Circulating A-FABP Exacerbates LPS-Induced Neurotoxicity by Crossing the Disrupted Blood–Brain Barrier and Promoting Neuronal Apoptosis

Circulating A-FABP Exacerbates LPS-Induced Neurotoxicity by Crossing the Disrupted Blood–Brain Barrier and Promoting Neuronal Apoptosis | 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 Circulating A-FABP Exacerbates LPS-Induced Neurotoxicity by Crossing the Disrupted Blood–Brain Barrier and Promoting Neuronal Apoptosis Muhammad Mustapha Ibrahim, Chunyan Li, Linhui Qiu, Shilun Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7369791/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Jan, 2026 Read the published version in Cell Communication and Signaling → Version 1 posted 8 You are reading this latest preprint version Abstract Sepsis-associated encephalopathy (SAE) is a critical complication of systemic inflammation with poorly understood mechanisms. This study identified adipocyte fatty acid-binding protein (A-FABP) as a key mediator linking peripheral inflammation to central nervous system damage. Using an LPS-induced endotoxemia model in wild-type and A-FABP knockout mice, we demonstrated that circulating A-FABP ( 1 ) crosses the compromised blood‒brain barrier (BBB), ( 2 ) accumulates in hippocampal neurons, and ( 3 ) synergizes with LPS to drive neuronal apoptosis. The monoclonal antibody 6H2, which neutralizes A-FABP, significantly reduced BBB leakage, attenuated neuroinflammation, and improved neuronal survival. In vitro studies confirmed that HT22 neurons internalize exogenous A-FABP, which amplifies LPS-induced late apoptosis without affecting early apoptotic pathways. These findings establish circulating A-FABP as both a biomarker and therapeutic target for SAE, revealing a novel periphery-to-CNS inflammatory cascade. A-FABP sepsis-associated encephalopathy blood‒brain barrier neuroinflammation LPS neuronal apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Severe systemic inflammation, often triggered by infections or traumatic events, can progress to sepsis, a life-threatening condition frequently associated with central nervous system (CNS) dysfunction, leading to sepsis-associated encephalopathy (SAE) ( 1 – 3 ). SAE is characterized by disruption of the blood‒brain barrier (BBB), neuroinflammation, neuronal dysfunction, and behavioral changes such as sickness or depression-like behaviors. Lipopolysaccharide (LPS), an endotoxin from gram-negative bacteria, plays a significant role in sepsis pathogenesis, as these infections are the most common cause of sepsis ( 4 ). Sepsis-related mortality remains high due to complex mechanisms and a lack of effective therapies ( 5 , 6 ). A dynamic equilibrium of pro- and anti-inflammatory factors regulates inflammation. Dysregulation of these mediators during infection or injury can lead to organ dysfunction, including brain injury, contributing to high mortality rates if uncontrolled in sepsis models. BBB disruption is closely linked to neuroinflammation, which is characterized by microglial activation and increased levels of proinflammatory cytokines in brain tissue ( 7 – 9 ). Proinflammatory cytokines such as TNFα, IL-1β, and IL-6, which are elevated in the bloodstream, act as immunological messengers that drive CNS inflammation ( 10 ). Sepsis-associated encephalopathy affects 50–70% of sepsis patients, contributing to long-term cognitive impairment and increased mortality ( 11 – 14 ). While systemic inflammation disrupts the blood‒brain barrier (BBB), the specific mediators that cross the CNS to drive neurodegeneration remain poorly characterized. Adipocyte fatty acid-binding protein (A-FABP), a lipid chaperone expressed in adipocytes and macrophages, regulates systemic metabolism and inflammation( 15 – 17 ). It is implicated in insulin resistance, metabolic syndrome, and cardiovascular diseases ( 18 – 21 ). A-FABP also plays a critical role in sepsis pathogenesis. Studies have shown that A-FABP inhibition, either genetically or pharmacologically, mitigates septic acute kidney injury by disrupting the TLR4/c-Jun signaling pathway, which increases inflammation and cell death ( 22 , 23 ). Bioinformatics analysis of sepsis patient data revealed that higher A-FABP levels correlate with increased mortality, suggesting its role as a mediator of sepsis severity and a potential biomarker for poor outcomes ( 24 ). Additionally, A-FABP is implicated in sepsis-induced acute respiratory distress syndrome (ARDS), where gut microbiota-derived acetic acid influences neutrophil apoptosis via A-FABP, exacerbating lung injury ( 25 , 26 ). However, the involvement of A-FABP in sepsis-associated neuroinflammation and SAE and whether it represents a therapeutic target remain unclear. Recent evidence suggests that A-FABP may bridge metabolic and inflammatory pathways in neurological disorders. In macrophages and microglia, A-FABP potentiates TLR4 signaling, the same pathway activated by LPS ( 27 – 29 ). Crucially, A-FABP levels are correlated with sepsis mortality ( 30 – 32 ), but whether A-FABP actively contributes to CNS injury or merely reflects disease severity is unresolved. This knowledge gap is significant because A-FABP neutralization improves outcomes in metabolic diseases, raising the possibility of repurposing this approach for neuroprotection. Our study addresses three fundamental questions: First, does circulating A-FABP infiltrate the brain during systemic inflammation? Second, what cellular populations internalize A-FABP? Third, does A-FABP interact with LPS to exacerbate neuronal injury? Using complementary in vivo (A-FABP KO mice) and in vitro (HT22 neurons) models, we identified a previously unrecognized "two-hit" mechanism: LPS primes BBB disruption and neuronal vulnerability, whereas blood-derived A-FABP executes neurotoxicity through synergistic apoptosis induction. These findings not only advance our understanding of SAE pathogenesis but also validate A-FABP neutralization as a mechanistically grounded therapeutic strategy. Methods Animals and treatments The mice were kept in a clean, pathogen-free environment with a standard 12-hour light and dark cycle. They had free access to food and water throughout the study. All animal handling and experimental procedures complied with the NIH guidelines for ethical animal care (NIH Publication No. 86 − 23, revised 1985) and were approved by the Institutional Animal Care and Use Committee (IACUC) at the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences. We used male C57BL/6J mice, aged 8–10 weeks, sourced from the Vital River Laboratory in Beijing, China. To induce endotoxemia, we injected LPS ( Escherichia coli O55:B5, Sigma, Cat. No. L2880) into the abdominal cavity at a dose of 25 mg/kg body weight. Thirty minutes after the LPS injection, the mice received an intravenous dose of either the mouse mAb 6H2 (3.6 mg/kg) or a control antibody (mouse IgG, Immuno-Diagnostics, Cat. No. 221116, RRID: AB_3073816) at the same concentration. Any mice that died before the scheduled endpoint were excluded from the analysis. The assignment of the mice to treatment groups was randomized to ensure unbiased results. Cell culture and treatments The murine hippocampal neuronal cell line HT22 (Merck, #SCC129) and the microglial cell line BV2 (Procell, #CL-0493) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Cytiva, #sh30243.01) supplemented with 10% fetal bovine serum (FBS; Gibco, #10270106) and 1% penicillin/streptomycin (Gibco, #15140122) at 37°C in a humidified 5% CO₂ incubator. For the experimental treatments, the cells were serum starved (HT22, 24 hours; BV2, 12 hours) and exposed to the indicated concentrations of lipopolysaccharide (LPS; Sigma, #L2880) or recombinant murine A-FABP (MCE, #HY-P75215). Treatments were administered for the indicated times in complete medium. The control groups received vehicle (PBS) only. Evaluation of blood‒brain barrier (BBB) permeability via Evans blue Four hours before sacrifice, anesthetized mice received an injection of Evans blue (EB) (2%, 4 mL/kg) via the tail vein. After anesthesia, the mice were perfused transcardially with 40 mL of ice-cold PBS to flush out any remaining dye in the bloodstream. The brain was then carefully removed and homogenized in 1 mL of 50% trichloroacetic acid. The homogenate was centrifuged at 12,000 rpm for 15 minutes, and the EB concentration in the supernatant was measured via a spectrophotometer (Thermo Fisher, MultiskanTM FC) at 620 nm. A standard curve was used to calculate the EB levels, which are expressed as ng per gram of brain tissue. Immunofluorescence Staining For tissue processing, deeply anesthetized mice were transcardially perfused with cold PBS, followed by brain extraction. The harvested tissues were fixed in 4% PFA for 1 hour at room temperature and then cryoprotected by sequential dehydration in 15% and 30% sucrose solutions at 4°C. After being embedded in OCT compound (Sakura, Cat. #4583), the tissues were sectioned into 20 µm-thick slices, air-dried, and washed with PBS. For A-FABP staining, the sections were subjected to acetone fixation at -20°C, SDS-mediated antigen retrieval, and blocking with a solution containing 10% donkey serum, 1% BSA, 1% Triton X-100, and 0.05% NaN3. Primary and secondary antibodies (Tables S1-S2) were applied overnight at 4°C or for 1 hour at RT, respectively, followed by incubation with DAPI (Cell Signaling Technology, Cat. #8961S). Fluorescence imaging was performed via a ZEISS Axio Imager Z2 microscope with ApoTome.2. Quantitative analysis included assessment of BBB leakage (target protein density/CD31 area), microglial activation (Iba1 + cells/area), and astrocyte reactivity (GFAP mean density), all of which were performed via ImageJ software. The hippocampus and cortex were specifically analyzed for these parameters to evaluate neurovascular and neuroinflammatory responses. RNA Extraction and RT‒qPCR Analysis The mice were deeply anesthetized and transcardially perfused with ice-cold PBS. The brain tissue was carefully dissected on ice and homogenized in RNA lysis buffer. For cultured cells, the medium was aspirated, and the cells were rinsed once with PBS. Total RNA was isolated via the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme), followed by cDNA synthesis with HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme) to ensure genomic DNA removal. For gene expression analysis, RT‒qPCR was conducted on a LightCycler 96 system (Roche) with the following thermal profile: initial denaturation at 95°C for 3 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. We quantified the expression levels of inflammatory markers (IL6, IL-1β, Ccl2, IL-17a, A-FABP, and TNF-α) via the use of β-actin as a housekeeping gene. All primers (Table S2 ) were designed with a 60°C annealing temperature and were verified via melt curve analysis. Relative gene expression was determined via normalization to β-actin via the ΔΔCt method. Western blot analysis Protein samples (10–15 µg per lane) were resolved via 10% SDS‒PAGE and subsequently transferred to PVDF membranes. Following transfer, the membranes were blocked with 5% nonfat milk in TBST for 1 hour at room temperature to prevent nonspecific binding. Primary antibodies (Table S2 ) were applied overnight at 4°C with gentle agitation to ensure optimal antigen‒antibody interactions. After extensive washing (X5 for 5 minutes in TBST), the membranes were incubated with appropriate HRP-conjugated secondary antibodies (Table S2 ) for 1 hour at room temperature. Following additional TBST washes, protein bands were visualized via a chemiluminescent HRP substrate (Millipore, Cat. #WBKLS0500) and imaged via the GelView 6000 M system (BioLight, GV6000). Quantitative analysis was performed via ImageJ software, with target protein expression levels normalized to those of β-actin or GAPDH as loading controls. Flow cytometric analysis of immune cells in the brain We started by carefully chopping the mouse brain tissue into fine pieces via a sterilized razor blade. The minced tissue was then broken down using an enzyme cocktail containing Type IV collagenase (400 U/ml), dispase (1.2 U/ml), and DNase I (32 U/ml) - all from Worthington - dissolved in 6 mL of calcium/magnesium-supplemented PBS. This digestion process ran for 30 minutes at 37°C, with gentle mixing every 10 minutes to help break the tissue. After digestion, we added 1 mL of FBS and 20 mL of chilled PBS to stop the reaction and then passed the mixture through 40 µm filters. The cells were subjected to several washing steps: first, they were spun at 400 × g for 5 minutes at 4°C; then, they were resuspended in 20% BSA solution for myelin removal at 1,000 × g (25 minutes, 4°C). We washed the cells twice in 0.3% FBS/PBS solution with centrifugation at 400× g each time. For surface marker analysis, the cells were first incubated with a mouse BD Fc block in 0.3% FBS/PBS for 10 minutes on ice to prevent nonspecific antibody binding. After being washed with PBS, the cells were stained with anti-CD11b-APC (BioLegend, cat. #101211) and PE-conjugated anti-CD45 (BD Biosciences, cat. #516087) for 45 minutes at 4°C in the dark. The stained cells were washed and resuspended in PBS for acquisition. Flow cytometric analysis was performed via a Sony MA900 flow cytometer equipped with a 100 µm nozzle, and CD45⁺/CD11b⁺ populations were identified for downstream analysis. Cell Apoptosis Assay The cells were digested with 0.25% trypsin (Gibco, #15050065) for 1 min at 37°C. The suspension was subsequently centrifuged at 300 × g for 5 minutes at 4°C. The cells were stained with the Annexin V-PE/7-AAD Apoptosis Detection Kit (Yeasen, Cat. No. 40310ES50) following the manufacturer’s protocol. Briefly, after treatment, the cells were washed twice with cold PBS