Astrocyte-Dependent Neuroinflammation Triggers Hippocampal Neuronal Apoptosis through the TLR4/NF-κB/NLRP3 Axis in Hepatic Encephalopathy

Astrocyte-Dependent Neuroinflammation Triggers Hippocampal Neuronal Apoptosis through the TLR4/NF-κB/NLRP3 Axis in Hepatic Encephalopathy | 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 Article Astrocyte-Dependent Neuroinflammation Triggers Hippocampal Neuronal Apoptosis through the TLR4/NF-κB/NLRP3 Axis in Hepatic Encephalopathy Ruijun Tang, Shuqi Zhang, Pengyin Nie, Yexi Tang, Qiao Lv, Chengcheng Huang, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9288348/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Cognitive impairment remains a significant neuropsychiatric challenge in hepatic encephalopathy (HE). Characterizing how astrocytes mediate ammonia-driven neuroinflammation may identify therapeutic targets for HE-related cognitive decline. We demonstrate that hippocampal neuronal apoptosis in HE is driven by astrocyte-mediated neuroinflammation through the TLR4/NF-κB/NLRP3 signaling axis. Both genetic deletion and pharmacological blockade of astrocytic TLR4 attenuated neuroinflammation and neuronal apoptosis. Notably, we identified the drug simvastatin as a potent inhibitor of this pathway, which curbed astrocyte reactivity and rescued cognitive function. These findings establish astrocytic TLR4/NF-κB/NLRP3 signaling as a key driver of HE-related cognitive decline and nominate simvastatin as a readily translatable inhibitor of this pathogenic cascade Biological sciences/Neuroscience/Cognitive neuroscience/Cognitive control Health sciences/Medical research/Drug development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome that develops in patients with acute or chronic liver dysfunction or portal shunt( 1 , 2 ), manifested by a spectrum of cognitive and consciousness dysfunction ranging from subtle attention loss to coma and death( 3 , 4 ). These symptoms are thought to arise from the combined effects of ammonia accumulation, oxidative stress, astrocyte swelling, cerebral edema, inflammation, and mitochondrial dysfunction. Current managements of HE targets lowering blood ammonia. Lactulose or Rifaximin is used to curb intestinal ammonia production and branched-chain amino acids or L-ornithine-L-aspartate is employed to enhance systemic ammonia removal( 5 ), yet clinical improvement is limited. Consequently, there is an urgent need to identify effective therapeutic targets for cognitive impairment in HE. Sustained hyperammonemia disrupts the structure and function of neurons, astrocytes, microglia, and endothelial cells in HE( 6 , 7 ). Previous studies have suggested that the neuropsychiatric symptoms of HE are mainly associated with impaired glial cell function( 8 ). Within the glial lineage, astrocytes are the first and principal cerebral targets under hyperammonemia because of the dominant potassium channels and glutamine synthetase( 9 , 10 ). Excessive ammonia leads to glutamine accumulation in astrocytes, which in turn increases intracellular osmotic pressure, causes astrocyte swelling and cerebral edema( 5 ). Additionally, astrocytes play a critical role in maintaining the blood-brain barrier (BBB). The distortion of swollen astrocytic endfeet compromises BBB integrity, thereby promoting HE pathogenesis( 11 ). More recently, accumulating research has revealed that astrocytes may also contribute to HE progression through the induction of neuroinflammation( 12 , 13 ). Specifically, excessive ammonia triggers astrocyte reactivity that generates reactive oxygen and nitrogen species which elevates IL-1β, IL-6 and prostaglandin E2, and exacerbates neuroinflammation and neurotoxicity( 5 ). Importantly, astrocytes are essential for normal neuronal function( 14 ). However, the mechanisms through which the reactive astrogliosis impairs neurons and ultimately contributes to cognitive impairment in HE remains elusive. Toll-like receptor 4 (TLR4), a key pattern recognition receptor in the innate immune system, is widely expressed in astrocytes and plays a pivotal role in initiating and amplifying inflammatory responses( 15 – 17 ). Activated TLR4 further triggers the nuclear factor-κB (NF-κB) signaling pathway, promoting the nuclear translocation of NF-κB p65 subunit and transcription of pro-inflammatory cytokine genes (e.g., IL-1β, IL-6). More importantly, TLR4/NF-κB activation can further induce the assembly and activation of the NLRP3 inflammasome, a key mediator of pro-inflammatory cytokine release, which amplifies astrocyte-derived neuroinflammation( 18 ). Despite the established role of the TLR4/NF-κB/NLRP3 axis in neurological disorders including intracerebral hemorrhage, Alzheimer's disease, and Parkinson's disease( 19 – 21 ), its precise role in hyperammonemia-driven reactive astrogliosis and the subsequent neuronal injury in HE remains unclear, which constitutes a critical research gap addressed in the present study. In this study, we established two classic models of HE, observing both cognitive deficits and significant hippocampal neuronal apoptosis. Mechanistically, hyperammonemia activates the reactive astrocytic TLR4/NF-κB/NLRP3 axis, triggering pro-inflammatory factor release and subsequent neuronal apoptosis. Through simulated molecular docking, we identified simvastatin as a TLR4-targeting agent. It effectively inhibited hyperammonemia-induced astrocyte activation and neuronal apoptosis in both in vitro and in vivo models. Thus, our findings establish hyperammonemia-driven TLR4/NF-κB/NLRP3 signaling in astrocytes as a key driver of hippocampal neuronal apoptosis and cognitive impairment in HE. Materials and Methods Animals Adult (6–8 weeks old) male wild-type C57BL/6 J mice, GFAP-Cre ; TLR4 flox/flox mice (generated by crossing GFAP-Cre mice with TLR4 flox/flox mice; Cyagen Biosciences Inc., Suzhou, China), TLR4 flox/flox mice, and Sprague–Dawley (SD) rats were used. Wild-type mice and SD rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animals were housed under specific pathogen-free (SPF) conditions (12-h light/dark cycle, 22 ± 2°C, 50 ± 5% relative humidity) with free access to standard chow and sterile water. All procedures using laboratory animals were approved by and conducted consistently the guidelines of the Laboratory AnimalWelfare and Ethics Committee Of the Army Medical University (approval No. AMUWEC20255435). Cell culture and treatment Mouse neuronal line HT22, astrocyte line C8-D1A (Procell Life Science & Technology Co., Ltd., Wuhan, China), and 293T cells (ATCC) were cultured in high-glucose DMEM (4.5 g/L glucose) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C/5% CO₂, passaged every 2–3 days at ~ 70% confluence. Primary astrocytes were isolated from newborn (≤ 48 h) C57BL/6 J mice or SD rats. Hippocampi were dissected, minced, digested with 0.25% trypsin-EDTA (15 min, 37°C), filtered (70 µm), and plated in DMEM/F-12 + 10% FBS. After 10–12 days, microglia and oligodendrocytes were removed by orbital shaking (200 rpm, 24 h). Astrocytes were trypsinised (0.05% trypsin-EDTA, 3 min) and re-seeded onto poly-D-lysine (PDL, 10 µg/ml)-coated plates, used within two passages. Primary neurons were isolated from E16–18 C57BL/6 J mouse or SD rat embryos. Hippocampi were processed as above, digested with 0.125% trypsin-EDTA (5 min), filtered (40 µm), and plated at 4–6 × 10⁴ cells/cm² on PDL-coated plates in Neurobasal-A + 2% B-27 + 1% GlutaMAX + 1% antibiotic–antimycotic. Half-medium changes were performed every 3 days. TAK-242 (TLR4 antagonist) was dissolved in 0.1% DMSO (250 µM) and diluted to 100 µM in culture medium. Astrocytic cultures (C8-D1A or primary) were pre-treated with 100 µM TAK-242 for 8 h, followed by 5 mM NH₄Cl for 48 h. Vehicle controls received 0.1% DMSO. Supernatants from treated astrocytes were diluted 1:1 with phenol-red-free Neurobasal-A + 2% B-27 and applied to HT22 or primary neurons for 24 h. For cytokine challenge, neurons were treated with 10 ng/ml IL-1β, IL-6, TNF-α, or vehicle for 24 h. All conditions were tested in triplicate. Establishment of HE animal models HE models were established by bile duct ligation (BDL) in mice or intraperitoneal (i.p.) injection of 150 mg/kg thioacetamide (TAA) in rats. For BDL: mice were anaesthetised with sevoflurane (3–4% in 1 L/min O₂), a mid-line laparotomy was performed, and the common bile duct was ligated with 4 − 0 silk (proximally and at the pancreatic border) without transection. Sham-operated mice underwent identical manipulation without ligation. In accordance with the experimental protocol, WT C57BL/6 J mice received intraperitoneal injections of TAK-242 (3 mg/kg every other day) or its vehicle. In parallel, GFAP-Cre ; TLR4 flox/flox mice or TLR4 flox/flox mice were treated for 4 weeks starting post-surgery: daily gavage with vehicle (PBS with 0.2% SDS), rifaximin (50 mg/kg), or daily intraperitoneal injection of simvastatin (0.2 mg·kg⁻¹), as well as the corresponding combined treatment. For TAA model: rats received TAA (in sterile 0.9% saline) once daily for 3 consecutive days; controls received saline. 5% glucose in 0.9% saline was administered orally (25 ml/kg) 12 h after the first TAA injection. Rats were pre-treated with TAK-242 (3 mg/kg i.p.) or vehicle every other day for 3 injections before TAA administration. H&E staining Animals were euthanized by cervical dislocation after sodium pentobarbital anaesthesia (100 mg/kg i.p.). Livers were fixed in 4% neutral-buffered formalin, dehydrated, cleared in xylene, and embedded in paraffin. 4-µm sections were stained with hematoxylin and eosin, examined under a bright-field microscope (Olympus, Japan). Nissl staining After behavioural tests, mice/rats (n = 5 per group) were transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4). Brains were post-fixed in 4% PFA (4°C, 24 h), dehydrated, cleared, and embedded in paraffin. 