study measured the expression levels of ferroptosis-related indicators, Fe\u003csup\u003e2+\u003c/sup\u003e, LPO, and GSH. In the pathway of ferroptosis, Fe\u003csup\u003e2+\u003c/sup\u003e promotes the generation of LPO through the Fenton reaction, which triggers membrane damage due to LPO accumulation, further releasing more Fe\u003csup\u003e2+\u003c/sup\u003e and ROS, thereby creating a vicious cycle. In this process, GPX4 uses GSH as a cofactor to resist lipid peroxidation, thus protecting membrane integrity. The results of this study indicate that CLP-induced sepsis leads to significant increases in Fe\u003csup\u003e2+\u003c/sup\u003e and LPO levels while downregulating GSH levels, thereby promoting the occurrence of ferroptosis. Conversely, SalB can downregulate Fe\u003csup\u003e2+\u003c/sup\u003e and LPO levels and promote GSH secretion, demonstrating its capacity to exert neuroprotective effects on septic mice by inhibiting oxidative stress damage induced by iron ions.\u003c/p\u003e\n\u003cp\u003eThrough protein chip experiments, it has been demonstrated that SalB can significantly and specifically bind to the ACSL4 protein. The ACSL4 pathway, as a key protein pathway for lipid peroxidation, plays an important role in the process of ferroptosis, as it is responsible for CoA esterification to free fatty acids in an ATP-dependent manner [29]. The formation of acyl-CoA activates the corresponding fatty acids for fatty acid oxidation or lipid biosynthesis, thereby further initiating the lipid peroxidation process. It, along with the GPX4 pathway, constitutes an important cyclic mechanism for the generation and degradation of lipid peroxides during physiological activities. Specifically, the ACSL4 pathway promotes the generation of lipid peroxides, while GPX4 is responsible for the degradation of these harmful peroxidized lipids. The two pathways complement each other, maintaining the balance of lipid metabolism within cells. This balance is crucial for preventing cellular damage caused by the accumulation of lipid peroxides, particularly evident in the occurrence and development of ferroptosis. Through this cyclic mechanism, the body can effectively regulate lipid peroxidation levels, thus influencing the process of ferroptosis. Therefore, we further performed protein-small molecule docking between SalB and ACSL4 using molecular docking technology, and we also docked CoA with ACSL4, comparing and analyzing the results of these two docking studies. The results showed that SalB shares the same binding sites with CoA at the amino acid residues Lys-572, Leu-574, and Ser-607 in the ACSL4 protein, and its binding effect is superior to that of CoA. Based on the experimental results, it is inferred that SalB may exert its pharmacological activity through competitive binding. Through structural analysis of the ACSL4 protein, we identified that the amino acid residues Lys-572, Leu-574, and Ser-607 are located within the C-terminal catalytic domain (approximately residues 500-711) and the Eukaryotic long-chain fatty acid CoA synthetase (LC-FACS) domain (residues 80-655), both of which constitute the core region governing substrate binding and enzymatic activity. Among these, the regions harboring Lys-572 and Leu-574 may guide drug molecules into the protein interior via hydrogen bonding, thereby altering local or global conformational states of the protein. Such structural changes could modify the geometry and properties of the substrate-binding pocket, ultimately disrupting ACSL4-mediated fatty acid activation. Notably, Ser-607, which also serves as a phosphorylation site, is implicated in this process. Given that phosphorylation of ACSL4 critically amplifies lipid peroxidation linked to ferroptosis [30], the formation of hydrogen bonds between SalB and Ser-607 may either obstruct phosphorylation or perturb post-phosphorylation signaling. This dual mechanism would regulate ACSL4-driven lipid metabolic pathways, thereby influencing the progression of ferroptosis. Finally, we returned to in vivo studies, measuring the expression levels of ACSL4 and GPX4 pathway proteins. The results indicated that in the CLP septic model mice, the protein level of the ACSL4 pathway was significantly elevated compared to the sham surgery group, while the expression of GPX4 pathway proteins was suppressed, suggesting that excessive lipid peroxidation had occurred in the septic mice, promoting ferroptosis. After administration of SalB, the protein level of the ACSL4 pathway was significantly downregulated, while the expression of GPX4 pathway proteins was significantly increased, indicating that SalB can effectively inhibit the occurrence of lipid peroxidation, regulate the generation of lipid peroxides, and inhibit ferroptosis (Fig 9). Notably, the ferroptosis inhibitor Fer-1 was less effective on the ACSL4 pathway protein compared to SalB, but it significantly regulated the expression of GPX4 pathway proteins, demonstrating its specific biological activity on the GPX4 pathway.\u003c/p\u003e"},{"header":"5 Conclusion ","content":"\u003cp\u003eIn summary, our study demonstrates that SalB significantly improves the survival rates of mice subjected to CLP-induced sepsis, reduces inflammatory responses, and alleviates neuronal damage in the hippocampus. Furthermore, SalB mediates its therapeutic effects by regulating ferroptosis processes through the ACSL4 pathway in the context of hippocampal injury caused by CLP-induced sepsis. Additionally, our protein arrays and molecular docking analysis further supports the hypothesis that SalB exerts its pharmacological activity via competitive inhibition with the substrate CoA of the ACSL4 protein. Our findings provide new insights into the use of SalB for treating sepsis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eACSL4, Long-chain acyl-CoA synthetase 4; BBB, Blood-brain barrier; CLP, Cecal ligation and puncture; CNS, Central nervous system; CoA, coenzyme A; Fer-1, Ferrostatin-1; GSH, glutathione; GPX4, Glutathione peroxidase 4; H\u0026amp;E, hematoxylin and eosin; IL, Interleukin; LPO, lipid peroxides; MDA, Malondialdehyde; MSS, Murine Sepsis Score; mNSS, modified Neurological Severity Score; OD, optical density; PVDF, polyvinylidene difluoride; ROS, Reactive oxygen species; SalB, Salvianolic acid B; SAE, Sepsis-associated encephalopathy; SNR, Signal-to-Noise Ratio; SOD, superoxide dismutase ; WB, Western blot.