and resuspended in Annexin V binding buffer. Annexin V-PE and 7-AAD were then added to the cell suspension and incubated for 15 minutes at room temperature in the dark. The samples were analyzed via flow cytometry (Beckman, CytoFLEX S) within one hour of staining. Viable, early apoptotic, and late apoptotic cells were distinguished on the basis of their staining profiles: Annexin V⁻/7-AAD⁻ (viable), Annexin V⁺/7-AAD⁻ (early apoptotic), and Annexin V⁺/7-AAD⁺ (late apoptotic). All flow cytometric data were analyzed via FlowJo software. Murine Sepsis Score Development In our LPS-induced sepsis experiments, mice were evaluated via a refined behavioral assessment to monitor disease progression and treatment effects. Following baseline observations, the animals were scored at regular intervals via a murine sepsis score (MSS) adapted for the LPS model. The MSS comprises six parameters, spontaneous movement, stimulus response, posture, respiratory rate, breathing pattern, and piloerection, each graded from 0 (normal) to 4 (severely impaired), yielding a total score indicative of illness severity. We conducted careful observation, ensuring high interrater reliability. Data were recorded on standardized sheets alongside rectal temperature and body weight measurements. The MSS provides a quantitative, reproducible framework for tracking sepsis severity, enabling comparisons across the sham, IgG-treated, and 6H2-treated groups. Validation against physiological markers, including hypothermia and weight loss, confirmed the system’s sensitivity and alignment with established models ( 33 – 35 ). Cell viability assay (CCK-8) HT22 cells were seeded into 96-well plates at a density of 5,000 cells/well in 100 µL of complete DMEM and incubated for 24 hours at 37°C in a humidified 5% CO₂ atmosphere to allow cell attachment. Following attachment, the cells were treated with lipopolysaccharide (LPS), recombinant A-FABP, or vehicle controls in complete medium for 6, 24, or 48 hours. After treatment, 10 µL of Cell Counting Kit-8 (Yeasen, Cat. No. 40203ES80) solution was added to each well and incubated for an additional 1 hour at 37°C. The absorbance was then measured at 450 nm via a microplate reader (Thermo Scientific, Multiskan GO). Quantitative analysis All quantitative data are presented as the mean ± standard error of the mean (SEM). Statistical analyses were performed via GraphPad Prism version 10 (GraphPad Software, CA, USA). For comparisons between two groups, unpaired two-tailed Student’s t tests were applied, whereas one-way analysis of variance (ANOVA) was used for comparisons involving more than two groups. A p value less than 0.05 was considered statistically significant. For the analysis of microglial morphology, an ImageJ-based skeletonization protocol was employed. Briefly, original fluorescence micrographs were preprocessed via fast Fourier transform (FFT) bandpass filtering and unsharp masking to increase image quality. The processed images were then binarized and skeletonized, followed by quantification of microglial branching structures via the Analyze Skeleton plugin. Cropped overlays were used to ensure the anatomical accuracy of segmentation. Endpoint counts derived from skeletons were normalized to the number of microglial somata per field to account for cellular density. Results 6H2 treatment lowers the Murine Sepsis Score (MSS) and reduces mortality in LPS-Induced endotoxemia In this study, 8-week-old male C57BL/6J wild-type mice were used to establish an LPS-induced (25 mg/kg body weight) sepsis model to evaluate the therapeutic effects of the monoclonal antibody 6H2 (3.6 mg/kg). The experimental groups included a saline control group (sham), an LPS + mIgG control group (LPS + mIgG), and an LPS + 6H2 treatment group (LPS + 6H2). Thirty minutes after intraperitoneal LPS injection, the mice received either mIgG or 6H2 via tail vein injection. Survival rates, body weight changes, body temperature fluctuations, and sepsis scores were assessed 24 hours later (Fig. 1 A). The results revealed survival rates of 100% in the control group, 70% in the mIgG group, and 80% in the 6H2 group (Fig. 1 B). All septic mice exhibited significant weight loss, hypothermia, and motor dysfunction (manifested as reduced spontaneous movement, jerky motions, abnormal gait, piloerection, partially closed eyelids, and ocular discharge) within 24 hours. Compared with the mIgG controls, 6H2 treatment failed to reverse the LPS-induced metabolic disturbances reflected by persistent weight loss and hypothermia (Fig. 1 C and D ). Sepsis scoring confirmed minimal septic manifestations in the sham group, severe sepsis in the LPS + mIgG group, and significant symptom alleviation in the LPS + 6H2 group (Fig. 1 E). Notably, compared with septic control mice, 6H2-treated mice presented reduced neurological manifestations, including less piloerection, improved eye opening, and decreased ocular discharge, suggesting specific neuroprotective effects independent of systemic metabolic recovery. These findings demonstrate that 6H2 effectively lowers the murine sepsis score and mortality and modulates LPS-induced immune dysregulation, as reflected by lower sepsis scores, highlighting its therapeutic potential in inflammatory diseases. 6H2 Treatment alleviates LPS-induced blood‒brain barrier disruption LPS-induced sepsis causes significant BBB leakage, neuroinflammation, and systemic inflammation, leading to severe neurological damage( 25 ). To evaluate the ability of 6H2 to protect against LPS-induced blood‒brain barrier (BBB) disruption, we employed complementary approaches. Evans blue extravasation was used to quantify global leakage, whereas endogenous IgG infiltration was used to assess molecular-level compromise. LPS-challenged mice presented increased Evans blue penetration into the brain parenchyma, and 6H2 treatment significantly attenuated Evans blue leakage (Fig. 2 A). Western blot analysis further supported this finding, as the LPS + mIgG group presented elevated IgG levels in brain tissue, whereas the LPS + 6H2 group presented reduced mIgG levels (Fig. 2 B). Additionally, co-immunofluorescence staining of plasma IgG and CD31 (endothelial markers) in the hippocampus and cortex revealed intense IgG staining in the LPS + mIgG group, whereas the LPS + 6H2 group presented significantly reduced IgG staining, further supporting the protective effects of 6H2 on BBB integrity (Fig. 2 C and 2 D). These complementary approaches consistently demonstrate that 6H2 preserves BBB integrity during sepsis. 6H2 treatment alleviates LPS-induced neuroinflammation LPS-induced sepsis not only causes substantial blood‒brain barrier (BBB) disruption but also elicits both vascular and neuroinflammatory responses. LPS causes neuroinflammation, as evidenced by the upregulation of proinflammatory cytokines and microglial and astrocyte activation ( 26 ). Next, we investigated whether 6H2 treatment could mitigate neuroinflammation. RT‒qPCR analysis revealed that LPS significantly increased the expression of proinflammatory cytokines (Il-6, TNF-α, Il-1β, Il-17a, and Ccl2) in the brain (Fig. 3 A), but 6H2 treatment notably reduced their expression, resulting in cytokine levels closer to those observed in the sham group. Immunofluorescence staining of Iba1, a microglial marker, in the hippocampus revealed that LPS-treated mice exhibited significant microglial activation, which was notably reduced by 6H2 treatment (Fig. 3 B). Microglial morphology varied among the groups: a resting phenotype in the Sham group (15 Endpoints/Cell), an activated phenotype in the IgG-treated group (~ 10 Endpoints/Cell, p < 0.0001 vs. Sham), and a recuperative phenotype in the 6H2-treated group (~ 20 Endpoints/Cell, p < 0.0001 vs. IgG). Quantitative analysis confirmed that 6H2 effectively diminished LPS-induced microglial activation (Fig. 3 C). Similarly, LPS-induced astrocyte activation is characterized by morphological changes, including increased cell size, complexity, and process density. These changes are indicative of a reactive state in astrocytes ( 27 ). GFAP staining, an astrocyte marker, revealed that 6H2 alleviated LPS-induced astrocytic activation, as characterized by increased size, complexity, and process density in the hippocampus (Fig. 3 D). Collectively, these findings suggest that 6H2 mitigates LPS-induced neuroinflammation by reducing proinflammatory cytokine expression and suppressing the activation of both microglia and astrocytes. 6H2 treatment improves neuronal survival in LPS-induced sepsis Given that 6H2 preserves BBB integrity and attenuates neuroinflammatory responses, we next investigated its neuroprotective potential upon LPS administration. To assess neuronal apoptosis, we conducted co-immunofluorescence staining for NeuN (a neuronal marker) and subsequently performed a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay in hippocampal tissue ( Fig. 4 A ) . Immunohistochemical analysis revealed significant differences in neuronal survival and apoptosis across the experimental groups ( Fig. 4 B ) . Compared with the sham group, which presented abundant NeuN + cells and minimal TUNEL + staining, indicating healthy neuronal populations with negligible apoptosis, the LPS + mIgG group presented severe neuronal damage characterized by a marked reduction in NeuN + cells and a substantial increase in TUNEL + cells. This pattern demonstrated extensive LPS-induced neuronal apoptosis. Importantly, the LPS + 6H2 treatment group showed significant neuroprotection, with preserved NeuN + cell counts and dramatically fewer TUNEL + cells than the LPS + mIgG group. Further analysis of NeuN + /TUNEL + double-labeled cells confirmed these findings, whereas the sham group presented virtually no apoptotic neurons, the LPS + mIgG group presented numerous double-positive cells, and this effect was significantly attenuated by 6H2 treatment. These results clearly demonstrate that 6H2 confers robust neuroprotection in the LPS-induced sepsis model by maintaining neuronal viability and reducing apoptosis, suggesting its potential therapeutic value for sepsis-associated neurological damage. 6H2 modulates leukocyte infiltration and attenuates A-FABP accumulation in microglia and neurons in LPS-induced sepsis We next investigated whether 6H2 treatment influences A-FABP levels in the brain upon LPS stimulation. First, we conducted FACS to evaluate whether 6H2 affects the infiltration of immune cells in the blood into the brain parenchyma. The sham group exhibited negligible leukocyte (lymphocyte and monocyte) infiltration due to the absence of inflammatory stimulation; the LPS + mIgG group exhibited significant leukocyte recruitment (Fig. 5 A and 5 B). The LPS + 6H2 group displayed reduced leukocyte infiltration, suggesting that the drug 6H2 mitigated LPS-driven inflammation and BBB disruption. Next, we assessed A-FABP mRNA expression and protein localization in the brain via RT‒qPCR and co-immunofluorescence staining, respectively, followed by quantitative analysis. First, we measured A-FABP mRNA expression levels in brain tissue via RT-qPCR. Quantitative RT‒PCR analysis revealed that the sham group presented the highest expression levels of A-FABP mRNA, reflecting baseline expression under normal physiological conditions (Fig. 5 C). In contrast, the LPS + mIgG group presented a marked decrease in A-FABP mRNA expression compared with the sham group, whereas the LPS + 6H2 group presented a further reduction in A-FABP mRNA levels. These findings suggest that LPS stimulation downregulates A-FABP transcription in the brain and that 6H2 treatment enhances this effect, highlighting its efficacy in modulating A-FABP levels in response to LPS-induced inflammation. To further investigate A-FABP protein localization under neuroinflammatory conditions, we performed co-immunofluorescence staining for A-FABP with either IBA1 or NeuN in hippocampal brain tissue. In the LPS + mIgG group, we observed pronounced A-FABP immunoreactivity in both NeuN + neurons (Fig. 5 D) and IBA1 + microglia (Fig. 5 G), indicating significant A-FABP protein accumulation despite the observed downregulation of A-FABP mRNA. This discrepancy suggests that the accumulated A-FABP protein may originate from the peripheral circulation, crossing the compromised BBB under inflammatory conditions and subsequently interacting with neuronal and microglia. In contrast, the LPS + 6H2 group presented attenuated A-FABP staining intensity in both cell types, suggesting that 6H2 treatment reduces A-FABP accumulation. Minimal A-FABP immunoreactivity was detected in the sham group, which served as a baseline control ( Fig. 5 E ). To quantify A-FABP accumulation in microglia, we analyzed the integrated density (IntDen) of A-FABP within IBA1 + cells, which was normalized to the total cell count (IBA1 + A-FABP + IntDen/Total Cell). The LPS + mIgG group presented the highest A-FABP + IBA1 + intensity, confirming substantial A-FABP deposition in microglia. Conversely, the LPS + 6H2 group presented a significantly lower A-FABP + IBA1 + intensity, further confirming that 6H2 treatment mitigated A-FABP accumulation in microglia (Fig. 5 F). Similarly, co-staining for A-FABP and NeuN revealed robust A-FABP immunoreactivity in the neurons of the LPS + mIgG-treated mice, whereas those in the LPS + 6H2 group presented a reduced neuronal A-FABP signal ( Fig. 5 G ). Quantification of A-FABP + NeuN + intensity further supported these observations, demonstrating that 6H2 treatment significantly decreased A-FABP accumulation in neurons ( Fig. 5 H ). These findings collectively demonstrate that while LPS-induced neuroinflammation suppresses A-FABP mRNA expression, it promotes A-FABP protein accumulation in both microglia and neurons, likely