5-µm coronal sections were deparaffinized, rehydrated, stained with 0.1% cresyl violet (Servicebio, G1036) for 8 min, differentiated in 95% ethanol, cleared in xylene, and coverslipped. Hippocampal CA1 neurons were imaged (×40 objective) and counted using Fiji ImageJ v1.53. Golgi staining Brains were dissected and immersed in Golgi-Cox OptimStain™ Kit solutions A + B (Hitobiotec, HTKNS1125) in the dark (RT, 14 days), then transferred to solution C (72 h, RT). 100-µm coronal sections were cut, air-dried, developed with solutions D:E:ddH₂O (1:1:2), dehydrated, cleared, and coverslipped. Neurons were imaged (200× for Sholl analysis, 1000× for spine density) with Fiji ImageJ (experimenter-blinded). NeuN immunohistochemistry Sections were prepared as for Nissl staining. Endogenous peroxidase was blocked with 3% H₂O₂ in methanol (15 min, RT). Antigen retrieval was performed in 10 mM sodium citrate (pH 6.0, 95°C, 10 min). Sections were blocked with 5% normal goat serum + 0.3% Triton X-100 (1 h, RT), incubated with rabbit anti-NeuN (4°C, overnight), followed by biotinylated goat anti-rabbit IgG (1 h, RT) and VECTASTAIN Elite ABC-HRP kit (Vector PK-6100). Immunoreactivity was visualized with 0.05% DAB + 0.01% H₂O₂. Sections were counter-stained with hematoxylin, dehydrated, and coverslipped. NeuN-positive cells were counted (×40 objective) using Fiji ImageJ. Immunofluorescence Paraffin sections were deparaffinized, rehydrated, fixed in 10% neutral buffered formalin (10 min), and antigen-retrieved (citrate buffer, pH 6.0, microwave). Sections were permeabilized with 0.3% Triton X-100 (30 min), blocked with 5% BSA (1 h, RT), and incubated with primary antibody pairs (4°C, overnight): rabbit anti-NeuN (Thermo Fisher Scientific, PA5-78499) + rabbit anti-cleaved Caspase-3 (Cell Signaling Technology, 9661); mouse anti-GFAP (ServiceBio, GB12096) + rabbit anti-TLR4 (Thermo Fisher Scientific, PA5-23124), p-P65 (Cell Signaling Technology, 3033) or NLRP3 (ServiceBio, GB114320). After washing, species-matched Alexa-Fluor-conjugated secondary antibodies (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488), ab150113; Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594), ab150080) were applied (1 h, RT, dark). Sections were counter-stained with DAPI (1 µg/ml, 10 min) and mounted. Images were acquired (Olympus BX53) and analysed (Fiji ImageJ, blinded). For six-colour multiplex immunofluorescence, 5-µm sections were stained with NeuN (Thermo Fisher Scientific, PA5-78499), cleaved Caspase 3 (Cell Signaling Technology, 9661), GFAP (ServiceBio, GB12096), C3 (Thermo Fisher Scientific, PA5-21349), S100A10 (Thermo Fisher Scientific, PA5-95505) and DAPI using the TG TSA Multiplex IHC Assay Kit (TissueGnostics, TGFP550) and imaged on a TissueFAXS Cytometry platform. For cell immunofluorescence, primary astrocytes and neurons on coverslips were fixed with 4% PFA (15 min, RT), permeabilized with 0.3% Triton X-100, blocked with 5% BSA (1 h), and incubated with primary antibody pairs (4°C, overnight): mouse anti-GFAP (ServiceBio, GB12096) + rabbit anti-TLR4 (Thermo Fisher Scientific, PA5-23124), p-P65 (Cell Signaling Technology, 3033) or NLRP3 (ServiceBio, GB114320); mouse anti-β3-Tubulin (Cell Signaling Technology, 4466), rabbit anti-cleaved Caspase-3 (Cell Signaling Technology, 9661). Secondary antibodies were applied (1 h, RT), nuclei counter-stained with DAPI, and images acquired on a Leica TCS SP8 confocal microscope (×40 oil-immersion objective). Transmission electron microscopy Hippocampal blocks (1 mm³) were fixed in 2.5% glutaraldehyde (4°C, overnight), post-fixed in 1% OsO₄ (1 h), dehydrated, and embedded in Epon-812. 60-nm ultrathin sections were stained with 3% uranyl acetate and lead citrate, observed under a JEM-1200EX transmission electron microscope (JEOL, Japan) at 8000–15000×. Western blotting Hippocampal tissues, cells, and organoids were lysed in RIPA buffer (Yeasen, 20101ES60) supplemented with 1 mM PMSF (ServiceBio, G2008) and 1× phosphatase inhibitor cocktail (Millipore Sigma, 4906837001) (100:1:1). Lysates were rotated (4°C, 30 min), centrifuged (12000×g, 30 min, 4°C), and protein concentration determined by BCA assay (Beyotime, P0010). 30 µg protein was separated by 10%/12% SDS-PAGE, transferred to 0.45 µm PVDF membranes (Merck Millipore, USA), blocked with 5% non-fat dry milk (1 h, RT), and incubated with primary antibodies (4°C, overnight): cleaved Caspase-3 (Cell Signaling Technology, 9661), Caspase-3 (Cell Signaling Technology, 9662), BAX (ServiceBio, GB15690), BCL2 (Cohesion Biosciences, CPA1095), α-Tubulin (Abclonal, A6830), TNF-α (ServiceBio, GB11188), IL-6 (Affinity Biosciences, DF6087), IL-10 (Abclonal, A2171), IL-1β (Abcam, ab283818), C3 (Thermo Fisher Scientific, PA5-21349), S100A10 (Thermo Fisher Scientific, PA5-95505), TLR4 (Thermo Fisher Scientific, PA5-23124), NF-κB p65 (Cell Signaling Technology, 8242), Phospho-NF-κB p65 (Cell Signaling Technology, 3033), NLRP3 (ServiceBio, GB114320). After washing, HRP-conjugated secondary antibodies (goat anti-mouse or goat anti-rabbit, 1:10000) were applied (1 h, RT). Bands were visualized with ECL reagent (Servicebio, G2014) and quantified with Fiji ImageJ. Liver function detection Orbital venous blood was collected after anaesthesia, kept on ice (4°C), clotted, and centrifuged (3000×g, 15 min, 4°C). Serum ALT (C009-2-1), AST (C010-2-1), TBIL (C019-1-1), and DBIL (C019-2-1) levels were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute) per manufacturer’s instructions. Ammonia level determination Blood and hippocampal homogenates (1:9 w/v in ice-cold saline) were centrifuged (3500×g, 15 min, 4°C). Ammonia levels in supernatants were assayed with the Ammonia/Ammonium Microplate Assay Kit (Absin, abs580164) per manufacturer’s instructions. ELISA Hippocampal tissues were homogenised in PBS + protease inhibitor cocktail, centrifuged (12000×g, 30 min, 4°C). Supernatant IL-1β, IL-6, IL-10, and TNF-α levels were measured using commercial ELISA kits (Mouse IL-1 beta: Thermo Fisher Scientific, BMS6002-2; Mouse IL-6: Thermo Fisher Scientific, BMS603-2HS; Mouse IL-10: Thermo Fisher Scientific, BMS614; Mouse TNF alpha: Thermo Fisher Scientific, BMS607-3; Rat IL-1β: Elabscience, E-EL-R0012; Rat IL-6: Elabscience, E-EL-R0015; Rat IL-10: Elabscience, E-EL-R0016; Rat TNF-α: Elabscience, E-EL-R2856) per manufacturer’s instructions, normalized to total protein content. Y-Maze test Y-maze test assessed cognitive function and spatial reference memory in HE models. The 120° three-arm maze included three phases: 30-min individual habituation, training (blocked arm; 10 min/rats or 8 min/mice exploration of accessible arms, 1-h interval), and testing (novel arm opened; 5-min exploration of all three arms from the original starting arm). Arm entry times were recorded and analyzed with a Smart Video Tracking System (Stoelting Co., USA) for memory evaluation. Novel object recognition test Novel Object Recognition test (exploiting rodents' novelty preference) assessed recognition memory and cognitive function. Setup included an open-field chamber and HD camera linked to EthoVision XT 17.0 (Noldus, Netherlands). After 3 days of 5-min habituation, animals underwent 5-min familiarization (two identical objects, A + A), followed by 1-h delay and 5-min test (one object replaced with a novel square, A + B). Exploration time/tracks were recorded via EthoVision. Recognition memory was quantified by recognition index [(Novel time/Total exploration time) ×100%] and discrimination index [(Novel−Familiar time)/Total exploration time×100%], with higher values indicating better memory. Flow-cytometric apoptosis assay HT22 neurons (2 × 10⁵ cells/well) were treated, harvested, centrifuged (300×g, 5 min, 4°C), and resuspended in 1× binding buffer (1 × 10⁶ cells/ml). 100 µl cell suspension was incubated with 5 µl APC-conjugated Annexin-V and 10 µl 7-AAD (Annexin V-APC/7-AAD Apoptosis Analysis Kit, Absin, abs50008) (15 min, RT, dark). 400 µl binding buffer was added, and samples analysed on a BD FACSCanto II flow cytometer. Apoptotic fractions (Annexin-V⁺/7-AAD⁻ + Annexin-V⁺/7-AAD⁺) were quantified with FlowJo v10. shRNA-mediated TLR4 knockdown Three shRNAs targeting mouse TLR4 (NM_021297) were designed: shTLR4-1 (5′-CCTGTAAGTTACCTGCATATT-3′), shTLR4-2 (5′-CCCTCCATAGACTTCAATTAT-3′), shTLR4#3 (5′-TAGAGGTAGTTCCTAATATTA-3′) and a non-targeting control shNC (5′-CCTAAGGTTAAGTCGCCCTCG-3′). Lentiviruses were produced for the two most effective shRNAs (shTLR4#1 and shTLR4#2) and shNC. C8-D1A astrocytes were transduced (MOI ≈ 5, 8 µg/ml polybrene, 12 h) and selected with 0.5 µg/ml puromycin (7 days). Knockdown efficiency was verified by Western blot. Bulk RNA-Seq and analysis Hippocampal tissues from Sham/BDL mice and Ctrl/TAA rats (n = 5 per group) were collected. Total RNA (RIN > 7.0, 28S:18S > 1.8) was isolated with TRIzol (Invitrogen). Poly(A) mRNA was enriched with NEB Next Poly(A) mRNA Magnetic Isolation Module, fragmented, and reverse transcribed. Libraries were constructed with NEB Next Ultra RNA Library Prep Kit for Illumina and sequenced on an Illumina NovaSeq 6000 (2 × 150 bp). Reads were trimmed with Trimmomatic v0.36, aligned to genomes (GRCm38/Rnor_6.0) with STAR, quantified by featureCounts, and filtered (average TPM 0.585, FDR < 0.05) and analysed by Metascape and GSEA (clusterProfiler). scRNA/snRNA-seq and analysis Hippocampal single-cell/single-nucleus suspensions were prepared for 10× Genomics Chromium capture. Libraries were constructed with Chromium Single Cell 3′ Reagent Kits v3.1 and sequenced on Illumina NovaSeq 6000. Data were processed with Seurat: genes expressed in ≥ 3 cells and cells with ≥ 500 features were retained; doublets removed with DoubletFinder; batches aligned with