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe graphical abstract figures were generated by FigDraw (www.figdraw.com). Animal Experiment Ethics Approval Number: BJTCM-M-2023-11-04. Clinical trial number: not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Key Research and Development Program of China (2022YFD1801102), National Natural Science Foundation of China (82474428, 82174348), Sanming Project of Medicine in Shenzhen (No.SZZYSM202411012).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conceived and designed by KF, XXL, and QQL. The initial draft of the manuscript was prepared by XYL, ZZM, and YMY, while the main experimental data were collected by XYL, CXZ, and SWZ. YL. further revised and refined the manuscript. YMY and ZZM were responsible for the analysis, interpretation, and visualization of the data. YXL. and LW. directly accessed and validated the data presented in the article. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSinger, M., Deutschman, C.S., Seymour, C.W., Shankar-Hari, M., Annane, D., Bauer, M., et al. (2016) The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA 315: 801-810. https://doi.org/10.1001/jama.2016.0287.\u003c/li\u003e\n\u003cli\u003eGofton, T.E., Young, G.B. (2012) Sepsis-associated encephalopathy. Nat Rev Neurol 8: 557-566. https://doi.org/10.1038/nrneurol.2012.183.\u003c/li\u003e\n\u003cli\u003eMazeraud, A., Righy, C., Bouchereau, E., Benghanem, S., Bozza, F.A., Sharshar, T. 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(2017) ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition. Nat Chem Biol 13: 91-98. https://doi.org/10.1038/nchembio.2239.\u003c/li\u003e\n\u003cli\u003eZhang, H.L., Hu, B.X., Li, Z.L., Du, T., Shan, J.L., Ye, Z.P., et al. (2022) PKCbetaII phosphorylates ACSL4 to amplify lipid peroxidation to induce ferroptosis. Nat Cell Biol 24: 88-98. https://doi.org/10.1038/s41556-021-00818-3.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Sepsis, sepsis-associated encephalopathy, Salvianolic acid B, Ferroptosis, Neuroinflammation","lastPublishedDoi":"10.21203/rs.3.rs-7925353/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7925353/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Sepsis frequently leads to multi-organ injury, with the highly metabolically active nervous system being particularly vulnerable, and ferroptosis has been implicated in driving disease progression. Although Salvianolic acid B (SalB), the most abundant water-soluble active component of Salvia miltiorrhiza Bunge., has demonstrated antioxidant and anti-inflammatory properties, its specific mechanisms in sepsis-associated hippocampal injury remain unclear. To investigate SalB's therapeutic potential against sepsis and its role in mitigating neural damage via ACSL4-mediated ferroptosis, a murine sepsis model was established by cecal ligation and puncture (CLP). SalB's efficacy was evaluated using 7-day survival rates, multi-organ biochemical markers, and critical treatment windows. Inflammatory cytokines were measured by ELISA, and hippocampal morphology was examined histologically. Mechanistic studies included Fe²⁺ staining and lipid peroxidation assays, while protein arrays and Western blotting clarified SalB's interaction with ACSL4, confirming its anti-ferroptotic role. Our results show that SalB significantly improved survival in CLP-induced septic mice, reduced levels of inflammatory factors, alleviated hippocampal neuronal damage, and preserved blood-brain barrier integrity. Data from biochemical assays and Western blot analysis indicated that SalB suppresses ferroptosis by modulating the ACSL4/GPX4 pathway, supporting its therapeutic role in septic hippocampal injury. Additionally, protein array and molecular docking studies provided evidence that SalB likely exerts its pharmacological activity by competitively inhibiting the substrate CoA binding to ACSL4 at amino acid residues LYS-572, LEU-574, and SER-607. In conclusion, SalB protects against sepsis-induced hippocampal injury by targeting ACSL4-mediated ferroptosis, offering a novel herbal-based strategic direction for sepsis treatment.","manuscriptTitle":"Salvianolic Acid B Inhibits Ferroptosis Through ACSL4 Pathway Regulation in sepsis-associated encephalopathy Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 16:28:13","doi":"10.21203/rs.3.rs-7925353/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-03T03:16:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-18T20:55:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-18T07:24:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-23T12:43:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5939749277082022481930554698738838285","date":"2025-11-21T20:53:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8497211194969423697692162046010032792","date":"2025-11-20T14:55:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326734182709059638480501640312740077755","date":"2025-11-20T05:37:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"129904681768791789470569896367023285050","date":"2025-11-18T08:05:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-07T07:17:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-25T10:08:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-25T10:07:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2025-10-22T15:56:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0b949879-dc76-4dc4-b063-79e66a49c0ca","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:00:32+00:00","versionOfRecord":{"articleIdentity":"rs-7925353","link":"https://doi.org/10.1007/s12035-026-05876-y","journal":{"identity":"molecular-neurobiology","isVorOnly":false,"title":"Molecular Neurobiology"},"publishedOn":"2026-04-22 15:56:58","publishedOnDateReadable":"April 22nd, 2026"},"versionCreatedAt":"2025-11-18 16:28:13","video":"","vorDoi":"10.1007/s12035-026-05876-y","vorDoiUrl":"https://doi.org/10.1007/s12035-026-05876-y","workflowStages":[]},"version":"v1","identity":"rs-7925353","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7925353","identity":"rs-7925353","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}