through the infiltration of circulating A-FABP across the disrupted BBB. Notably, 6H2 treatment not only further reduced A-FABP transcription but also attenuated A-FABP protein deposition in these cell populations. This dual regulatory effect suggests that the modulation of A-FABP levels, both at the transcriptional and protein accumulation levels, contributes to the anti-inflammatory and neuroprotective properties of 6H2. A-FABP synergizes with LPS to drive late-stage neuronal apoptosis in vitro Our study revealed an intriguing phenomenon: while LPS stimulation dramatically downregulated A-FABP mRNA expression in the brain, A-FABP protein accumulated prominently in neurons. This discrepancy suggests that neuronal A-FABP accumulation may not stem from endogenous production but rather from the uptake of circulating A-FABP, which crosses the compromised BBB during systemic inflammation. To investigate this hypothesis, we examined the direct effects of A-FABP on neuronal cells via the HT22 hippocampal neuronal line and evaluated its effects on A-FABP expression, protein accumulation, cell viability, and apoptosis in the presence or absence of LPS. Our quantitative PCR analysis revealed that recombinant A-FABP significantly suppressed A-FABP mRNA expression in HT22 cells at baseline (0 µg/ml LPS). Upon stimulation with LPS (1 µg/ml), the endogenous A-FABP mRNA was already downregulated, and exogenous A-FABP further decreased its transcriptional level ( Fig. 6 A ). These results suggest that extracellular A-FABP contributes to a feedback mechanism that reduces its gene expression, potentially modulating cellular responses to stress. Western blotting analysis revealed that HT22 cells did not express A-FABP, at least at low levels, while A-FABP administration led to a robust A-FABP band, independent of the presence of LPS. Quantification confirmed that A-FABP protein levels decreased with increasing LPS concentration and that LPS itself could not induce A-FABP expression in neurons ( Fig. 6 B ) . These results revealed that HT22 hippocampal neurons exhibit negligible endogenous A-FABP expression, yet they rapidly accumulate A-FABP protein when exposed to exogenous A-FABP, regardless of LPS stimulation. This finding strongly supports our hypothesis that neuronal A-FABP accumulation in vivo results primarily from the uptake of circulating A-FABP rather than its de novo synthesis. To determine whether internalized A-FABP directly affects neuronal viability, we treated HT22 cells with increasing concentrations of recombinant A-FABP (10–1000 ng/ml) in the presence or absence of LPS. In contrast, low concentrations of A-FABP alone did not improve cell viability (Fig. 6 C). Instead, higher doses (≥ 100 ng/ml) promoted HT22 survival in a dose-dependent manner, suggesting a neuroprotective effect of exogenous A-FABP under basal conditions. Strikingly, when A-FABP was combined with LPS (0.5 or 1 µg/ml), A-FABP synergistically amplified LPS-induced cytotoxicity. This effect was most pronounced at higher A-FABP concentrations (500–1000 ng/ml), where cell viability decreased significantly compared with that of the cells treated with LPS alone. Time-course experiments further revealed that prolonged exposure (48 hours) to A-FABP and LPS exacerbated neuronal death, highlighting a time- and dose-dependent neurotoxic synergy between these two factors (Fig. 6 C). To investigate the synergistic neurotoxic effects of A-FABP and LPS, we quantified the degree of apoptosis in HT22 neurons via Annexin V-PE/7-AAD staining and flow cytometry (Fig. 6 D). While neither A-FABP (0.5 µg/ml) nor LPS (1 µg/ml) alone significantly increased apoptosis compared with that of the controls, their combination resulted in a marked increase in late apoptotic cells (Annexin V+/7-AAD+; p < 0.01 vs LPS alone). Notably, early apoptosis (Annexin V+/7-AAD-) remained unaffected across all treatment groups (Fig. 6 E). The selective increase in late apoptosis suggests that A-FABP exacerbates terminal cell death pathways after LPS primes for neuronal damage. Collectively, our findings demonstrate a coordinated mechanism of neuronal damage involving A-FABP and LPS. This synergistic interaction provides a mechanistic explanation for the exacerbated neuronal injury observed under inflammatory conditions, where both systemic A-FABP elevation and LPS exposure occur concurrently. A-FABP potentiates LPS-induced neuronal apoptosis in vivo To validate our in vitro observations of the synergistic neurotoxic effects of A-FABP and LPS, we performed complementary in vivo experiments in A-FABP knockout (KO) mice. Eight-week-old male A-FABP KO mice were intraperitoneally injected with either LPS (25 mg/kg) to induce endotoxemia or saline as a control. Thirty minutes later, the mice received an intravenous injection of either recombinant A-FABP (3 µg) or saline through the tail vein (Fig. 7 A). This carefully controlled experimental design enabled us to specifically investigate the impact of exogenously administered A-FABP in the absence of endogenous A-FABP expression, thereby isolating the effects of circulating A-FABP on LPS-induced neurotoxicity. Assessment of sepsis severity via the murine sepsis score revealed distinct patterns among the treatment groups. Both the saline control groups (Saline + Saline and Saline + A-FABP) maintained low sepsis scores, indicating minimal systemic illness. In contrast, the mice that received LPS (LPS + Saline and LPS + A-FABP groups) presented significantly elevated sepsis scores, confirming the successful induction of severe systemic inflammation regardless of A-FABP status. These findings demonstrated that the knockout of A-FABP did not prevent the development of sepsis symptoms following LPS challenge. Notably, while both LPS-treated groups (LPS + Saline and LPS + A-FABP) presented marked illness, the LPS + A-FABP group presented even higher sepsis scores than the LPS + Saline group did (Fig. 7 B). This dose-dependent worsening of clinical symptoms with A-FABP administration suggests that circulating A-FABP not only fails to protect against LPS-induced sepsis but actually exacerbates the systemic inflammatory response and associated behavioral manifestations. These findings provide compelling in vivo evidence that A-FABP plays an active role in amplifying the pathological consequences of endotoxemia rather than being a passive biomarker of disease severity. Immunofluorescence analysis of hippocampal tissue provided crucial insights into A-FABP distribution and neuronal apoptosis (Fig. 7 C). In saline-treated A-FABP KO mice (Saline + Saline and Saline + A-FABP groups), we observed negligible A-FABP immunoreactivity, demonstrating that circulating A-FABP cannot cross the intact blood‒brain barrier. Interestingly, the LPS + Saline group presented a detectable A-FABP signal, likely reflecting either residual expression from incomplete knockout or minimal basal expression in certain cell types under inflammatory conditions. Most strikingly, the LPS + A-FABP group demonstrated intense A-FABP immunostaining that prominently localized to hippocampal neurons (Fig. 7 D). This dramatic increase in A-FABP signaling specifically in LPS-treated mice receiving exogenous A-FABP provides direct visual evidence that ( 1 ) LPS-induced systemic inflammation compromises blood‒brain barrier integrity and that ( 2 ) circulating A-FABP readily crosses the disrupted barrier to accumulate within vulnerable neuronal populations. Neuronal viability assessments yielded striking results. The saline control groups presented abundant NeuN-positive neurons with minimal TUNEL staining, reflecting healthy neuronal populations with negligible apoptosis. LPS administration alone caused significant neuronal damage, as evidenced by an increase in TUNEL-positive neuronal cells. Most importantly, the combination of LPS and A-FABP resulted in substantially greater neuronal apoptosis than did LPS alone, with a marked increase in NeuN+/TUNEL + double-labeled cells (Fig. 7 E). This in vivo observation directly corroborated our in vitro findings of synergistic neurotoxicity. These results collectively demonstrate that circulating A-FABP gains access to the CNS during systemic inflammation when the blood‒brain barrier becomes compromised. Once in the brain parenchyma, A-FABP is internalized by neurons, where it potentiates LPS-induced apoptotic pathways. Our findings position A-FABP as both a key mediator of neuroinflammatory injury and a promising therapeutic target for preventing sepsis-associated neurodegeneration. Discussion Our study demonstrated that neutralizing circulating A-FABP with the monoclonal antibody 6H2 effectively mitigated LPS-induced endotoxemia by preserving blood‒brain barrier (BBB) integrity, reducing neuroinflammation, and attenuating neuronal apoptosis. These findings highlight the pivotal role of A-FABP in sepsis-associated encephalopathy (SAE) and underscore its potential as a therapeutic target for neuroinflammatory conditions. The protective effects of 6H2 on BBB integrity were evident through reduced Evans blue leakage and decreased IgG extravasation in brain tissue. These results align with previous studies showing that systemic inflammation disrupts the BBB, allowing harmful substances to infiltrate the central nervous system (CNS)( 39 – 41 ). By neutralizing circulating A-FABP, 6H2 likely prevents its pathological accumulation in the brain, thereby preserving BBB function and limiting neuroinflammatory responses. Neuroinflammation, characterized by microglial and astrocyte activation, is a hallmark of SAE. Our data showed that 6H2 treatment significantly reduced the expression of proinflammatory cytokines (TNF-α, IL-1β, IL-6, IL-17a, and Ccl2) and attenuated glial activation. Notably, microglial morphology shifted from an activated, amoeboid state in LPS-treated mice to a more ramified, homeostatic phenotype in 6H2-treated animals. These findings suggest that A-FABP neutralization not only dampens inflammatory signaling but also promotes the restoration of glial homeostasis. The observed downregulation of A-FABP mRNA in LPS-treated brains, coupled with increased A-FABP protein accumulation in neurons and microglia, points to a dual mechanism: while LPS suppresses A-FABP transcription, peripheral A-FABP infiltrates the CNS due to BBB disruption. 6H2 mitigates this effect by neutralizing circulating A-FABP, reducing its deleterious effects on neural cells. The neuroprotective effects of 6H2 were further corroborated by reduced neuronal apoptosis, as evidenced by decreased TUNEL + staining and preserved NeuN + cell counts. Our in vitro experiments revealed that A-FABP synergizes with LPS to exacerbate neuronal apoptosis, particularly in the late stages. The in vivo findings in A-FABP knockout mice further support this finding, as the absence of A-FABP attenuated LPS-induced neuronal damage, whereas exogenous A-FABP worsened outcomes. These integrated in vitro and in vivo findings reveal a novel mechanism by which circulating A-FABP exacerbates neuronal injury during systemic inflammation. Although neurons normally maintain minimal endogenous A-FABP expression, they actively take up circulating A-FABP when the blood-brain barrier becomes compromised during endotoxemia, leading to synergistic neurotoxicity with LPS. The immunofluorescence data from A-FABP KO mice provide compelling evidence for this pathological cascade. The complete absence of the A-FABP signal in saline-treated controls confirms both the effectiveness of our knockout model and the impermeability of the intact blood-brain barrier to circulating A-FABP. The faint A-FABP signal observed in LPS + Saline KO mice suggests either residual expression in certain cell types under inflammatory conditions or potential limitations in knockout efficiency. Most importantly, the dramatic A-FABP accumulation specifically in the hippocampal neurons of LPS + A-FABP-treated mice visually demonstrates how systemic inflammation enables peripheral A-FABP to access the CNS parenchyma. These spatial distribution patterns correlate precisely with our observations of neuronal apoptosis. The robust A-FABP immunoreactivity in the hippocampal neurons of the LPS + A-FABP mice coincided with significantly increased TUNEL staining, supporting a direct neurotoxic role of blood-derived A-FABP. This finding extends our in vitro results showing that while neither A-FABP nor LPS alone strongly induces apoptosis in HT22 neurons, their combination synergistically promotes late-stage apoptotic pathways. The in vivo data confirmed that this synergy occurs through a sequential mechanism: LPS first disrupts BBB integrity and primes neuronal vulnerability, then circulating A-FABP enters the CNS and amplifies apoptotic signaling in susceptible neurons. The behavioral and histological outcomes further strengthen this interpretation. The exacerbated sepsis scores in the LPS + A-FABP group compared with those in the LPS + Saline control group indicate that circulating A-FABP, rather than simply serving as a passive biomarker, actively worsens clinical outcomes during endotoxemia. The selective vulnerability of hippocampal neurons to this combined insult may explain the cognitive impairments frequently observed in sepsis survivors, suggesting that A-FABP-mediated neurotoxicity could contribute to long-term neurological sequelae. Our findings position A-FABP as a crucial mediator crossing traditional boundaries between peripheral inflammation and central neurodegeneration. The demonstration that blood-derived A-FABP can infiltrate the brain during systemic inflammation and cooperate with LPS to drive neuronal apoptosis reveals a previously underappreciated pathway in SAE. This work suggests that therapeutic strategies