Harmony. PCA, graph-based clustering, and UMAP embedding were performed. Cluster markers were identified with FindAllMarkers, and GSEA was conducted with clusterProfiler. Human brain transcriptome analysis GSE41919 and GSE57193 datasets were downloaded from the Gene Expression Omnibus (GEO). GSE41919 initially contained 8 post-mortem brain samples from HE patients and 8 non-cirrhosis controls (GPL14550). Applying the sample-exclusion criteria of Hsu et al., 6 specimens that crossed the phenotypic boundary in hierarchical clustering and PCA (GSM1027454, GSM1027458, GSM1027462, GSM1027465, GSM1027467, GSM1027468) were removed, leaving 6 HE and 4 control samples. GSE57193 included 4 HE and 4 control brains (same platform) and was analysed separately without additional sample exclusion. DEGs were identified with limma (adjusted P 0.585). GSEA was performed with hallmark gene sets. Neuroinflammatory and apoptotic signatures were quantified by GSVA v1.46, and Pearson correlation was computed. Organoid culture and differentiation hPSCs were differentiated into cerebral organoids. EBs were formed in 96-well round-bottom plates (9,000 cells/well) with EB differentiation medium + 10 µM Y-27632. Neuroectodermal induction was performed on day 5, EBs embedded in Matrigel (Corning, 354253) on days 7–10, and maturation initiated on day 10 with orbital shaking (65 rpm). Mature organoids (day 40) were exposed to 5 mM NH₄Cl or 5 µM simvastatin. Organoids were released with Cell Recovery Solution (Corning, 354253) for protein extraction. Drug prediction, docking and molecular dynamics TLR4 was uploaded to DSigDB via Enrichr to identify candidate drugs (FDA/EMA-approved, blood-brain-barrier permeable, adjusted p-value < 0.01). TLR4 structure (PDB) and compound structures (PubChem) were downloaded. Docking was performed with AutoDock Vina and compounds with a binding energy < -5 kcal/mol were selected for subsequent molecular dynamics simulations. Molecular dynamics of TLR4-candesartan/simvastatin complexes were evaluated with GROMACS 2020.6 (100 ns production run). Complex stability was analysed with VMD and PyMOL. Statistical analysis Quantitative data were analysed with GraphPad Prism 10, presented as mean ± SD. Two-group comparisons used unpaired t -test; multi-group comparisons used one-way ANOVA followed by Dunnett’s or Holm–Šídák post-test. Significance levels are indicated in figure legends. Sample sizes were determined by independent experiments with multiple replicates. Results Convergent Evidence from BDL and TAA Models Identifies Hippocampal Neuronal Apoptosis as a Hallmark of HE We established a classic bile duct ligation (BDL) mouse model of HE in C57BL/6J mice, in which chronic hyperammonemia induced by surgical bile duct ligation led to HE after four weeks. (Fig. 1 A). Histological analysis of BDL model livers revealed severe structural injury, including extensive necrosis, steatosis, and inflammatory infiltrates (Fig. 1 B). Consistent with this structural damage, hepatic function was significantly compromised, as indicated by marked increases in AST, ALT, TBil, and DBil (Fig. 1 C). Furthermore, the systemic and central effects of the model were confirmed by elevated ammonia levels in both the blood and the brain (Fig. 1 D). Cognitive impairment is a well-established hallmark of HE. To assess this in our model, we subjected BDL mice to Y-maze and novel-object recognition (NOR) tests. The BDL mice exhibited significant spatial working memory deficits in the Y-maze, as evidenced by reduced entries into and less time spent in the novel arm, indicating a decreased preference for novelty (Fig. 1 E). Consistently, in the NOR test, the recognition and discrimination indices were significantly lower in BDL mice, confirming a general recognition memory deficit (Fig. 1 F). These results collectively demonstrate that BDL mice develop robust cognitive impairment. The hippocampus serves as a central hub for cognitive processing, and damage to hippocampal neurons is a critical factor in the development of cognitive impairment( 22 ). Nissl staining of our BDL model hippocampus showed neuronal loss and damage features, including neuronal shrinkage, darkened staining, and irregular contours (Fig. 1 G), as well as the similar phenotype was observed in NeuN immunohistochemistry (Fig. 1 H). Structural integrity was further compromised at the single neuron level, as Golgi staining revealed simplified dendritic arbors and decreased spine density (Fig. 1 I- 1 K), collectively pointing to a profound disruption of neuronal connectivity that underlies the observed cognitive deficits. Ultrastructural evidence of neuronal apoptosis was observed via transmission electron microscopy (TEM), as BDL hippocampal neurons displayed classic morphological changes, including electron-dense cytoplasm, clumped chromatin margination, and cellular shrinkage (Fig. 1 L). Consistent with these morphological findings, we demonstrated a robust activation of the intrinsic apoptotic pathway, characterized by increased abundance of cleaved Caspase-3 and BAX, and a decrease in BCL2 in BDL hippocampus (Fig. 1 M). Further supporting this, immunofluorescence revealed a significant decrease in NeuN signal intensity concomitant with an increase in cleaved Caspase-3 intensity within the hippocampus (Fig. 1 N). Our results indicated ongoing apoptosis and neuronal loss in BDL mice. To extend these findings, we generated another complementary HE model in SD rats via intraperitoneal injection of thioacetamide (TAA) (Figure S1 A). This paradigm induced severe hepatocellular injury, as shown by extensive necrosis and inflammatory infiltration (Figure S1 B), elevated serum markers of liver damage (AST, ALT, TBil, DBil), and increased hippocampal ammonia (Figure S1 C and S1D). TAA-exposed rats recapitulated the cognitive deficits observed in BDL mice, exhibiting impaired performance in both Y-maze and NOR tests (Figure S1 E and S1F). Mirroring the hippocampal pathology, NeuN immunostaining confirmed neuronal loss in the hippocampus of TAA‑treated rats (Figure S1 G). Ultrastructural analysis by TEM further revealed apoptotic morphology in hippocampal neurons, including electron-dense cytoplasm and chromatin condensation (Figure S1 H). Consistently, immunofluorescence demonstrated decreased NeuN signal alongside increased cleaved Caspase-3 intensity (Figure S1 I). Together, the convergent findings from BDL and TAA models robustly establish hippocampal neuronal apoptosis as a conserved pathological hallmark in HE. A1 Reactive Astrocytes Drive Hippocampal Neuronal Apoptosis in HE To identify key pathways driving neuronal apoptosis in HE, we conducted bulk RNA-seq on hippocampi from mice model. The enrichment pathways of differentially expressed genes highlighted significant involvement of the acute inflammatory response, neuronal apoptosis regulation, and interleukin-6-family signaling in BDL model (Fig. 2 A and Table S1 ). Notably, gene set enrichment analysis (GSEA) revealed significant up-regulation of inflammatory and neuroinflammation gene sets, alongside down-regulation of neuron development and projection signatures in BDL hippocampi (Fig. 2 B and Table S2 ), indicating prominent neuroinflammation. Consistent with this transcriptomic profile, western blot and ELISA confirmed elevated pro-inflammatory cytokines IL-1β, IL-6, TNF-α and reduced anti-inflammatory IL-10 in BDL hippocampi (Fig. 2 C and 2 D), underscoring a neuroinflammatory microenvironment. Astrocytes, the most abundant glial cells in the central nervous system, rapidly undergo molecular and structural remodeling when the brain microenvironment is disturbed, a process known as reactive astrogliosis( 23 , 24 ). These reactive astrocytes secrete a spectrum of factors such as IL-1β, IL-6, TNF-α, etc. , and severely reactive astrocytes are capable of triggering neuronal apoptosis and cognitive decline in acute ischemic and hemorrhagic stroke( 25 ). To investigate the role of this process in HE, we employed single-cell/nucleus RNA sequencing on hippocampal tissue from mice model (Fig. 2 E and 2 F). Our results revealed a marked expansion of the reactive astrocyte cluster alongside an increased proportion of apoptotic neurons in the BDL group (Fig. 2 G and 2 H). In addition, TEM of hippocampal astrocytes revealed the characteristic profile of reactive astrogliosis in BDL mice, as shown by swollen cell bodies and mitochondrial swelling with fragmented cristae (Fig. 2 J). These hypertrophic astrocytes frequently surrounded neurons with condensed nuclei and shrunken cytoplasm, indicating that reactive astrocytes spatially associate with apoptotic neurons (Fig. 2 I). These results indicated that astrocyte activation is a pivotal driver of neuroinflammation and neuronal apoptosis in BDL mice. Reactive astrocytes in mammals are broadly categorized into two types: A1 astrocytes secrete complement C3 and pro-inflammatory cytokines, exerting neurotoxic effects that impair synapses and promote neuronal death. In contrast, A2 astrocytes release trophic factors such as BDNF and S100A10, along with anti-inflammatory mediators, thereby supporting neuronal survival and repair( 26 – 28 ). As expected, immunofluorescence staining showed markedly more cleaved Caspase-3 positive neurons in BDL hippocampi compared to sham groups (Fig. 2 K). In addition, a striking spatial relationship was observed: cleaved Caspase-3 positive neurons in BDL mice were closely surrounded by A1 astrocytes, whereas neurons in sham mice were associated with resting or A2 astrocytes (Fig. 2 K). Collectively, these observations suggest that the shift of astrocytes toward a pro-inflammatory A1 phenotype creates a deleterious microenvironment that promotes hippocampal neuronal apoptosis in the BDL model. Interrogating the TAA rat