targeting either A-FABP neutralization or blockade of its neuronal uptake could mitigate neuroinflammatory damage during systemic infections. Several important questions remain for future investigations. The molecular mechanisms enabling neuronal A-FABP uptake require clarification, as do the specific intracellular pathways through which A-FABP potentiates LPS toxicity. Longitudinal studies examining how early A-FABP neutralization affects long-term cognitive outcomes could strengthen the clinical relevance of these findings. Nevertheless, the current results provide compelling evidence that circulating A-FABP serves as both a biomarker and an active participant in the neuroinflammatory cascade during systemic infection. Declarations Acknowledgments We are grateful for the technical support from the Animal Facilities of Shenzhen Institutes of Advanced Technology (SIAT), the Chinese Academy of Sciences (CAS) and for the support from ANSO Scholarship for Young Talents. Author contributions Muhammad Mustapha Ibrahim and Chunyan Li, performed the research and wrote the manuscript. Linhui Qiu bred and prepared the animals. Muhammad Mustapha Ibrahim and Cheng Fang analyzed the experimental data. Cheng Fang, Junlei Chang, and Shilun Yang conceived the study and supervised the laboratory experiments. All the authors reviewed and approved the manuscript. Funding This work was supported by the National Natural Science Foundation of China (31900704 to C.F., 32170985, 81771293 to J.C. and 82273923 to Y.M.), the National Key Research and Development Program of China (2023YFE0202200 and 2021YFA0910000 to J.C.), the Guangdong Province Basic and Applied Basic Research Grant (2023A1515010467 to C.F., 2024A1515013262 to S.Y., 2021Β1515120089 to J.C.), the Shenzhen Science and Technology Program (JCYJ20230807140721043 to C.F., JCYJ20220531100203008 to Y.M.), the Shenzhen Medical Research Fund (D2403002 to J.C.), the International Collaboration Project of Chinese Academy of Sciences (172644KYSB20200045 to J.C.), Joint Laboratory between Guangdong and Hong Kong on Metabolic Diseases (2025B1212150002 to J.C.), the CAS-Croucher Funding Scheme for Joint Laboratories, and the Guangdong Innovation Platform of Translational Research for Cerebrovascular Diseases. The funders were not involved in the study design, data collection, data analysis, manuscript preparation or publication decisions. Data availability No datasets were generated or analyzed during the current study. Ethics approval and consent to participate All animal work was approved by the Institutional Animal Care and Use Committee of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences. Consent for publication All the authors agreed on the content of this manuscript before its submission. Conflict of interest The authors declare no conflicts of interest. References Huang X, Wei P, Fang C, Yu M, Yang S, Qiu L, et al. Compromised endothelial Wnt / β-catenin signaling mediates the blood-brain barrier disruption and leads to neuroinflammation in endotoxemia. 2024; Pan S, Lv Z, Wang R, Shu H, Yuan S, Yu Y. Review Article Sepsis-Induced Brain Dysfunction : Pathogenesis , Diagnosis , and Treatment. 2022;2022. Wang R, Bi W, Huang S, Han Q, Deng J, Wang Z, et al. 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Supplementary Files SupplementaryFile.docx SupplementaryFile.pdf Cite Share Download PDF Status: Published Journal Publication published 23 Jan, 2026 Read the published version in Cell Communication and Signaling → Version 1 posted Editorial decision: Revision requested 12 Oct, 2025 Reviews received at journal 09 Sep, 2025 Reviewers agreed at journal 26 Aug, 2025 Reviewers agreed at journal 26 Aug, 2025 Reviewers invited by journal 24 Aug, 2025 Editor assigned by journal 21 Aug, 2025 Submission checks completed at journal 21 Aug, 2025 First submitted to journal 14 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7369791","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":507612237,"identity":"740bcb39-f4cf-40a6-adbf-1af1c017200c","order_by":0,"name":"Muhammad Mustapha Ibrahim","email":"","orcid":"","institution":"Shenzhen Institutes of Advanced Technology, Chinese Academy of Science","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Mustapha","lastName":"Ibrahim","suffix":""},{"id":507612238,"identity":"4a1274de-ac92-494b-82de-f38dea638877","order_by":1,"name":"Chunyan Li","email":"","orcid":"","institution":"Shenzhen Institutes of Advanced Technology, Chinese Academy of Science","correspondingAuthor":false,"prefix":"","firstName":"Chunyan","middleName":"","lastName":"Li","suffix":""},{"id":507612239,"identity":"fe983acc-21a2-4785-9bbb-f7f53b2f1455","order_by":2,"name":"Linhui Qiu","email":"","orcid":"","institution":"Shenzhen Institutes of Advanced Technology, Chinese Academy of Science","correspondingAuthor":false,"prefix":"","firstName":"Linhui","middleName":"","lastName":"Qiu","suffix":""},{"id":507612240,"identity":"999a2289-9cf0-491f-a3bd-6e6d59f955c3","order_by":3,"name":"Shilun Yang","email":"","orcid":"","institution":"Shenzhen Institutes of Advanced Technology, Chinese Academy of Science","correspondingAuthor":false,"prefix":"","firstName":"Shilun","middleName":"","lastName":"Yang","suffix":""},{"id":507612241,"identity":"0a0e0ebb-288a-4bae-8a19-53445f43d5fa","order_by":4,"name":"Junlei Chang","email":"","orcid":"","institution":"Shenzhen Institutes of Advanced Technology, Chinese Academy of Science","correspondingAuthor":false,"prefix":"","firstName":"Junlei","middleName":"","lastName":"Chang","suffix":""},{"id":507612242,"identity":"4e487f6e-3906-4a3d-bf4b-9265034a901d","order_by":5,"name":"Cheng Fang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYDACCTApB8TMBxgSSNBiDMRsCUAtBiRp4QEpJ0IL/+zmYw+/MBjIm/Ov+fzhQc0fBv72A4yfC/BZcudYurEMg4Hhzhlvt0kkHDNgkDiTwCw9A48WA4kcM2kJhj+MG26c3caQwAZ02A0GNmYevFryvwG1GNhvuHHm8YeEfwYM8oS15LBJfmAwSNxwvodBIrHNgMGAkBaJG2lm0gwGBskbbrCZSST2GfMYnklslsanhX9G8jPJHxUGthvOH3788cc3OTm544cPfsanBQSYwTEikQDmABUzNhDQAFTyA2zfAYIKR8EoGAWjYIQCABePRvZv7SdqAAAAAElFTkSuQmCC","orcid":"","institution":"Shenzhen Institutes of Advanced Technology, Chinese Academy of Science","correspondingAuthor":true,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Fang","suffix":""}],"badges":[],"createdAt":"2025-08-14 04:53:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7369791/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7369791/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12964-026-02680-y","type":"published","date":"2026-01-23T15:59:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90352281,"identity":"b10777e2-5c02-4012-9b7f-0f60d99132a9","added_by":"auto","created_at":"2025-09-01 18:16:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":249679,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe A-FABP mAb 6H2 reduces mortality and improves the murine sepsis scorein mice with LPS-inducedendotoxemia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Experimental design of LPS-induced endotoxemia (25 mg/kg) and treatment. (B) Improved 24 h survival with 6H2 (20% mortality) vs LPS+mIgG (70%). (C) 6H2 attenuated LPS-induced weight loss. (D) Partial prevention of hypothermia by 6H2. (E) Murine sepsis scores were lower(14.0±1.5) in theLPS+mIgG group than in the LPS+mIgG group (17.5±1.2).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7369791/v1/31d80f0dcff331241d414e0d.png"},{"id":90352285,"identity":"c6471aff-f503-4d6a-b35e-05f1923eb4d6","added_by":"auto","created_at":"2025-09-01 18:16:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":865341,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA-FABP mAb 6H2 treatment protects blood‒brain barrier integrity in endotoxemia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Quantitative Evans blue assay results showing that 6H2 significantly reduces LPS-induced BBB leakage (***p\u0026lt;0.001 vs LPS+mIgG). (B) Western blot analysis demonstrated that 6H2 decreases extravasated IgG levels in brain tissue (*p\u0026lt;0.05). (C-D) Immunofluorescence images and quantification revealed that 6H2 reduces IgG leakage in the CD31\u003csup\u003e+\u003c/sup\u003e vasculature of the hippocampal and cortical regions. Scale bars: 50 μm. The data are presented as the means ± SEMs; one-way ANOVA with Tukey's post hoc test.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7369791/v1/01f46339d9091a0e224d1ed9.png"},{"id":90352286,"identity":"3494a6c3-0da5-48a2-8576-e32a87541a4a","added_by":"auto","created_at":"2025-09-01 18:16:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":789838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe A-FABP mAb 6H2 attenuates neuroinflammation and glial activation in LPS-induced endotoxemia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Cortical mRNA levels of proinflammatory mediators (IL6, TNF-α, IL-1β, IL-17A, and CCL2) show that 6H2 significantly reduces LPS-induced cytokine/chemokine upregulation (p\u0026lt;0.05, p\u0026lt;0.01 vs LPS+mIgG). (B) Representative images of IBA1+ microglia: sham mice with a ramified morphology (resting state), LPS+mIgG mice with hypertrophic processes (activated state), and LPS+6H2 mice with an intermediate morphology. Scale bar: 50 μm. (C) Quantitative analysis of microglial complexity (endpoints/cell) confirmed the significant reduction in LPS-induced activation induced by 6H2 (p\u0026lt;0.01 vs sham; p\u0026lt;0.01 vs LPS+mIgG). (D) GFAP\u003csup\u003e+\u003c/sup\u003e astrocyte images reveal the following: sham group with fine processes, LPS+mIgG group with thickened branches (reactive astrogliosis), and LPS+6H2 group showing partial recovery. Scale bar: 50 μm. The data are presented as the means ± SEMs; one-way ANOVA with Tukey's post hoc test.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7369791/v1/968558dc137fc7c228c78f9f.png"},{"id":90352774,"identity":"0b667897-a1d4-487c-b928-8e8653d8fec3","added_by":"auto","created_at":"2025-09-01 18:24:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":545293,"visible":true,"origin":"","legend":"\u003cp\u003eThe A-FABP mAb 6H2 protects against LPS-induced neuronal apoptosis.\u003c/p\u003e\n\u003cp\u003e(A-B) Triple immunofluorescence staining of hippocampal sections revealed that sham-treated mice exhibited healthy NeuN\u003csup\u003e+\u003c/sup\u003e (neurons) with minimal TUNEL\u003csup\u003e+ \u003c/sup\u003eapoptosis and ramified IBA1\u003csup\u003e+\u003c/sup\u003e microglia, whereas LPS+mIgG-treated mice exhibited extensive TUNEL\u003csup\u003e+\u003c/sup\u003e neuronal death alongside amoeboid-shaped, activated microglia. The LPS+6H2 group demonstrated significant neuroprotection, with a reduction in the number of apoptotic neurons and an intermediate change in microglial morphology. Compared with the sham treatment, the LPS+mIgG treatment increased the number of TUNEL\u003csup\u003e+ \u003c/sup\u003eand NeuN\u003csup\u003e-\u003c/sup\u003e cells (p\u0026lt;0.001), with the 6H2 treatment resulting in a reduction in apoptosis (p\u0026lt;0.01). Microglial activation scores followed the pattern of LPS+mIgG \u0026gt; 6H2 \u0026gt; sham (all p\u0026lt;0.01). Scale bars: 50 μm (overviews), 20 μm (insets). The data are presented as the means ± SEMs (n=7 mice/group); one-way ANOVA with Tukey's post hoc test.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7369791/v1/0379d9566d9094297e44418b.png"},{"id":90352287,"identity":"0016a4d3-9723-4e18-8db6-9f0eb516db37","added_by":"auto","created_at":"2025-09-01 18:16:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":988655,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe A-FABP mAb 6H2 modulates A-FABP-mediated neuroinflammation through multiple mechanisms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlow cytometry analysis (A-B) revealed that 6H2 reduced LPS-induced CD45\u003csup\u003e+\u003c/sup\u003e leukocyte infiltration (p\u0026lt;0.001 vs LPS+mIgG). RT‒qPCR (C) revealed that LPS downregulated brain A-FABP mRNA (p\u0026lt;0.01 vs sham), with 6H2 causing further reduction (p\u0026lt;0.05). Immunofluorescence staining (D-E) demonstrated that LPS+mIgG induced robust A-FABP accumulation in IBA1 microglia (p\u0026lt;0.001), whereas 6H2 attenuated this accumulation (p\u0026lt;0.01), with representative images showing reduced A-FABP in the hippocampus. Scale bars: 100 μm. The data are presented as the mean ± SEM (n=5- mice7/group). (F-G) LPS+mIgG induced robust A-FABP accumulation in neurons (p\u0026lt;0.001), whereas 6H2 attenuated this accumulation (p\u0026lt;0.01), with representative images showing reduced A-FABP in the hippocampus. Scale bars: 50 μm. The data are presented as the means ± SEMs (n=5‒7 mice/group); one-way ANOVA with Tukey's post hoc test. These findings demonstrate the multiple actions of 6H2 in limiting leukocyte infiltration and microglial and neuronal A-FABP uptake during endotoxemia.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7369791/v1/e570be84bc1d67db6c24ef58.png"},{"id":90352776,"identity":"c53d3263-a06a-4364-9c1d-b43d2f393500","added_by":"auto","created_at":"2025-09-01 18:24:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":688842,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA-FABP modulates A-FABP expression and enhances LPS-induced cytotoxicity in HT22 neurons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) qPCR analysis of endogenous A-FABP mRNA in HT22 cells treated with or without 0.5 μg/ml recombinant A-FABP and LPS (0 or 1 μg/ml). (B) Western blot quantification of A-FABP protein levels normalized to those of β-actin. (C) Cell viability (OD450) was measured over time via a CCK-8 assay in cells treated with various concentrations of recombinant A-FABP. (D) Representative flow cytometry plots showing Annexin V‒PE and 7-AAD staining of HT22 cells under the indicated conditions. (E) Quantification of early apoptosis (annexin V+/7-AAD−) and late apoptosis (annexin V+/7-AAD+).