model, hippocampal bulk RNA-seq revealed a pro-inflammatory gene signature—encompassing apoptosis, inflammatory response, TNF-α/NF-κB, and IL-6/JAK-STAT3 pathways—that closely mirrored the BDL profile (Figure S2 A and Table S3 ). This transcriptional conservation was reinforced at the protein level, with marked increases in IL-1β, IL-6, and TNF-α and a decrease in IL-10 (Figure S2 B and S2C). In addition, our immunofluorescence demonstrated that apoptotic neurons in TAA rats were flanked by A1-type astrocytes, while control neurons neighbored resting or A2-type cells (Figure S2 D). These convergent results from both models establish that HE-induced astrocytic activation and A1-mediated neuroinflammation are key drivers of hippocampal neuronal apoptosis. Ammonia Drives Astrocyte Reactivation and Neuroinflammation to Induce Neuronal Apoptosis To further confirm that ammonia stimulates astrocyte reactivation, leading to the release of pro-inflammatory factors and subsequent hippocampal neuronal apoptosis, we triggered primary mouse astrocytes with 5 mM NH 4 Cl for 48 h to mimic HE-associated hyperammonemia, which induced a clear pro-inflammatory shift. Western blot analysis showed upregulated levels of C3, IL-1β, IL-6, and TNF-α concomitant with downregulation of IL-10 and S100A10 (Fig. 3 A). Consistent with this, immunofluorescence staining demonstrated enhanced GFAP and C3 expression but reduced S100A10 in ammonia-treated astrocytes (Fig. 3 B and 3 C), corroborating their activation toward a neurotoxic phenotype. In addition, we treated primary hippocampal neurons with conditioned medium from NH₄Cl-exposed astrocytes (AC-CM). AC-CM, compared to control medium (Ctrl-CM), increased cleaved Caspase-3 expression and induced neuronal injury characterized by somal disruption and axonal breakage (Fig. 3 D and 3 E). This neurotoxicity was directly replicated by treating neurons with IL-1β, IL-6, or TNF-α individually (Fig. 3 F and 3 G), pinpointing these cytokines as key mediators. We next sought to validate these findings in a rat model. Ammonia exposure (5 mM NH₄Cl, 48 h) induced a consistent pro-inflammatory phenotype in rat primary astrocytes, evidenced by upregulated pro-inflammatory markers (C3, IL-1β, IL-6, TNF-α) and downregulated anti-inflammatory markers (IL-10, S100A10) on western blot (Figure S3 A) and immunofluorescence (Figure S3 B and S3C). Conditioned medium from these rat astrocytes (AC-CM) was potently neurotoxic, elevating cleaved Caspase-3 and causing somal disruption and axonal fragmentation in rat hippocampal neurons (Figure S3 D and S3E). This neurotoxicity was directly attributable to specific cytokines, as individual application of IL-1β, IL-6, or TNF-α to neurons replicated the apoptotic damage (Figure S3 F and S3G), confirming the conserved role of these astrocyte-derived factors. To ensure experimental consistency and further validate our findings in a defined system, we employed the C8-D1A astrocyte cell line. Treatment with 5 mM NH₄Cl for 48 h induced a pro-inflammatory shift in these cells, marked by increased C3, IL-1β, IL-6, and TNF-α and decreased IL-10 and S100A10 (Fig. 3 H). Conditioned medium from these cells (AC-CM) triggered significant apoptosis in neuronal HT22 cells, shown by a higher apoptotic cell proportion (Fig. 3 I) and elevated pro-apoptotic protein levels (BAX, cleaved Caspase-3) with reduced BCL2 (Fig. 3 J). Direct treatment with IL-1β, IL-6, or TNF-α reproduced this apoptotic effect (Fig. 3 K), confirming that astrocyte-derived cytokines are sufficient to induce neuronal apoptosis across experimental models. Astrocytic TLR4/NF-κB/NLRP3 Signaling Drives Neuroinflammation and Neuronal Apoptosis in HE Pattern-recognition receptors on astrocytes enable rapid detection of tissue damage( 29 , 30 ). TLR4, a key member of this family, amplifies neuroinflammation by disrupting the blood-brain barrier and boosting pro-inflammatory factors( 16 , 17 ). Although microglia are the primary source of TLR4 in the healthy brain( 31 – 33 ), whether hyperammonemia induces TLR4 upregulation and activation in astrocytes is not known. Previous work in LPS and trauma models shows that astrocytic TLR4 signaling activates NF-κB, upregulates NLRP3, and increases IL-1β, IL-6, and TNF-α production( 34 , 35 ). We proposed a mechanism that hyperammonemia engages the TLR4/NF-κB/NLRP3 cascade in astrocytes, driving astrocytic reactivation and subsequent neuronal apoptosis in HE (Fig. 4 A). Immunofluorescence of hippocampal tissue in BDL mice revealed that increased co-expression of GFAP with TLR4, NF-κB, and NLRP3, indicating activation of this pathway in vivo (Fig. 4 B- 4 D). This was recapitulated in vitro , treating primary murine astrocytes with 5 mM NH₄Cl for 48 hours significantly increased protein levels of TLR4, NF-κB, and NLRP3 (Fig. 4 E). Critically, pretreatment with the TLR4 inhibitor TAK-242 abolished these increases and reversed the inflammatory profile, reducing NF-κB, NLRP3, C3, IL-1β, IL-6, and TNF-α while restoring IL-10 and S100A10 (Fig. 4 F). Having validated this pathway in vitro , we next investigated its role in established HE models. In BDL model treated with TAK-242 (3 mg kg⁻¹ every 48 h for four weeks), hippocampal astrocytes showed significant decrease fluorescence intensity of GFAP, NF-κB, and NLRP3 (Fig. 4 G and 4 H). Western blot and ELISA analyses of hippocampal tissue confirmed parallel decreases in IL-1β, IL-6, and TNF-α, alongside a recovery of IL-10 (Fig. 4 I and 4 J). These data establish astrocytic TLR4/NF-κB/NLRP3 signaling as a key driver of neuroinflammation in the BDL model. As expected, preemptive administration of TAK-242 similarly reduced hippocampal levels of NF-κB, NLRP3, and GFAP (Figure S4 A and S4B), and decreased IL-1β, IL-6, and TNF-α while increasing IL-10 in TAA rat model (Figure S4 C). Collectively, these results demonstrate that pharmacological TLR4 inhibition suppresses the astrocytic NF-κB/NLRP3 axis and attenuates neuroinflammation in both murine and rat models of HE. To determine whether blocking the astrocytic TLR4 pathway protects neurons, we exposed mouse primary neurons to conditioned medium from astrocytes. When astrocytes were pretreated with the TLR4 inhibitor TAK-242 before NH 4 Cl treatment, the resulting medium failed to increase cleaved Caspase-3 in neurons, unlike medium from vehicle-pretreated astrocytes (Fig. 4 K). Similarly, conditioned medium from C8-D1A astrocytes with stable TLR4 shRNA knockdown, under the same ammonium exposure, caused a marked reduction in HT22 neuronal apoptosis compared to control shRNA, as shown by flow cytometry (Fig. 4 L). These data demonstrate that inhibiting astrocytic TLR4 signaling effectively attenuates neuronal apoptosis in vitro . Dual Modulation of Astrocytic TLR4 Alleviates Neuroinflammation and Cognitive Deficits in HE To determine whether pharmacological blockade of astrocytic TLR4 protects neurons in vivo , we administered TAK-242 (3 mg kg⁻¹, i.p. every 48 h) or vehicle to BDL mice for four weeks. Administration of TAK-242 to BDL mice significantly reduced hippocampal neuronal loss and apoptosis (Fig. 5 A and 5 D). These changes were coincided with decreased GFAP and C3 together with elevated S100A10 fluorescence, indicating a shift in astrocytes from a pro-inflammatory A1 to a protective A2 phenotype (Fig. 5 B). This was accompanied by an anti-apoptotic molecular profile including decreased BAX/cleaved Caspase-3 and increased BCL2 (Fig. 5 C). Crucially, these improvements extended to cognitive function, with treated mice performing better in Y-maze and novel object recognition tests (Fig. 5 E and 5 F). Identical treatment in TAA rats yielded equivalent protection across histopathological, molecular, and behavioral readouts (Figure S5 A-S5F). To complement the pharmacological approach, we genetically ablated TLR4 specifically in astrocytes using GFAP - Cre ; TLR4 fl/fl mice. After BDL surgery, these mice showed preserved hippocampal neurons (Fig. 5 G) and attenuated neuroinflammation (reduced NLRP3, NF-κB, IL-1β, IL-6, TNF-α; increased IL-10) and apoptosis (reduced cleaved Caspase-3, BAX) at the molecular level (Fig. 5 H and 5 I). The finding supported by immunofluorescence and TEM (Fig. 5 J and 5 K). Paralleling the TAK-242 treatment, cognitive deficits were also rescued in the knockout mice (Fig. 5 L and 5 M). Together, these data demonstrate that targeting astrocytic TLR4—either pharmacologically or genetically—effectively dampens neuroinflammation, prevents neuronal apoptosis, and restores cognitive function in HE. Drug Repurposing of Simvastatin via Astrocytic TLR4 Inhibition Rescues Cognition in HE While our results demonstrated that ammonia elicits pro-inflammatory cytokine release from astrocytes, leading to neuronal injury, its relevance to human HE remained unclear. To translate these findings, we analyzed public transcriptomic data from post-mortem HE patient brains (GSE57193 and GSE41919). GSEA of GSE57193 showed marked enrichment of gene sets related to apoptosis, inflammatory response, and key signaling pathways such as IL-6–JAK–STAT3, TNF-α via NF-κB and complement (Fig. 6 A and Table S4 ). Furthermore, a positive correlation was observed between neuroinflammatory and neuronal apoptosis gene signatures across patient samples (Fig. 6 B), confirming concomitant neuroinflammation and apoptosis in human HE. Our results indicated that the TLR4/NF-κB/NLRP3 axis may play a critical role in neuroinflammation in patients with HE. Building on the identification of TLR4 as a potential therapeutic target, we aimed to repurpose clinically approved drugs that target TLR4 to alleviate HE-related cognitive impairment. We first performed molecular docking of compounds from several drug classes (e.g., statins, ARBs, NSAIDs, Table S5 ). Four compounds—candesartan, simvastatin, dexamethasone, and methylprednisolone—showed promising binding affinity (Fig. 6 C). Based on potential hepatotoxicity, we focused on candesartan and simvastatin for further analysis. Molecular dynamics simulations revealed that simvastatin had a more stable binding mode with TLR4 (Fig. 6 D). We then experimentally validated these candidates, in ammonia-treated C8-D1A astrocytes, only simvastatin pretreatment effectively reduced markers of reactive astrogliosis and pro-inflammatory cytokine expression (Fig. 6 E). Notably, in human iPSC-derived cerebral organoids, simvastatin treatment suppressed ammonia-induced upregulation of key inflammatory mediators-C3, NF-κB, NLRP3, IL-1β, IL-6, TNF-α (Fig. 6 F). Thus, our integrated screening identifies simvastatin as a TLR4-targeting agent capable of dampening astrocyte-driven neuroinflammation, positioning it for therapeutic repurposing in HE. To validate the therapeutic potential of simvastatin in vivo , we administered it every other day for four weeks to our BDL model. Simvastatin treatment significantly improved cognitive function, increasing novel arm entries in the Y-maze and the recognition index in the novel object recognition test (Fig. 6 G and 6 H). Its efficacy was comparable to rifaximin, the classical HE-therapy drug (Fig. 6 G and 6 H). Interestingly, rifaximin showed a more pronounced effect in GFAP Cre ; TLR4 fl/fl mice, prompting us to test a combination therapy. Co-administration of simvastatin and rifaximin resulted in superior amelioration of cognitive impairment compared to either drug alone (Fig. 6 G and 6 H). Collectively, these results establish​ astrocytic TLR4 as a validated​ therapeutic target for HE. They further nominate​ simvastatin, either as monotherapy or in synergistic combination with rifaximin, as a ready-to-repurpose​ treatment strategy to alleviate cognitive impairment. Discussion Hepatic encephalopathy, a severe neuropsychiatric complication of liver disease, manifests as progressive cognitive impairment. Existing treatments focusing on ammonia lowering offer only modest and unsustained efficacy, underscoring the critical need for novel central nervous system-targeted interventions. Astrocytes, owing to their unique ammonia metabolism, are critically involved in HE pathogenesis. Yet, how hyperammonemia precisely triggers these cells to propagate neuroinflammation and subsequent neuronal damage is unclear, representing a key knowledge gap. In this study, we define a pathogenic cascade wherein hyperammonemia activates the TLR4/NF-κB/NLRP3 axis in astrocytes, leading to neuroinflammation, neuronal apoptosis, and cognitive dysfunction.​ Furthermore, we repurpose the lipid-lowering agent simvastatin as a TLR4 antagonist that effectively disrupts this cascade and rescues cognition, offering a readily translatable therapeutic strategy (Fig. 6 I). A key remaining question is how hyperammonemia initiates the astrocytic TLR4/NF-κB/NLRP3 cascade. Prior work in endothelial cells established that ammonia rapidly elevates TLR4 levels via a Ca 2+ -dependent ROS burst( 36 ). The conservation of this rapid Ca 2+ -oxidant response in astrocytes suggests a similar pathway may upregulate TLR4( 37 , 38 ). Mitochondrial damage provides a potential source of ligands to engage TLR4. Specifically, damage-associated molecular patterns (mtDAMPs) released from mitochondria can act as potent agonists for TLR4, including mtDNA, cardiolipin and cytochrome C( 39 ). Accumulating evidence has demonstrated that hyperammonemia induces mitochondrial injury in astrocytes during the pathogenesis of HE( 40 , 41 ). Consistent with this, the TME showed that astrocytic mitochondria exhibited marked injury in our BDL model, characterized by swelling and cristae fragmentation (Fig. 2 J). Notably, each of these mtDAMP components has been identified as an TLR4 ligand( 42 – 45 ), with the capacity to initiate downstream NF-κB and NLRP3 signaling cascades. Moreover, mtDAMPs released upon mitochondrial damage can act on recipient cells via autocrine, paracrine, or vesicle-mediated secretory pathways( 46 ) , ( 47 ). We propose a mechanism for HE: ammonia may first enhance astrocytic TLR4 expression via Ca 2+ -ROS signaling, and the receptor is then activated by mtDAMPs released from damaged mitochondria, triggering the TLR4/NF-κB/NLRP3 axis and astrocytic type A1 reactivity. Simvastatin, a widely used lipid-lowering agent with known certain neuroprotective properties and established clinical application in cerebrovascular accidents( 48 ), emerged from our drug screening as a promising TLR4 modulator. Additionally, simvastatin can scavenge superoxide anions and suppress NADH and NADPH oxidase-mediated reactive oxygen species generation, thereby reducing lipid peroxidation in liver and brain tissues and indirectly alleviating oxidative stress-related exacerbation of HE( 49 ). Beyond these metabolic and antioxidant effects, accumulating evidence indicates that statins confer broad anti-inflammatory actions in the brain by down-regulating TLR4( 50 , 51 ). In models of intracerebral hemorrhage, simvastatin lowers TLR4 expression, blunts NF-κB activation, and decreases subsequent production of IL-1β and TNF-α, resulting in attenuated neuronal damage and improved neurological outcome( 52 ). Given its established clinical safety profile and low cost, simvastatin is uniquely positioned for rapid therapeutic repurposing. Our data support the progression to dose-finding and proof-of-concept trials in HE patients. Notably, simvastatin co-administered with rifaximin yielded synergistic cognitive benefits, likely through complementary actions on the gut-liver-brain axis. This combination strategy, simultaneously reducing peripheral toxin load and central neuroinflammation, presents a promising translational avenue for alleviating HE-related cognitive impairment. Several limitations warrant consideration. A key limitation is the indirect evidence for astrocyte-specific ammonia handling; direct assessment of NH₄⁺ flux across neural cell types is required. In addition, the precise molecular steps connecting ammonia to astrocytic TLR4 activation remain incompletely resolved. Specifically, the roles of the Ca²⁺-ROS cascade in receptor upregulation and of mtDAMPs as TLR4 ligands are plausible yet unverified; targeted experiments in astrocytes would help clarify these mechanisms. Third, the specific role of astrocyte-derived factors requires further delineation. Although astrocytic cytokines were associated with neuronal apoptosis, a comprehensive profiling of the astrocyte secretome was not performed, and we did not disrupt potential pro-inflammatory crosstalk from microglia( 17 , 53 ). Consequently, the individual contributions of astrocyte-versus microglia-mediated neurotoxicity remain unresolved. Furthermore, all efficacy data for simvastatin are derived from animal studies. Its clinical potential must be confirmed through subsequent dose-finding, pharmacokinetic, and long-term safety trials in HE patients. In summary, our study defines a mechanism in hepatic encephalopathy where hyperammonemia elicits astrocyte reactivity via the TLR4/NF-κB/NLRP3 pathway, driving neuroinflammation, neuronal apoptosis, and cognitive decline. Targeting this pathway with simvastatin reverses these deficits, offering a directly repurposable therapy for patients with HE. Declarations Conflict of Interest The authors declare no conflict of interest. Funding This work was supported by the Chongqing Medical Leading Talent Program [grant YXLJ202521 to L.Z.];the Discipline Excellence Talent Program [grant 2023XKRC005 to L.Z.]; the Chongqing Technology Innovation and Application Development General Program [grant CSTB2023TIAD-GPX0013 to L.Z.];the National Natural Science Foundation of China [grant 8257116374 to X.X.]; the Chongqing Natural Science Foundation [grant CSTB2024NSCQ-KJFZZDX0016 to X.X.];and the National Natural Science Foundation of China [grant 32500501 to R.T.]. Author Contributions S.Z., P.N., R.T., X.X., and L.Z. designed the study; L.Z., R.T., and S.Z. wrote the manuscript and prepared the figures; S.Z. performed most experiments; P.N. assisted with animal models and primary cell isolation; Y.T. conducted bioinformatics analysis and drug screening; Q.L. performed flow cytometry-based apoptosis detection; C.H., S.Z., and X.J. assisted with behavioral tests and animal drug administration; J.Y., X.H. and Z.W. provided support for immunofluorescence staining experiments; W.L., Y.H. and N.Y. performed the evaluation of clinical public datasets related to HE; all authors reviewed and edited the manuscript. Acknowledgements We gratefully acknowledge Drs. Chaoqun Wang, Ruihao Yang, Senlin Li, Bin Han, Zhengfei Bi and Cyagen Biosciences, Beijing Vital River Laboratory Animal Technology, Yeasen biotechnology for inspiring discussions or reagents. The graphical illustrations in this work were created using the BioGDP platform (BioGDP.com), developed as the Generic Diagramming Platform (GDP) (Jiang S et al., Nucleic Acids Res, 2025). Availability of Data and Materials Sequencing data in this study have been deposited in the OMIX (National Genomics Data Center, China) under the accession numbers OMIX014552 (mouse bulk RNA-seq), OMIX014554 (rat bulk RNA-seq), and OMIX014555 (mouse scRNA-seq and snRNA-seq). The datasets are currently under restricted access and a permanent public access will be enabled via https://ngdc.cncb.ac.cn/omix after publication. Human bulk RNA-sequencing data were obtained from public repositories (GEO: GSE57193 and GEO: GSE41919). References Tsai CY, Wu JCC, Wu CJ, Chan SHH. Protective role of VEGF/VEGFR2 signaling against high fatality associated with hepatic encephalopathy via sustaining mitochondrial bioenergetics functions. J Biomed Sci. 2022;29(1):47. 