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7369791/v1/9d180edf041e552ab786528c.png"},{"id":90352777,"identity":"e934d70c-551d-434c-b6b2-3070d477466c","added_by":"auto","created_at":"2025-09-01 18:24:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":630963,"visible":true,"origin":"","legend":"\u003cp\u003eA-FABP and LPS synergistically drive neural apoptosis in vivo\u003c/p\u003e\n\u003cp\u003e(A) Experimental design of LPS-induced endotoxemia (25 mg/kg) in A-FABP KO mice. (B) Murine sepsis scores. (C) Triple immunofluorescence staining of hippocampal sections showing that saline+saline/saline+A-FABP-treated mice exhibit healthy NeuN\u003csup\u003e+\u003c/sup\u003e (neurons) with minimal TUNEL\u003csup\u003e+ \u003c/sup\u003eapoptosis and no A-FABP expression, whereas LPS+saline/LPS+A-FABP-treated mice exhibit extensive TUNEL\u003csup\u003e+\u003c/sup\u003e neuronal death alongside amoeboid-shaped death. (D-E) Quantitative analysis revealed that saline+Saline/Saline+A-FABP and LPS+Saline/LPS+A-FABP caused increases in TUNEL\u003csup\u003e+ \u003c/sup\u003eand NeuN\u003csup\u003e-\u003c/sup\u003e cells compared with the control (p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7369791/v1/bf21081eb6eb9c3c9a39764e.png"},{"id":101151913,"identity":"6d02dab3-07bf-470f-8b90-02196eddc40c","added_by":"auto","created_at":"2026-01-26 16:07:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5855024,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7369791/v1/fffe032e-7f1b-453f-bd5b-d9f2d34bdfed.pdf"},{"id":90352773,"identity":"00769ea1-8879-4ae9-bd55-2587a03a16d8","added_by":"auto","created_at":"2025-09-01 18:24:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18694,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7369791/v1/7e97088fb9494cc340b9984e.docx"},{"id":90352283,"identity":"568b82a8-bab9-41db-adae-93e6cfa84fe8","added_by":"auto","created_at":"2025-09-01 18:16:04","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":82418,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7369791/v1/48b9dc7bd0adb1c62ff04293.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Circulating A-FABP Exacerbates LPS-Induced Neurotoxicity by Crossing the Disrupted Blood–Brain Barrier and Promoting Neuronal Apoptosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSevere systemic inflammation, often triggered by infections or traumatic events, can progress to sepsis, a life-threatening condition frequently associated with central nervous system (CNS) dysfunction, leading to sepsis-associated encephalopathy (SAE) (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). SAE is characterized by disruption of the blood‒brain barrier (BBB), neuroinflammation, neuronal dysfunction, and behavioral changes such as sickness or depression-like behaviors. Lipopolysaccharide (LPS), an endotoxin from gram-negative bacteria, plays a significant role in sepsis pathogenesis, as these infections are the most common cause of sepsis (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Sepsis-related mortality remains high due to complex mechanisms and a lack of effective therapies (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). A dynamic equilibrium of pro- and anti-inflammatory factors regulates inflammation. Dysregulation of these mediators during infection or injury can lead to organ dysfunction, including brain injury, contributing to high mortality rates if uncontrolled in sepsis models. BBB disruption is closely linked to neuroinflammation, which is characterized by microglial activation and increased levels of proinflammatory cytokines in brain tissue (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Proinflammatory cytokines such as TNFα, IL-1β, and IL-6, which are elevated in the bloodstream, act as immunological messengers that drive CNS inflammation (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Sepsis-associated encephalopathy affects 50\u0026ndash;70% of sepsis patients, contributing to long-term cognitive impairment and increased mortality (\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). While systemic inflammation disrupts the blood‒brain barrier (BBB), the specific mediators that cross the CNS to drive neurodegeneration remain poorly characterized.\u003c/p\u003e\u003cp\u003eAdipocyte fatty acid-binding protein (A-FABP), a lipid chaperone expressed in adipocytes and macrophages, regulates systemic metabolism and inflammation(\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). It is implicated in insulin resistance, metabolic syndrome, and cardiovascular diseases (\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). A-FABP also plays a critical role in sepsis pathogenesis. Studies have shown that A-FABP inhibition, either genetically or pharmacologically, mitigates septic acute kidney injury by disrupting the TLR4/c-Jun signaling pathway, which increases inflammation and cell death (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Bioinformatics analysis of sepsis patient data revealed that higher A-FABP levels correlate with increased mortality, suggesting its role as a mediator of sepsis severity and a potential biomarker for poor outcomes (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Additionally, A-FABP is implicated in sepsis-induced acute respiratory distress syndrome (ARDS), where gut microbiota-derived acetic acid influences neutrophil apoptosis via A-FABP, exacerbating lung injury (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). However, the involvement of A-FABP in sepsis-associated neuroinflammation and SAE and whether it represents a therapeutic target remain unclear.\u003c/p\u003e\u003cp\u003eRecent evidence suggests that A-FABP may bridge metabolic and inflammatory pathways in neurological disorders. In macrophages and microglia, A-FABP potentiates TLR4 signaling, the same pathway activated by LPS (\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Crucially, A-FABP levels are correlated with sepsis mortality (\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), but whether A-FABP actively contributes to CNS injury or merely reflects disease severity is unresolved. This knowledge gap is significant because A-FABP neutralization improves outcomes in metabolic diseases, raising the possibility of repurposing this approach for neuroprotection.\u003c/p\u003e\u003cp\u003eOur study addresses three fundamental questions: First, does circulating A-FABP infiltrate the brain during systemic inflammation? Second, what cellular populations internalize A-FABP? Third, does A-FABP interact with LPS to exacerbate neuronal injury? Using complementary in vivo (A-FABP KO mice) and in vitro (HT22 neurons) models, we identified a previously unrecognized \"two-hit\" mechanism: LPS primes BBB disruption and neuronal vulnerability, whereas blood-derived A-FABP executes neurotoxicity through synergistic apoptosis induction. These findings not only advance our understanding of SAE pathogenesis but also validate A-FABP neutralization as a mechanistically grounded therapeutic strategy.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals and treatments\u003c/h2\u003e\u003cp\u003eThe mice were kept in a clean, pathogen-free environment with a standard 12-hour light and dark cycle. They had free access to food and water throughout the study. All animal handling and experimental procedures complied with the NIH guidelines for ethical animal care (NIH Publication No. 86\u0026thinsp;\u0026minus;\u0026thinsp;23, revised 1985) and were approved by the Institutional Animal Care and Use Committee (IACUC) at the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences. We used male C57BL/6J mice, aged 8\u0026ndash;10 weeks, sourced from the Vital River Laboratory in Beijing, China. To induce endotoxemia, we injected LPS (\u003cem\u003eEscherichia coli\u003c/em\u003e O55:B5, Sigma, Cat. No. L2880) into the abdominal cavity at a dose of 25 mg/kg body weight. Thirty minutes after the LPS injection, the mice received an intravenous dose of either the mouse mAb 6H2 (3.6 mg/kg) or a control antibody (mouse IgG, Immuno-Diagnostics, Cat. No. 221116, RRID: AB_3073816) at the same concentration. Any mice that died before the scheduled endpoint were excluded from the analysis. The assignment of the mice to treatment groups was randomized to ensure unbiased results.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell culture and treatments\u003c/h3\u003e\n\u003cp\u003eThe murine hippocampal neuronal cell line HT22 (Merck, #SCC129) and the microglial cell line BV2 (Procell, #CL-0493) were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; Cytiva, #sh30243.01) supplemented with 10% fetal bovine serum (FBS; Gibco, #10270106) and 1% penicillin/streptomycin (Gibco, #15140122) at 37\u0026deg;C in a humidified 5% CO₂ incubator. For the experimental treatments, the cells were serum starved (HT22, 24 hours; BV2, 12 hours) and exposed to the indicated concentrations of lipopolysaccharide (LPS; Sigma, #L2880) or recombinant murine A-FABP (MCE, #HY-P75215). Treatments were administered for the indicated times in complete medium. The control groups received vehicle (PBS) only.\u003c/p\u003e\n\u003ch3\u003eEvaluation of blood‒brain barrier (BBB) permeability via Evans blue\u003c/h3\u003e\n\u003cp\u003eFour hours before sacrifice, anesthetized mice received an injection of Evans blue (EB) (2%, 4 mL/kg) via the tail vein. After anesthesia, the mice were perfused transcardially with 40 mL of ice-cold PBS to flush out any remaining dye in the bloodstream. The brain was then carefully removed and homogenized in 1 mL of 50% trichloroacetic acid. The homogenate was centrifuged at 12,000 rpm for 15 minutes, and the EB concentration in the supernatant was measured via a spectrophotometer (Thermo Fisher, MultiskanTM FC) at 620 nm. A standard curve was used to calculate the EB levels, which are expressed as ng per gram of brain tissue.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence Staining\u003c/h3\u003e\n\u003cp\u003eFor tissue processing, deeply anesthetized mice were transcardially perfused with cold PBS, followed by brain extraction. The harvested tissues were fixed in 4% PFA for 1 hour at room temperature and then cryoprotected by sequential dehydration in 15% and 30% sucrose solutions at 4\u0026deg;C. After being embedded in OCT compound (Sakura, Cat. #4583), the tissues were sectioned into 20 \u0026micro;m-thick slices, air-dried, and washed with PBS. For A-FABP staining, the sections were subjected to acetone fixation at -20\u0026deg;C, SDS-mediated antigen retrieval, and blocking with a solution containing 10% donkey serum, 1% BSA, 1% Triton X-100, and 0.05% NaN3. Primary and secondary antibodies (Tables S1-S2) were applied overnight at 4\u0026deg;C or for 1 hour at RT, respectively, followed by incubation with DAPI (Cell Signaling Technology, Cat. #8961S).\u003c/p\u003e\u003cp\u003eFluorescence imaging was performed via a ZEISS Axio Imager Z2 microscope with ApoTome.2. Quantitative analysis included assessment of BBB leakage (target protein density/CD31 area), microglial activation (Iba1\u003csup\u003e+\u003c/sup\u003e cells/area), and astrocyte reactivity (GFAP mean density), all of which were performed via ImageJ software. The hippocampus and cortex were specifically analyzed for these parameters to evaluate neurovascular and neuroinflammatory responses.\u003c/p\u003e\n\u003ch3\u003eRNA Extraction and RT‒qPCR Analysis\u003c/h3\u003e\n\u003cp\u003eThe mice were deeply anesthetized and transcardially perfused with ice-cold PBS. The brain tissue was carefully dissected on ice and homogenized in RNA lysis buffer. For cultured cells, the medium was aspirated, and the cells were rinsed once with PBS. Total RNA was isolated via the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme), followed by cDNA synthesis with HiScript III RT SuperMix for qPCR (+\u0026thinsp;gDNA wiper) (Vazyme) to ensure genomic DNA removal.\u003c/p\u003e\u003cp\u003eFor gene expression analysis, RT‒qPCR was conducted on a LightCycler 96 system (Roche) with the following thermal profile: initial denaturation at 95\u0026deg;C for 3 minutes, followed by 40 cycles of 95\u0026deg;C for 15 seconds and 60\u0026deg;C for 1 minute. We quantified the expression levels of inflammatory markers (IL6, IL-1β, Ccl2, IL-17a, A-FABP, and TNF-α) via the use of β-actin as a housekeeping gene. All primers (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) were designed with a 60\u0026deg;C annealing temperature and were verified via melt curve analysis. Relative gene expression was determined via normalization to β-actin via the ΔΔCt method.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot analysis\u003c/h2\u003e\u003cp\u003eProtein samples (10\u0026ndash;15 \u0026micro;g per lane) were resolved via 10% SDS‒PAGE and subsequently transferred to PVDF membranes. Following transfer, the membranes were blocked with 5% nonfat milk in TBST for 1 hour at room temperature to prevent nonspecific binding. Primary antibodies (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) were applied overnight at 4\u0026deg;C with gentle agitation to ensure optimal antigen‒antibody interactions.\u003c/p\u003e\u003cp\u003eAfter extensive washing (X5 for 5 minutes in TBST), the membranes were incubated with appropriate HRP-conjugated secondary antibodies (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) for 1 hour at room temperature. Following additional TBST washes, protein bands were visualized via a chemiluminescent HRP substrate (Millipore, Cat. #WBKLS0500) and imaged via the GelView 6000 M system (BioLight, GV6000). Quantitative analysis was performed via ImageJ software, with target protein expression levels normalized to those of β-actin or GAPDH as loading controls.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eFlow cytometric analysis of immune cells in the brain\u003c/h3\u003e\n\u003cp\u003eWe started by carefully chopping the mouse brain tissue into fine pieces via a sterilized razor blade. The minced tissue was then broken down using an enzyme cocktail containing Type IV collagenase (400 U/ml), dispase (1.2 U/ml), and DNase I (32 U/ml) - all from Worthington - dissolved in 6 mL of calcium/magnesium-supplemented PBS. This digestion process ran for 30 minutes at 37\u0026deg;C, with gentle mixing every 10 minutes to help break the tissue.\u003c/p\u003e\u003cp\u003eAfter digestion, we added 1 mL of FBS and 20 mL of chilled PBS to stop the reaction and then passed the mixture through 40 \u0026micro;m filters. The cells were subjected to several washing steps: first, they were spun at 400 \u0026times; g for 5 minutes at 4\u0026deg;C; then, they were resuspended in 20% BSA solution for myelin removal at 1,000 \u0026times; g (25 minutes, 4\u0026deg;C). We washed the cells twice in 0.3% FBS/PBS solution with centrifugation at 400\u0026times; g each time.\u003c/p\u003e\u003cp\u003eFor surface marker analysis, the cells were first incubated with a mouse BD Fc block in 0.3% FBS/PBS for 10 minutes on ice to prevent nonspecific antibody binding. After being washed with PBS, the cells were stained with anti-CD11b-APC (BioLegend, cat. #101211) and PE-conjugated anti-CD45 (BD Biosciences, cat. #516087) for 45 minutes at 4\u0026deg;C in the dark. The stained cells were washed and resuspended in PBS for acquisition. Flow cytometric analysis was performed via a Sony MA900 flow cytometer equipped with a 100 \u0026micro;m nozzle, and CD45⁺/CD11b⁺ populations were identified for downstream analysis.\u003c/p\u003e\n\u003ch3\u003eCell Apoptosis Assay\u003c/h3\u003e\n\u003cp\u003eThe cells were digested with 0.25% trypsin (Gibco, #15050065) for 1 min at 37\u0026deg;C. The suspension was subsequently centrifuged at 300 \u0026times; g for 5 minutes at 4\u0026deg;C. The cells were stained with the Annexin V-PE/7-AAD Apoptosis Detection Kit (Yeasen, Cat. No. 40310ES50) following the manufacturer\u0026rsquo;s protocol. Briefly, after treatment, the cells were washed twice with cold PBS and resuspended in Annexin V binding buffer. Annexin V-PE and 7-AAD were then added to the cell suspension and incubated for 15 minutes at room temperature in the dark. The samples were analyzed via flow cytometry (Beckman, CytoFLEX S) within one hour of staining. Viable, early apoptotic, and late apoptotic cells were distinguished on the basis of their staining profiles: Annexin V⁻/7-AAD⁻ (viable), Annexin V⁺/7-AAD⁻ (early apoptotic), and Annexin V⁺/7-AAD⁺ (late apoptotic). All flow cytometric data were analyzed via FlowJo software.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMurine Sepsis Score Development\u003c/h2\u003e\u003cp\u003eIn our LPS-induced sepsis experiments, mice were evaluated via a refined behavioral assessment to monitor disease progression and treatment effects. Following baseline observations, the animals were scored at regular intervals via a murine sepsis score (MSS) adapted for the LPS model. The MSS comprises six parameters, spontaneous movement, stimulus response, posture, respiratory rate, breathing pattern, and piloerection, each graded from 0 (normal) to 4 (severely impaired), yielding a total score indicative of illness severity.\u003c/p\u003e\u003cp\u003eWe conducted careful observation, ensuring high interrater reliability. Data were recorded on standardized sheets alongside rectal temperature and body weight measurements. The MSS provides a quantitative, reproducible framework for tracking sepsis severity, enabling comparisons across the sham, IgG-treated, and 6H2-treated groups. Validation against physiological markers, including hypothermia and weight loss, confirmed the system\u0026rsquo;s sensitivity and alignment with established models (\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eCell viability assay (CCK-8)\u003c/h2\u003e\u003cp\u003eHT22 cells were seeded into 96-well plates at a density of 5,000 cells/well in 100 \u0026micro;L of complete DMEM and incubated for 24 hours at 37\u0026deg;C in a humidified 5% CO₂ atmosphere to allow cell attachment. Following attachment, the cells were treated with lipopolysaccharide (LPS), recombinant A-FABP, or vehicle controls in complete medium for 6, 24, or 48 hours. After treatment, 10 \u0026micro;L of Cell Counting Kit-8 (Yeasen, Cat. No. 40203ES80) solution was added to each well and incubated for an additional 1 hour at 37\u0026deg;C. The absorbance was then measured at 450 nm via a microplate reader (Thermo Scientific, Multiskan GO).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative analysis\u003c/h2\u003e\u003cp\u003eAll quantitative data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical analyses were performed via GraphPad Prism version 10 (GraphPad Software, CA, USA). For comparisons between two groups, unpaired two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e tests were applied, whereas one-way analysis of variance (ANOVA) was used for comparisons involving more than two groups. A \u003cem\u003ep\u003c/em\u003e value less than 0.05 was considered statistically significant.\u003c/p\u003e\u003cp\u003eFor the analysis of microglial morphology, an ImageJ-based skeletonization protocol was employed. Briefly, original fluorescence micrographs were preprocessed via fast Fourier transform (FFT) bandpass filtering and unsharp masking to increase image quality. The processed images were then binarized and skeletonized, followed by quantification of microglial branching structures via the Analyze Skeleton plugin. Cropped overlays were used to ensure the anatomical accuracy of segmentation. Endpoint counts derived from skeletons were normalized to the number of microglial somata per field to account for cellular density.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003e6H2 treatment lowers the Murine Sepsis Score (MSS) and reduces mortality in LPS-Induced endotoxemia\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn this study, 8-week-old male C57BL/6J wild-type mice were used to establish an LPS-induced (25 mg/kg body weight) sepsis model to evaluate the therapeutic effects of the monoclonal antibody 6H2 (3.6 mg/kg). The experimental groups included a saline control group (sham), an LPS\u0026thinsp;+\u0026thinsp;mIgG control group (LPS\u0026thinsp;+\u0026thinsp;mIgG), and an LPS\u0026thinsp;+\u0026thinsp;6H2 treatment group (LPS\u0026thinsp;+\u0026thinsp;6H2). Thirty minutes after intraperitoneal LPS injection, the mice received either mIgG or 6H2 via tail vein injection. Survival rates, body weight changes, body temperature fluctuations, and sepsis scores were assessed 24 hours later (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The results revealed survival rates of 100% in the control group, 70% in the mIgG group, and 80% in the 6H2 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). All septic mice exhibited significant weight loss, hypothermia, and motor dysfunction (manifested as reduced spontaneous movement, jerky motions, abnormal gait, piloerection, partially closed eyelids, and ocular discharge) within 24 hours. Compared with the mIgG controls, 6H2 treatment failed to reverse the LPS-induced metabolic disturbances reflected by persistent weight loss and hypothermia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC \u003cb\u003eand D\u003c/b\u003e). Sepsis scoring confirmed minimal septic manifestations in the sham group, severe sepsis in the LPS\u0026thinsp;+\u0026thinsp;mIgG group, and significant symptom alleviation in the LPS\u0026thinsp;+\u0026thinsp;6H2 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Notably, compared with septic control mice, 6H2-treated mice presented reduced neurological manifestations, including less piloerection, improved eye opening, and decreased ocular discharge, suggesting specific neuroprotective effects independent of systemic metabolic recovery. These findings demonstrate that 6H2 effectively lowers the murine sepsis score and mortality and modulates LPS-induced immune dysregulation, as reflected by lower sepsis scores, highlighting its therapeutic potential in inflammatory diseases.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e6H2 Treatment alleviates LPS-induced blood‒brain barrier disruption\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLPS-induced sepsis causes significant BBB leakage, neuroinflammation, and systemic inflammation, leading to severe neurological damage(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). To evaluate the ability of 6H2 to protect against LPS-induced blood‒brain barrier (BBB) disruption, we employed complementary approaches. Evans blue extravasation was used to quantify global leakage, whereas endogenous IgG infiltration was used to assess molecular-level compromise. LPS-challenged mice presented increased Evans blue penetration into the brain parenchyma, and 6H2 treatment significantly attenuated Evans blue leakage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Western blot analysis further supported this finding, as the LPS\u0026thinsp;+\u0026thinsp;mIgG group presented elevated IgG levels in brain tissue, whereas the LPS\u0026thinsp;+\u0026thinsp;6H2 group presented reduced mIgG levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Additionally, co-immunofluorescence staining of plasma IgG and CD31 (endothelial markers) in the hippocampus and cortex revealed intense IgG staining in the LPS\u0026thinsp;+\u0026thinsp;mIgG group, whereas the LPS\u0026thinsp;+\u0026thinsp;6H2 group presented significantly reduced IgG staining, further supporting the protective effects of 6H2 on BBB integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These complementary approaches consistently demonstrate that 6H2 preserves BBB integrity during sepsis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e6H2 treatment alleviates LPS-induced neuroinflammation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLPS-induced sepsis not only causes substantial blood‒brain barrier (BBB) disruption but also elicits both vascular and neuroinflammatory responses. LPS causes neuroinflammation, as evidenced by the upregulation of proinflammatory cytokines and microglial and astrocyte activation (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Next, we investigated whether 6H2 treatment could mitigate neuroinflammation. RT‒qPCR analysis revealed that LPS significantly increased the expression of proinflammatory cytokines (Il-6, TNF-α, Il-1β, Il-17a, and Ccl2) in the brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), but 6H2 treatment notably reduced their expression, resulting in cytokine levels closer to those observed in the sham group. Immunofluorescence staining of Iba1, a microglial marker, in the hippocampus revealed that LPS-treated mice exhibited significant microglial activation, which was notably reduced by 6H2 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Microglial morphology varied among the groups: a resting phenotype in the Sham group (15 Endpoints/Cell), an activated phenotype in the IgG-treated group (~\u0026thinsp;10 Endpoints/Cell, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 vs. Sham), and a recuperative phenotype in the 6H2-treated group (~\u0026thinsp;20 Endpoints/Cell, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 vs. IgG). Quantitative analysis confirmed that 6H2 effectively diminished LPS-induced microglial activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Similarly, LPS-induced astrocyte activation is characterized by morphological changes, including increased cell size, complexity, and process density. These changes are indicative of a reactive state in astrocytes (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). GFAP staining, an astrocyte marker, revealed that 6H2 alleviated LPS-induced astrocytic activation, as characterized by increased size, complexity, and process density in the hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Collectively, these findings suggest that 6H2 mitigates LPS-induced neuroinflammation by reducing proinflammatory cytokine expression and suppressing the activation of both microglia and astrocytes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e6H2 treatment improves neuronal survival in LPS-induced sepsis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven that 6H2 preserves BBB integrity and attenuates neuroinflammatory responses, we next investigated its neuroprotective potential upon LPS administration. To assess neuronal apoptosis, we conducted co-immunofluorescence staining for NeuN (a neuronal marker) and subsequently performed a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay in hippocampal tissue \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Immunohistochemical analysis revealed significant differences in neuronal survival and apoptosis across the experimental groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Compared with the sham group, which presented abundant NeuN\u003csup\u003e+\u003c/sup\u003e cells and minimal TUNEL\u003csup\u003e+\u003c/sup\u003e staining, indicating healthy neuronal populations with negligible apoptosis, the LPS\u0026thinsp;+\u0026thinsp;mIgG group presented severe neuronal damage characterized by a marked reduction in NeuN\u003csup\u003e+\u003c/sup\u003e cells and a substantial increase in TUNEL\u003csup\u003e+\u003c/sup\u003e cells. This pattern demonstrated extensive LPS-induced neuronal apoptosis. Importantly, the LPS\u0026thinsp;+\u0026thinsp;6H2 treatment group showed significant neuroprotection, with preserved NeuN\u003csup\u003e+\u003c/sup\u003e cell counts and dramatically fewer TUNEL\u003csup\u003e+\u003c/sup\u003e cells than the LPS\u0026thinsp;+\u0026thinsp;mIgG group. Further analysis of NeuN\u003csup\u003e+\u003c/sup\u003e/TUNEL\u003csup\u003e+\u003c/sup\u003e double-labeled cells confirmed these findings, whereas the sham group presented virtually no apoptotic neurons, the LPS\u0026thinsp;+\u0026thinsp;mIgG group presented numerous double-positive cells, and this effect was significantly attenuated by 6H2 treatment. These results clearly demonstrate that 6H2 confers robust neuroprotection in the LPS-induced sepsis model by maintaining neuronal viability and reducing apoptosis, suggesting its potential therapeutic value for sepsis-associated neurological damage.