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University","correspondingAuthor":false,"prefix":"","firstName":"Shuqi","middleName":"","lastName":"Zhang","suffix":""},{"id":627503670,"identity":"982e46b6-1267-4041-a42e-c8e67101a35b","order_by":2,"name":"Pengyin Nie","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Pengyin","middleName":"","lastName":"Nie","suffix":""},{"id":627503672,"identity":"7acbf4d1-d6bb-4f63-919d-e6f510225310","order_by":3,"name":"Yexi Tang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yexi","middleName":"","lastName":"Tang","suffix":""},{"id":627503674,"identity":"010ea283-8a18-4b19-9273-a18bad518b6b","order_by":4,"name":"Qiao Lv","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical 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07:45:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9288348/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9288348/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108495285,"identity":"e88e4450-90ea-41c2-9049-57882b0a683f","added_by":"auto","created_at":"2026-05-05 10:09:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1343645,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeuronal apoptosis occurs in the hippocampus of HE BDL model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic representation of the BDL model in mice. (B) Liver H\u0026amp;E staining of sham and BDL mice. Bar: 50 µm. (C) Serum levels of ALT, AST, TBil, and DBil in sham and BDL mice. **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 by unpaired \u003cem\u003et\u003c/em\u003e test. (D) Blood and brain ammonia levels in sham and BDL mice. **, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 by unpaired \u003cem\u003et\u003c/em\u003etest. (E) \u003cem\u003eLeft\u003c/em\u003e: Representative Y-maze exploration traces for sham and BDL mice (novel arm indicated by red box); \u003cem\u003eRight\u003c/em\u003e: percentage of entries and time spent in the novel arm. **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 by unpaired \u003cem\u003et\u003c/em\u003etest. (F) \u003cem\u003eLeft\u003c/em\u003e: Representative NOR test traces for sham and BDL mice (novel object indicated by square); \u003cem\u003eRight\u003c/em\u003e: recognition index and discrimination index. ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 by unpaired \u003cem\u003et\u003c/em\u003e test. (G) Representative images of Nissl staining and quantitative analysis of viable neurons in the hippocampal CA1 region. Bar: 200 µm. ****,\u003cem\u003e p \u003c/em\u003e\u0026lt; 0.0001 by unpaired \u003cem\u003et\u003c/em\u003etest. (H) Representative images of NeuN immunohistochemistry and quantitative analysis of NeuN-positive neurons in the hippocampal CA1 region. Bar: 200 µm. ****,\u003cem\u003e p \u003c/em\u003e\u0026lt; 0.0001 by unpaired \u003cem\u003et\u003c/em\u003etest. (I-J) Representative images at 200× magnification and Sholl analysis of Golgi–Cox-stained hippocampal neurons showing dendritic length (I) and number of intersections (J). ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 by unpaired t test. (K) High-magnification (1000×) images of dendritic spines and quantification of spine density (spines/10 µm). Bar: 10 µm; ***, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 by unpaired \u003cem\u003et\u003c/em\u003e test. (L) Representative TEM images of hippocampal neurons showing ultrastructural changes indicative of apoptosis. Bar: 5 µm. (M) \u003cem\u003eLeft\u003c/em\u003e: Western-blot images of hippocampal lysates probed for cleaved Caspase-3, BAX and BCL2; \u003cem\u003eRight\u003c/em\u003e: band-density quantification. ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 by unpaired \u003cem\u003et\u003c/em\u003e test. (N) \u003cem\u003eLeft\u003c/em\u003e: Representative images of immunofluorescence staining for NeuN (green) and cleaved Caspase-3 (red) in the hippocampus for sham and BDL mice. Bar: 200 µm; \u003cem\u003eRight\u003c/em\u003e: quantification of mean fluorescence intensity for NeuN and cleaved Caspase-3. ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001 by unpaired \u003cem\u003et\u003c/em\u003e test.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9288348/v1/cbb4d22157804970cbc00806.png"},{"id":108495717,"identity":"73609ea9-dbed-429d-b5ee-b4259790b843","added_by":"auto","created_at":"2026-05-05 10:10:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1493226,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrocyte-mediated neuroinflammation induces neuronal apoptosis in HE BDL model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Bar charts showing enriched pathways for DEGs in hippocampus of BDL versus sham mice. (B) Gene Set Enrichment Analysis (GSEA) of hippocampal RNA-seq data from BDL versus sham mice using the M5 gene-set collection. (C-D) Western blot and ELISA quantification of IL-1β, IL-6, TNF-α and IL-10 proteins in hippocampus of BDL and sham groups. *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 by unpaired \u003cem\u003et\u003c/em\u003e test. (E) UMAP visualization of single-cell and single-nucleus RNA-seq data from hippocampus of BDL and sham mice. (F) Bubble plot showing canonical marker genes used for major cell-type annotation. (G) Apoptosis score distribution in hippocampal neurons of BDL and sham models. \u003cem\u003eMann-Whitney\u003c/em\u003e test.(H) Reactive astrocyte score distribution in hippocampus of BDL and sham models. \u003cem\u003eMann-Whitney\u003c/em\u003e test. (I) Representative TEM images of astrocytes showing reactive features (swollen soma, dark cytoplasm), and a reactive astrocyte adjacent to an apoptotic neuron (condensed chromatin, dark cytoplasm) in BDL hippocampus. Bar: 5 µm. (J) Representative TEM images of hippocampal astrocytes in sham and BDL groups. (K) Multiplex immunofluorescence of hippocampus from sham and BDL mice showing GFAP (white), NeuN (orange), cleaved Caspase-3 (red), complement C3 (yellow), and S100A10 (green). Bar: 50 µm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9288348/v1/c530cb4c1d662ff40ebe705b.png"},{"id":108496387,"identity":"58d63dae-8d0d-4699-9842-9ecf1bfdf9ae","added_by":"auto","created_at":"2026-05-05 10:11:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1213911,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAmmonia triggers astrocyte reactivation and neuroinflammation to promote neuronal apoptosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot of complement C3, TNF-α, IL-6, IL-10, IL-1β and S100A10 in primary mouse hippocampal astrocytes after 48 h NH₄Cl treatment.\u003cstrong\u003e \u003c/strong\u003e(B) Representative images of mouse hippocampal astrocytes co-immunostained for GFAP (white) with C3 (red) or S100A10 (green). Bar: 20 µm.\u003cstrong\u003e \u003c/strong\u003e(C) Quantification of mean fluorescence intensity for GFAP, C3 and S100A10 in Fig.3B. ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 by unpaired \u003cem\u003et\u003c/em\u003e test.\u003cstrong\u003e \u003c/strong\u003e(D) Representative images of primary mouse hippocampal neurons exposed for 24 h to astrocyte-conditioned medium from NH₄Cl-treated or control astrocytes, co-stained for β3-Tubulin (yellow) and cleaved-Caspase-3 (purple). Bar: 10 µm.\u003cstrong\u003e \u003c/strong\u003e(E) Quantification of mean fluorescence intensity for cleaved Caspase-3 in Fig.3D. ****, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 by unpaired \u003cem\u003et\u003c/em\u003e test.\u003cstrong\u003e \u003c/strong\u003e(F) Representative images of primary mouse hippocampal neurons treated with IL-1β, IL-6, TNF-α or medium alone for 24 h, co-stained for β3-Tubulin (yellow) and cleaved Caspase-3 (purple). Bar: 10 µm\u003cstrong\u003e. \u003c/strong\u003e(G) Quantification of mean fluorescence intensity for cleaved Caspase-3 in Fig.3F. ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 by one-way ANOVA test followed by Dunnett’s post-test.\u003cstrong\u003e \u003c/strong\u003e(H) Western blot of complement C3, TNF-α, IL-6, IL-10, IL-1β and S100A10 in C8-D1A astrocytic cells.\u003cstrong\u003e \u003c/strong\u003e(I) HT22 mouse neuronal cell line exposed to C8-D1A conditioned medium from NH₄Cl-treated or control astrocytes; apoptosis quantified by Annexin-V-APC/7-AAD flow cytometry and apoptotic fractions were defined as Annexin-V⁺/7-AAD⁻ (early) plus Annexin-V⁺/7-AAD⁺ (late). **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 by unpaired \u003cem\u003et\u003c/em\u003etest.\u003cstrong\u003e \u003c/strong\u003e(J) Western blot analysis for cleavedCaspase-3, BAX and BCL2 in HT22 cells after conditioned medium treatment.\u003cstrong\u003e \u003c/strong\u003e(K) The abundance of apoptosis markers in HT22 cells treated with IL-1β, IL-6, TNF-α or medium alone.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9288348/v1/b05bc67557658ffa129c349c.png"},{"id":108492334,"identity":"a6274ca8-d12c-4899-89a1-8165c2dba9de","added_by":"auto","created_at":"2026-05-05 09:57:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":22156153,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTLR4/NF-κB/NLRP3 in astrocytes triggers neuroinflammation and neuronal apoptosis in HE.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic summary showing that ammonia triggers the TLR4/NF-κB/NLRP3 cascade in reactive astrocytes, amplifying neuroinflammation and promoting neuronal apoptosis in HE\u003cstrong\u003e. \u003c/strong\u003e(B–D) Hippocampus from sham or BDL mice co-stained for GFAP with TLR4 (B), p-P65 (C) or NLRP3 (D). \u003cem\u003eLeft\u003c/em\u003e: representative images. Bar: 50µm; \u003cem\u003eRight\u003c/em\u003e: quantification of mean fluorescence intensity for GFAP, TLR4, p-P65, and NLRP3. **, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001; ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 by unpaired \u003cem\u003et\u003c/em\u003etest.