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e6H2 modulates leukocyte infiltration and attenuates A-FABP accumulation in microglia and neurons in LPS-induced sepsis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe next investigated whether 6H2 treatment influences A-FABP levels in the brain upon LPS stimulation. First, we conducted FACS to evaluate whether 6H2 affects the infiltration of immune cells in the blood into the brain parenchyma. The sham group exhibited negligible leukocyte (lymphocyte and monocyte) infiltration due to the absence of inflammatory stimulation; the LPS\u0026thinsp;+\u0026thinsp;mIgG group exhibited significant leukocyte recruitment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The LPS\u0026thinsp;+\u0026thinsp;6H2 group displayed reduced leukocyte infiltration, suggesting that the drug 6H2 mitigated LPS-driven inflammation and BBB disruption.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we assessed A-FABP mRNA expression and protein localization in the brain via RT‒qPCR and co-immunofluorescence staining, respectively, followed by quantitative analysis. First, we measured A-FABP mRNA expression levels in brain tissue via RT-qPCR. Quantitative RT‒PCR analysis revealed that the sham group presented the highest expression levels of A-FABP mRNA, reflecting baseline expression under normal physiological conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In contrast, the LPS\u0026thinsp;+\u0026thinsp;mIgG group presented a marked decrease in A-FABP mRNA expression compared with the sham group, whereas the LPS\u0026thinsp;+\u0026thinsp;6H2 group presented a further reduction in A-FABP mRNA levels. These findings suggest that LPS stimulation downregulates A-FABP transcription in the brain and that 6H2 treatment enhances this effect, highlighting its efficacy in modulating A-FABP levels in response to LPS-induced inflammation.\u003c/p\u003e\u003cp\u003eTo further investigate A-FABP protein localization under neuroinflammatory conditions, we performed co-immunofluorescence staining for A-FABP with either IBA1 or NeuN in hippocampal brain tissue. In the LPS\u0026thinsp;+\u0026thinsp;mIgG group, we observed pronounced A-FABP immunoreactivity in both NeuN\u003csup\u003e+\u003c/sup\u003e neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) and IBA1\u003csup\u003e+\u003c/sup\u003e microglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), indicating significant A-FABP protein accumulation despite the observed downregulation of A-FABP mRNA. This discrepancy suggests that the accumulated A-FABP protein may originate from the peripheral circulation, crossing the compromised BBB under inflammatory conditions and subsequently interacting with neuronal and microglia. In contrast, the LPS\u0026thinsp;+\u0026thinsp;6H2 group presented attenuated A-FABP staining intensity in both cell types, suggesting that 6H2 treatment reduces A-FABP accumulation. Minimal A-FABP immunoreactivity was detected in the sham group, which served as a baseline control \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE\u003cb\u003e).\u003c/b\u003e To quantify A-FABP accumulation in microglia, we analyzed the integrated density (IntDen) of A-FABP within IBA1\u003csup\u003e+\u003c/sup\u003e cells, which was normalized to the total cell count (IBA1\u003csup\u003e+\u003c/sup\u003e A-FABP\u003csup\u003e+\u003c/sup\u003e IntDen/Total Cell). The LPS\u0026thinsp;+\u0026thinsp;mIgG group presented the highest A-FABP\u003csup\u003e+\u003c/sup\u003eIBA1\u003csup\u003e+\u003c/sup\u003e intensity, confirming substantial A-FABP deposition in microglia. Conversely, the LPS\u0026thinsp;+\u0026thinsp;6H2 group presented a significantly lower A-FABP\u003csup\u003e+\u003c/sup\u003eIBA1\u003csup\u003e+\u003c/sup\u003e intensity, further confirming that 6H2 treatment mitigated A-FABP accumulation in microglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Similarly, co-staining for A-FABP and NeuN revealed robust A-FABP immunoreactivity in the neurons of the LPS\u0026thinsp;+\u0026thinsp;mIgG-treated mice, whereas those in the LPS\u0026thinsp;+\u0026thinsp;6H2 group presented a reduced neuronal A-FABP signal \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG\u003cb\u003e).\u003c/b\u003e Quantification of A-FABP\u003csup\u003e+\u003c/sup\u003eNeuN\u003csup\u003e+\u003c/sup\u003e intensity further supported these observations, demonstrating that 6H2 treatment significantly decreased A-FABP accumulation in neurons \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThese findings collectively demonstrate that while LPS-induced neuroinflammation suppresses A-FABP mRNA expression, it promotes A-FABP protein accumulation in both microglia and neurons, likely through the infiltration of circulating A-FABP across the disrupted BBB. Notably, 6H2 treatment not only further reduced A-FABP transcription but also attenuated A-FABP protein deposition in these cell populations. This dual regulatory effect suggests that the modulation of A-FABP levels, both at the transcriptional and protein accumulation levels, contributes to the anti-inflammatory and neuroprotective properties of 6H2.\u003c/p\u003e\u003cp\u003e\u003cb\u003eA-FABP synergizes with LPS to drive late-stage neuronal apoptosis\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur study revealed an intriguing phenomenon: while LPS stimulation dramatically downregulated \u003cem\u003eA-FABP\u003c/em\u003e mRNA expression in the brain, A-FABP protein accumulated prominently in neurons. This discrepancy suggests that neuronal A-FABP accumulation may not stem from endogenous production but rather from the uptake of circulating A-FABP, which crosses the compromised BBB during systemic inflammation. To investigate this hypothesis, we examined the direct effects of A-FABP on neuronal cells via the HT22 hippocampal neuronal line and evaluated its effects on A-FABP expression, protein accumulation, cell viability, and apoptosis in the presence or absence of LPS.\u003c/p\u003e\u003cp\u003eOur quantitative PCR analysis revealed that recombinant A-FABP significantly suppressed A-FABP mRNA expression in HT22 cells at baseline (0 \u0026micro;g/ml LPS). Upon stimulation with LPS (1 \u0026micro;g/ml), the endogenous A-FABP mRNA was already downregulated, and exogenous A-FABP further decreased its transcriptional level \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e These results suggest that extracellular A-FABP contributes to a feedback mechanism that reduces its gene expression, potentially modulating cellular responses to stress. Western blotting analysis revealed that HT22 cells did not express A-FABP, at least at low levels, while A-FABP administration led to a robust A-FABP band, independent of the presence of LPS. Quantification confirmed that A-FABP protein levels decreased with increasing LPS concentration and that LPS itself could not induce A-FABP expression in neurons \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. These results revealed that HT22 hippocampal neurons exhibit negligible endogenous A-FABP expression, yet they rapidly accumulate A-FABP protein when exposed to exogenous A-FABP, regardless of LPS stimulation. This finding strongly supports our hypothesis that neuronal A-FABP accumulation in vivo results primarily from the uptake of circulating A-FABP rather than its de novo synthesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine whether internalized A-FABP directly affects neuronal viability, we treated HT22 cells with increasing concentrations of recombinant A-FABP (10\u0026ndash;1000 ng/ml) in the presence or absence of LPS. In contrast, low concentrations of A-FABP alone did not improve cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Instead, higher doses (\u0026ge;\u0026thinsp;100 ng/ml) promoted HT22 survival in a dose-dependent manner, suggesting a neuroprotective effect of exogenous A-FABP under basal conditions. Strikingly, when A-FABP was combined with LPS (0.5 or 1 \u0026micro;g/ml), A-FABP synergistically amplified LPS-induced cytotoxicity. This effect was most pronounced at higher A-FABP concentrations (500\u0026ndash;1000 ng/ml), where cell viability decreased significantly compared with that of the cells treated with LPS alone. Time-course experiments further revealed that prolonged exposure (48 hours) to A-FABP and LPS exacerbated neuronal death, highlighting a time- and dose-dependent neurotoxic synergy between these two factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eTo investigate the synergistic neurotoxic effects of A-FABP and LPS, we quantified the degree of apoptosis in HT22 neurons via Annexin V-PE/7-AAD staining and flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). While neither A-FABP (0.5 \u0026micro;g/ml) nor LPS (1 \u0026micro;g/ml) alone significantly increased apoptosis compared with that of the controls, their combination resulted in a marked increase in late apoptotic cells (Annexin V+/7-AAD+; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs LPS alone). Notably, early apoptosis (Annexin V+/7-AAD-) remained unaffected across all treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). The selective increase in late apoptosis suggests that A-FABP exacerbates terminal cell death pathways after LPS primes for neuronal damage.\u003c/p\u003e\u003cp\u003eCollectively, our findings demonstrate a coordinated mechanism of neuronal damage involving A-FABP and LPS. This synergistic interaction provides a mechanistic explanation for the exacerbated neuronal injury observed under inflammatory conditions, where both systemic A-FABP elevation and LPS exposure occur concurrently.\u003c/p\u003e\u003cp\u003e\u003cb\u003eA-FABP potentiates LPS-induced neuronal apoptosis\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo validate our in vitro observations of the synergistic neurotoxic effects of A-FABP and LPS, we performed complementary in vivo experiments in A-FABP knockout (KO) mice. Eight-week-old male A-FABP KO mice were intraperitoneally injected with either LPS (25 mg/kg) to induce endotoxemia or saline as a control. Thirty minutes later, the mice received an intravenous injection of either recombinant A-FABP (3 \u0026micro;g) or saline through the tail vein (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). This carefully controlled experimental design enabled us to specifically investigate the impact of exogenously administered A-FABP in the absence of endogenous A-FABP expression, thereby isolating the effects of circulating A-FABP on LPS-induced neurotoxicity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAssessment of sepsis severity via the murine sepsis score revealed distinct patterns among the treatment groups. Both the saline control groups (Saline\u0026thinsp;+\u0026thinsp;Saline and Saline\u0026thinsp;+\u0026thinsp;A-FABP) maintained low sepsis scores, indicating minimal systemic illness. In contrast, the mice that received LPS (LPS\u0026thinsp;+\u0026thinsp;Saline and LPS\u0026thinsp;+\u0026thinsp;A-FABP groups) presented significantly elevated sepsis scores, confirming the successful induction of severe systemic inflammation regardless of A-FABP status. These findings demonstrated that the knockout of A-FABP did not prevent the development of sepsis symptoms following LPS challenge. Notably, while both LPS-treated groups (LPS\u0026thinsp;+\u0026thinsp;Saline and LPS\u0026thinsp;+\u0026thinsp;A-FABP) presented marked illness, the LPS\u0026thinsp;+\u0026thinsp;A-FABP group presented even higher sepsis scores than the LPS\u0026thinsp;+\u0026thinsp;Saline group did (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). This dose-dependent worsening of clinical symptoms with A-FABP administration suggests that circulating A-FABP not only fails to protect against LPS-induced sepsis but actually exacerbates the systemic inflammatory response and associated behavioral manifestations. These findings provide compelling in vivo evidence that A-FABP plays an active role in amplifying the pathological consequences of endotoxemia rather than being a passive biomarker of disease severity.\u003c/p\u003e\u003cp\u003eImmunofluorescence analysis of hippocampal tissue provided crucial insights into A-FABP distribution and neuronal apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). In saline-treated A-FABP KO mice (Saline\u0026thinsp;+\u0026thinsp;Saline and Saline\u0026thinsp;+\u0026thinsp;A-FABP groups), we observed negligible A-FABP immunoreactivity, demonstrating that circulating A-FABP cannot cross the intact blood‒brain barrier. Interestingly, the LPS\u0026thinsp;+\u0026thinsp;Saline group presented a detectable A-FABP signal, likely reflecting either residual expression from incomplete knockout or minimal basal expression in certain cell types under inflammatory conditions. Most strikingly, the LPS\u0026thinsp;+\u0026thinsp;A-FABP group demonstrated intense A-FABP immunostaining that prominently localized to hippocampal neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). This dramatic increase in A-FABP signaling specifically in LPS-treated mice receiving exogenous A-FABP provides direct visual evidence that (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) LPS-induced systemic inflammation compromises blood‒brain barrier integrity and that (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) circulating A-FABP readily crosses the disrupted barrier to accumulate within vulnerable neuronal populations.\u003c/p\u003e\u003cp\u003eNeuronal viability assessments yielded striking results. The saline control groups presented abundant NeuN-positive neurons with minimal TUNEL staining, reflecting healthy neuronal populations with negligible apoptosis. LPS administration alone caused significant neuronal damage, as evidenced by an increase in TUNEL-positive neuronal cells. Most importantly, the combination of LPS and A-FABP resulted in substantially greater neuronal apoptosis than did LPS alone, with a marked increase in NeuN+/TUNEL\u0026thinsp;+\u0026thinsp;double-labeled cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). This in vivo observation directly corroborated our in vitro findings of synergistic neurotoxicity.\u003c/p\u003e\u003cp\u003eThese results collectively demonstrate that circulating A-FABP gains access to the CNS during systemic inflammation when the blood‒brain barrier becomes compromised. Once in the brain parenchyma, A-FABP is internalized by neurons, where it potentiates LPS-induced apoptotic pathways. Our findings position A-FABP as both a key mediator of neuroinflammatory injury and a promising therapeutic target for preventing sepsis-associated neurodegeneration.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study demonstrated that neutralizing circulating A-FABP with the monoclonal antibody 6H2 effectively mitigated LPS-induced endotoxemia by preserving blood‒brain barrier (BBB) integrity, reducing neuroinflammation, and attenuating neuronal apoptosis. These findings highlight the pivotal role of A-FABP in sepsis-associated encephalopathy (SAE) and underscore its potential as a therapeutic target for neuroinflammatory conditions. The protective effects of 6H2 on BBB integrity were evident through reduced Evans blue leakage and decreased IgG extravasation in brain tissue. These results align with previous studies showing that systemic inflammation disrupts the BBB, allowing harmful substances to infiltrate the central nervous system (CNS)(\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). By neutralizing circulating A-FABP, 6H2 likely prevents its pathological accumulation in the brain, thereby preserving BBB function and limiting neuroinflammatory responses. Neuroinflammation, characterized by microglial and astrocyte activation, is a hallmark of SAE. Our data showed that 6H2 treatment significantly reduced the expression of proinflammatory cytokines (TNF-α, IL-1β, IL-6, IL-17a, and Ccl2) and attenuated glial activation. Notably, microglial morphology shifted from an activated, amoeboid state in LPS-treated mice to a more ramified, homeostatic phenotype in 6H2-treated animals. These findings suggest that A-FABP neutralization not only dampens inflammatory signaling but also promotes the restoration of glial homeostasis. The observed downregulation of A-FABP mRNA in LPS-treated brains, coupled with increased A-FABP protein accumulation in neurons and microglia, points to a dual mechanism: while LPS suppresses A-FABP transcription, peripheral A-FABP infiltrates the CNS due to BBB disruption. 6H2 mitigates this effect by neutralizing circulating A-FABP, reducing its deleterious effects on neural cells. The neuroprotective effects of 6H2 were further corroborated by reduced neuronal apoptosis, as evidenced by decreased TUNEL\u003csup\u003e+\u003c/sup\u003e staining and preserved NeuN\u003csup\u003e+\u003c/sup\u003e cell counts.\u003c/p\u003e\u003cp\u003eOur in vitro experiments revealed that A-FABP synergizes with LPS to exacerbate neuronal apoptosis, particularly in the late stages. The in vivo findings in A-FABP knockout mice further support this finding, as the absence of A-FABP attenuated LPS-induced neuronal damage, whereas exogenous A-FABP worsened outcomes. These integrated in vitro and in vivo findings reveal a novel mechanism by which circulating A-FABP exacerbates neuronal injury during systemic inflammation. Although neurons normally maintain minimal endogenous A-FABP expression, they actively take up circulating A-FABP when the blood-brain barrier becomes compromised during endotoxemia, leading to synergistic neurotoxicity with LPS. The immunofluorescence data from A-FABP KO mice provide compelling evidence for this pathological cascade. The complete absence of the A-FABP signal in saline-treated controls confirms both the effectiveness of our knockout model and the impermeability of the intact blood-brain barrier to circulating A-FABP. The faint A-FABP signal observed in LPS\u0026thinsp;+\u0026thinsp;Saline KO mice suggests either residual expression in certain cell types under inflammatory conditions or potential limitations in knockout efficiency. Most importantly, the dramatic A-FABP accumulation specifically in the hippocampal neurons of LPS\u0026thinsp;+\u0026thinsp;A-FABP-treated mice visually demonstrates how systemic inflammation enables peripheral A-FABP to access the CNS parenchyma. These spatial distribution patterns correlate precisely with our observations of neuronal apoptosis. The robust A-FABP immunoreactivity in the hippocampal neurons of the LPS\u0026thinsp;+\u0026thinsp;A-FABP mice coincided with significantly increased TUNEL staining, supporting a direct neurotoxic role of blood-derived A-FABP. This finding extends our in vitro results showing that while neither A-FABP nor LPS alone strongly induces apoptosis in HT22 neurons, their combination synergistically promotes late-stage apoptotic pathways. The in vivo data confirmed that this synergy occurs through a sequential mechanism: LPS first disrupts BBB integrity and primes neuronal vulnerability, then circulating A-FABP enters the CNS and amplifies apoptotic signaling in susceptible neurons. The behavioral and histological outcomes further strengthen this interpretation. The exacerbated sepsis scores in the LPS\u0026thinsp;+\u0026thinsp;A-FABP group compared with those in the LPS\u0026thinsp;+\u0026thinsp;Saline control group indicate that circulating A-FABP, rather than simply serving as a passive biomarker, actively worsens clinical outcomes during endotoxemia. The selective vulnerability of hippocampal neurons to this combined insult may explain the cognitive impairments frequently observed in sepsis survivors, suggesting that A-FABP-mediated neurotoxicity could contribute to long-term neurological sequelae.\u003c/p\u003e\u003cp\u003eOur findings position A-FABP as a crucial mediator crossing traditional boundaries between peripheral inflammation and central neurodegeneration. The demonstration that blood-derived A-FABP can infiltrate the brain during systemic inflammation and cooperate with LPS to drive neuronal apoptosis reveals a previously underappreciated pathway in SAE. This work suggests that therapeutic strategies targeting either A-FABP neutralization or blockade of its neuronal uptake could mitigate neuroinflammatory damage during systemic infections. Several important questions remain for future investigations. The molecular mechanisms enabling neuronal A-FABP uptake require clarification, as do the specific intracellular pathways through which A-FABP potentiates LPS toxicity. Longitudinal studies examining how early A-FABP neutralization affects long-term cognitive outcomes could strengthen the clinical relevance of these findings. Nevertheless, the current results provide compelling evidence that circulating A-FABP serves as both a biomarker and an active participant in the neuroinflammatory cascade during systemic infection.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful for the technical support from the Animal Facilities of Shenzhen Institutes of Advanced Technology (SIAT), the Chinese Academy of Sciences (CAS) and for the support from ANSO Scholarship for Young Talents.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003econtributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMuhammad Mustapha Ibrahim and Chunyan Li, \u0026nbsp;performed the research and wrote the manuscript. Linhui Qiu bred and prepared the animals. Muhammad Mustapha Ibrahim and Cheng Fang analyzed the experimental data. Cheng Fang, Junlei Chang, and Shilun Yang conceived the study and supervised the laboratory experiments. All the authors reviewed and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (31900704 to C.F., 32170985, 81771293 to J.C. and 82273923 to Y.M.), the National Key Research and Development Program of China\u0026nbsp;(2023YFE0202200 and 2021YFA0910000\u0026nbsp;to J.C.), the Guangdong Province Basic and Applied Basic Research Grant (2023A1515010467 to C.F., 2024A1515013262 to S.Y., 2021\u0026Beta;1515120089 to J.C.), the Shenzhen Science and Technology Program (JCYJ20230807140721043 to C.F., JCYJ20220531100203008 to Y.M.), the Shenzhen Medical Research Fund (D2403002 to J.C.), the International\u0026nbsp;Collaboration Project\u0026nbsp;of Chinese Academy of Sciences (172644KYSB20200045 to J.C.), Joint Laboratory between Guangdong and Hong Kong on Metabolic Diseases (2025B1212150002 to J.C.), the CAS-Croucher Funding Scheme for Joint Laboratories, and the Guangdong Innovation Platform of Translational Research for Cerebrovascular Diseases.\u0026nbsp;The funders were not involved in the study design, data collection, data analysis, manuscript preparation or publication decisions.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eavailability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analyzed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal work was approved by the Institutional Animal Care and Use\u0026nbsp;Committee\u0026nbsp;of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConsent\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003efor\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the\u0026nbsp;authors\u0026nbsp;agreed on the content of this manuscript before its submission.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHuang X, Wei P, Fang C, Yu M, Yang S, Qiu L, et al. 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Springer Nature; 2021. p. 2489\u0026ndash;501. \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":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"A-FABP, sepsis-associated encephalopathy, blood‒brain barrier, neuroinflammation, LPS, neuronal apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-7369791/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7369791/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSepsis-associated encephalopathy (SAE) is a critical complication of systemic inflammation with poorly understood mechanisms. This study identified adipocyte fatty acid-binding protein (A-FABP) as a key mediator linking peripheral inflammation to central nervous system damage. Using an LPS-induced endotoxemia model in wild-type and A-FABP knockout mice, we demonstrated that circulating A-FABP (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) crosses the compromised blood‒brain barrier (BBB), (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) accumulates in hippocampal neurons, and (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) synergizes with LPS to drive neuronal apoptosis. The monoclonal antibody 6H2, which neutralizes A-FABP, significantly reduced BBB leakage, attenuated neuroinflammation, and improved neuronal survival. In vitro studies confirmed that HT22 neurons internalize exogenous A-FABP, which amplifies LPS-induced late apoptosis without affecting early apoptotic pathways. These findings establish circulating A-FABP as both a biomarker and therapeutic target for SAE, revealing a novel periphery-to-CNS inflammatory cascade.\u003c/p\u003e","manuscriptTitle":"Circulating A-FABP Exacerbates LPS-Induced Neurotoxicity by Crossing the Disrupted Blood–Brain Barrier and Promoting Neuronal Apoptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-01 18:15:59","doi":"10.21203/rs.3.rs-7369791/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-12T16:46:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-09T22:09:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"230659835422883104109387088347408220668","date":"2025-08-26T19:16:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"168452300372078379964461660889757655757","date":"2025-08-26T09:34:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-24T18:47:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-21T13:59:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-21T13:58:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Communication and Signaling","date":"2025-08-14T04:43:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5f2270dc-d086-454c-9b04-35b9b2421738","owner":[],"postedDate":"September 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T16:04:10+00:00","versionOfRecord":{"articleIdentity":"rs-7369791","link":"https://doi.org/10.1186/s12964-026-02680-y","journal":{"identity":"cell-communication-and-signaling","isVorOnly":false,"title":"Cell Communication and Signaling"},"publishedOn":"2026-01-23 15:59:04","publishedOnDateReadable":"January 23rd, 2026"},"versionCreatedAt":"2025-09-01 18:15:59","video":"","vorDoi":"10.1186/s12964-026-02680-y","vorDoiUrl":"https://doi.org/10.1186/s12964-026-02680-y","workflowStages":[]},"version":"v1","identity":"rs-7369791","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7369791","identity":"rs-7369791","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}