\u003cstrong\u003e \u003c/strong\u003e(E) \u003cem\u003eLeft\u003c/em\u003e: Representative immunofluorescence images ofmouse primary astrocytes treated with or without 5 mM NH₄Cl for 48h and stained for GFAP together with TLR4, p-P65 or NLRP3. Bar: 50µm; \u003cem\u003eRight\u003c/em\u003e: quantification of mean fluorescence intensity for TLR4, p-P65, and NLRP3. ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 by unpaired \u003cem\u003et \u003c/em\u003etest.\u003cstrong\u003e \u003c/strong\u003e(F) Astrocytes pre-incubated 8h with 100µM TAK-242 followed by NH₄Cl; Western blots for C3, NLRP3, p-P65, TNF-α, IL-6, IL-1β, IL-10 and S100A10. (G) Representative images of hippocampus from BDL mice given vehicle or TAK-242, co-stained for GFAP with p-P65 or NLRP3. Bar: 50µm.(H) Quantification of mean fluorescence intensity for GFAP, p-P65, and NLRP3. **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 by unpaired \u003cem\u003et\u003c/em\u003e test. (I) ELISA quantification of IL-1β, IL-6, TNF-α and IL-10 of hippocampus from BDL model given vehicle or TAK-242 treatment. *, \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 by t test. **, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 by unpaired \u003cem\u003et\u003c/em\u003e test. (J) The abundance of TNF-α, IL-6, IL-10 and IL-1β in hippocampus of BDL model given vehicle or TAK-242 treatment. (K) Representative images of mouse primary hippocampal neurons labeled for β3-Tubulin (yellow) and cleaved Caspase-3 (purple) and quantification of mean fluorescence intensity for cleaved Caspase-3. Bar: 10µm; ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 by unpaired \u003cem\u003et\u003c/em\u003e test. (L) Flow-cytometric apoptosis detection of HT22 neurons exposed to conditioned medium from C8-D1A astrocytes expressing non-target (NC) or two independent TLR4-shRNAs that had been stimulated with NH₄Cl. ***, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 by one-way ANOVA test followed by Dunnett’s post-test.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9288348/v1/48ee605fa324cc9e5cef80e7.png"},{"id":108494401,"identity":"7b1847bb-4f6b-4047-b3e6-fdec5c3ef0b1","added_by":"auto","created_at":"2026-05-05 10:04:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":19693050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrocytic TLR4 inhibition mitigated neuronal apoptosis and cognitive decline in HE.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) \u003cem\u003eLeft\u003c/em\u003e: Representative images of NeuN immunohistochemistry and Nissl staining of hippocampus from BDL+DMSO and BDL+TAK-242 mice. Bar: 200 µm; \u003cem\u003eRight\u003c/em\u003e: Quantification of neuronal density based on NeuN immunohistochemistry. **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 by unpaired \u003cem\u003et \u003c/em\u003etest. (B) Immunofluorescence of hippocampus from BDL+DMSO and BDL+TAK-242 mice showing GFAP (white), NeuN (orange), cleaved Caspase-3 (red), complement C3 (yellow), and S100A10 (green). Bar: 50 µm. (C) Western blot analysis of hippocampal lysates for cleaved Caspase-3, BAX, and BCL2 in BDL+DMSO and BDL+TAK-242 mice.\u003cstrong\u003e \u003c/strong\u003e(D) \u003cem\u003eLeft\u003c/em\u003e: Representative images of immunofluorescence co-staining of NeuN (green) and cleaved Caspase-3 (red) in hippocampus of BDL+DMSO and BDL+TAK-242 mice. Bar: 200 µm; \u003cem\u003eRight\u003c/em\u003e: Quantification of mean fluorescence intensity for NeuN and cleaved Caspase-3. ***, \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001; ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 by unpaired \u003cem\u003et\u003c/em\u003e test.\u003cstrong\u003e \u003c/strong\u003e(E) \u003cem\u003eLeft\u003c/em\u003e: Representative Y-maze exploration traces for BDL+DMSO and BDL+TAK-242 mice (novel arm indicated by red box); \u003cem\u003eRight\u003c/em\u003e: percentage of entries in the novel arm. ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 by unpaired \u003cem\u003et\u003c/em\u003e test.\u003cstrong\u003e \u003c/strong\u003e(F) \u003cem\u003eLeft\u003c/em\u003e: Representative NOR test traces for BDL+DMSO and BDL+TAK-242 mice (novel object indicated by square); \u003cem\u003eRight\u003c/em\u003e: Bar plot of recognition index. ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 by unpaired \u003cem\u003et\u003c/em\u003e test.\u003cstrong\u003e \u003c/strong\u003e(G) \u003cem\u003eLeft\u003c/em\u003e: Representative images of NeuN immunohistochemistry and Nissl staining of hippocampus from \u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand\u003cem\u003e GFAP-Cre;TLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice after 4-week BDL. Bar: 200 µm; \u003cem\u003eRight\u003c/em\u003e: Quantification of neuronal density based on NeuN immunohistochemistry. ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 by unpaired \u003cem\u003et\u003c/em\u003e test. (H) The abundance of NLRP3, p-P65, TNF-α, IL-6, IL-10, IL-1β in hippocampal lysates from \u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eGFAP-Cre;TLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice.\u003cstrong\u003e \u003c/strong\u003e(I) The abundance of cleaved Caspase-3, BAX and BCL2 in hippocampal lysates from \u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eGFAP-Cre;TLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice.\u003cstrong\u003e \u003c/strong\u003e(J) \u003cem\u003eLeft\u003c/em\u003e: Representative image of immunofluorescence co-staining of NeuN (green) and cleaved Caspase-3 (red) in hippocampus of \u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand\u003cem\u003e GFAP-Cre;TLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice after 4-week BDL. Bar: 200 µm; \u003cem\u003eRight\u003c/em\u003e: Quantification of mean fluorescence intensity for NeuN and cleaved Caspase-3. **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 by unpaired \u003cem\u003et\u003c/em\u003e test.\u003cstrong\u003e \u003c/strong\u003e(K) Representative TEM images of hippocampal astrocytes and neurons in \u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand\u003cem\u003e GFAP-Cre;TLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice after 4-week BDL. Bar: 5 µm.\u003cstrong\u003e \u003c/strong\u003e(L) Representative Y-maze exploration and NOR test traces for \u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand\u003cem\u003e GFAP-Cre;TLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice after 4-week BDL (novel arm indicated by red box and novel object indicated by square).\u003cstrong\u003e \u003c/strong\u003e(M) Bar plot of percentage of entries in the novel arm and recognition index. **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 by unpaired \u003cem\u003et\u003c/em\u003e test.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9288348/v1/6a313b808450edc7efc40a14.png"},{"id":108494603,"identity":"d02fe4d4-13ab-4f59-a9d0-43bdb9cc9f45","added_by":"auto","created_at":"2026-05-05 10:05:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1025351,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSimvastatin targets astrocytic TLR4 to attenuate neuroinflammation and rescue HE cognition.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Gene Set Enrichment Analysis (GSEA) of RNA-seq data from HE patients versus healthy controls (dataset GSE57193) using the HALLMARK gene-set collection.\u003cstrong\u003e \u003c/strong\u003e(B) Pearson correlation between neuroinflammatory response and neuronal apoptosis gene-set GSVA scores in HE patients (datasets GSE57193 and GSE41919). \u003cem\u003ep\u003c/em\u003e = 0.015 by two-tailed cor.test.\u003cstrong\u003e \u003c/strong\u003e(C) Molecular docking of human TLR4 with dexamethasone, methylprednisolone, candesartan, and simvastatin.\u003cstrong\u003e \u003c/strong\u003e(D) Molecular-dynamics simulation of human TLR4 with candesartan and simvastatin.\u003cstrong\u003e \u003c/strong\u003e(E) The abundance of C3, NLRP3, p-P65, TNF-α, IL-6, and IL-1β in C8-D1A astrocytes following treatment with NH\u003csub\u003e4\u003c/sub\u003eCl and subsequent exposure to candesartan or simvastatin.\u003cstrong\u003e \u003c/strong\u003e(F) The abundance of C3, NLRP3, p-P65, TNF-α, IL-6, and IL-1β in human cerebral organoids following treatment with NH\u003csub\u003e4\u003c/sub\u003eCl and subsequent exposure to simvastatin.\u003cstrong\u003e \u003c/strong\u003e(G) Representative Y-maze exploration and NOR test traces for\u003cem\u003e TLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e + simvastatin, \u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e + rifaximin, \u003cem\u003eGFAP-Cre;TLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003csup\u003e\u0026nbsp;\u003c/sup\u003e+ rifaximin, and \u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e + simvastatin + rifaximin mice after 4-week BDL.\u003cstrong\u003e \u003c/strong\u003e(H) Bar plot of percentage of entries in the novel arm and recognition index. *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 by one-way \u003cem\u003eANOVA\u003c/em\u003e followed by Holm–Šídák post-test.\u003cstrong\u003e \u003c/strong\u003e(I) The working model in this study. \u003cem\u003eTop:\u003c/em\u003e Excessive ammonia activates the TLR4/NF-κB/NLRP3 axis in hippocampal astrocytes, triggering reactive astrogliosis and inflammatory cytokine release that precipitate neuronal apoptosis and cognitive decline. \u003cem\u003eBottom:\u003c/em\u003e Simvastatin disrupts this cascade by dampening astrocyte reactivity, thereby reducing neuroinflammation and rescuing cognitive impairment in the HE model.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-9288348/v1/b9d2f5167186900b0997207f.png"},{"id":108497926,"identity":"c3771c3e-2cbd-49d5-922a-17d6ee384bf1","added_by":"auto","created_at":"2026-05-05 10:14:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":56355688,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9288348/v1/c796e7cd-2365-456f-ac1f-5f1bb2da2261.pdf"},{"id":108494404,"identity":"c95af22c-ed4f-4241-884e-bd677519b3ec","added_by":"auto","created_at":"2026-05-05 10:05:04","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16405,"visible":true,"origin":"","legend":"Table S3","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9288348/v1/e9f3398c532d86dbf8599df1.xlsx"},{"id":108494910,"identity":"a4da15d9-9168-489f-bfad-f850769003d2","added_by":"auto","created_at":"2026-05-05 10:07:56","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":24304,"visible":true,"origin":"","legend":"Table S5","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9288348/v1/7894fcfbd1f0c14af91ede73.xlsx"},{"id":108494629,"identity":"b4071a9f-1fdb-4f69-8fa0-f3ce1a9830b5","added_by":"auto","created_at":"2026-05-05 10:06:09","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":43167,"visible":true,"origin":"","legend":"Table S1","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9288348/v1/e10b90f1805f203ad8633228.xlsx"},{"id":108492337,"identity":"f7777feb-7af2-4f96-8b26-08f1e7cc6cd0","added_by":"auto","created_at":"2026-05-05 09:57:30","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":19290,"visible":true,"origin":"","legend":"Table S4","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9288348/v1/29e94b5b278e0461d5f26c59.xlsx"},{"id":108494461,"identity":"1ce95d34-8bd4-4118-8a96-7f9b56351733","added_by":"auto","created_at":"2026-05-05 10:05:31","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1224034,"visible":true,"origin":"","legend":"supplementary figures","description":"","filename":"supplementaryfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-9288348/v1/b1ffeb0d8a77ef98b1c6995e.docx"},{"id":108494456,"identity":"db0361ec-43cb-4e68-afef-cd899a3d3585","added_by":"auto","created_at":"2026-05-05 10:05:27","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":59329,"visible":true,"origin":"","legend":"Table S2","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9288348/v1/9845a8c94991e56f8d1819bf.xlsx"},{"id":108494616,"identity":"604ef900-f472-46df-ab6b-fb0dd29a4963","added_by":"auto","created_at":"2026-05-05 10:06:05","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":10699,"visible":true,"origin":"","legend":"Table S6","description":"","filename":"TableS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9288348/v1/3f4c161a91a967bfc9ec4c0b.xlsx"},{"id":108492361,"identity":"f9509a91-183f-4b9c-9731-af043977d1f4","added_by":"auto","created_at":"2026-05-05 09:57:35","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":64883732,"visible":true,"origin":"","legend":"full uncropped Gels and Blots images","description":"","filename":"fulluncroppedGelsandBlotsimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9288348/v1/5a7df7a7f4ca9766671bddb2.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Astrocyte-Dependent Neuroinflammation Triggers Hippocampal Neuronal Apoptosis through the TLR4/NF-κB/NLRP3 Axis in Hepatic Encephalopathy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatic encephalopathy (HE) is a complex neuropsychiatric syndrome that develops in patients with acute or chronic liver dysfunction or portal shunt(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), manifested by a spectrum of cognitive and consciousness dysfunction ranging from subtle attention loss to coma and death(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). These symptoms are thought to arise from the combined effects of ammonia accumulation, oxidative stress, astrocyte swelling, cerebral edema, inflammation, and mitochondrial dysfunction. Current managements of HE targets lowering blood ammonia. Lactulose or Rifaximin is used to curb intestinal ammonia production and branched-chain amino acids or L-ornithine-L-aspartate is employed to enhance systemic ammonia removal(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), yet clinical improvement is limited. Consequently, there is an urgent need to identify effective therapeutic targets for cognitive impairment in HE.\u003c/p\u003e \u003cp\u003eSustained hyperammonemia disrupts the structure and function of neurons, astrocytes, microglia, and endothelial cells in HE(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Previous studies have suggested that the neuropsychiatric symptoms of HE are mainly associated with impaired glial cell function(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Within the glial lineage, astrocytes are the first and principal cerebral targets under hyperammonemia because of the dominant potassium channels and glutamine synthetase(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Excessive ammonia leads to glutamine accumulation in astrocytes, which in turn increases intracellular osmotic pressure, causes astrocyte swelling and cerebral edema(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Additionally, astrocytes play a critical role in maintaining the blood-brain barrier (BBB). The distortion of swollen astrocytic endfeet compromises BBB integrity, thereby promoting HE pathogenesis(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). More recently, accumulating research has revealed that astrocytes may also contribute to HE progression through the induction of neuroinflammation(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Specifically, excessive ammonia triggers astrocyte reactivity that generates reactive oxygen and nitrogen species which elevates IL-1β, IL-6 and prostaglandin E2, and exacerbates neuroinflammation and neurotoxicity(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Importantly, astrocytes are essential for normal neuronal function(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). However, the mechanisms through which the reactive astrogliosis impairs neurons and ultimately contributes to cognitive impairment in HE remains elusive.\u003c/p\u003e \u003cp\u003eToll-like receptor 4 (TLR4), a key pattern recognition receptor in the innate immune system, is widely expressed in astrocytes and plays a pivotal role in initiating and amplifying inflammatory responses(\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Activated TLR4 further triggers the nuclear factor-κB (NF-κB) signaling pathway, promoting the nuclear translocation of NF-κB p65 subunit and transcription of pro-inflammatory cytokine genes (e.g., IL-1β, IL-6). More importantly, TLR4/NF-κB activation can further induce the assembly and activation of the NLRP3 inflammasome, a key mediator of pro-inflammatory cytokine release, which amplifies astrocyte-derived neuroinflammation(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Despite the established role of the TLR4/NF-κB/NLRP3 axis in neurological disorders including intracerebral hemorrhage, Alzheimer's disease, and Parkinson's disease(\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), its precise role in hyperammonemia-driven reactive astrogliosis and the subsequent neuronal injury in HE remains unclear, which constitutes a critical research gap addressed in the present study.\u003c/p\u003e \u003cp\u003eIn this study, we established two classic models of HE, observing both cognitive deficits and significant hippocampal neuronal apoptosis. Mechanistically, hyperammonemia activates the reactive astrocytic TLR4/NF-κB/NLRP3 axis, triggering pro-inflammatory factor release and subsequent neuronal apoptosis. Through simulated molecular docking, we identified simvastatin as a TLR4-targeting agent. It effectively inhibited hyperammonemia-induced astrocyte activation and neuronal apoptosis in both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models. Thus, our findings establish hyperammonemia-driven TLR4/NF-κB/NLRP3 signaling in astrocytes as a key driver of hippocampal neuronal apoptosis and cognitive impairment in HE.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eAdult (6\u0026ndash;8 weeks old) male wild-type C57BL/6 J mice, \u003cem\u003eGFAP-Cre\u003c/em\u003e; \u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice (generated by crossing \u003cem\u003eGFAP-Cre\u003c/em\u003e mice with \u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice; Cyagen Biosciences Inc., Suzhou, China), \u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice, and Sprague\u0026ndash;Dawley (SD) rats were used. Wild-type mice and SD rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animals were housed under specific pathogen-free (SPF) conditions (12-h light/dark cycle, 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity) with free access to standard chow and sterile water. All procedures using laboratory animals were approved by and conducted consistently the guidelines of the Laboratory AnimalWelfare and Ethics Committee Of the Army Medical University (approval No. AMUWEC20255435).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture and treatment\u003c/h3\u003e\n\u003cp\u003eMouse neuronal line HT22, astrocyte line C8-D1A (Procell Life Science \u0026amp; Technology Co., Ltd., Wuhan, China), and 293T cells (ATCC) were cultured in high-glucose DMEM (4.5 g/L glucose) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37\u0026deg;C/5% CO₂, passaged every 2\u0026ndash;3 days at ~\u0026thinsp;70% confluence.\u003c/p\u003e \u003cp\u003ePrimary astrocytes were isolated from newborn (\u0026le;\u0026thinsp;48 h) C57BL/6 J mice or SD rats. Hippocampi were dissected, minced, digested with 0.25% trypsin-EDTA (15 min, 37\u0026deg;C), filtered (70 \u0026micro;m), and plated in DMEM/F-12\u0026thinsp;+\u0026thinsp;10% FBS. After 10\u0026ndash;12 days, microglia and oligodendrocytes were removed by orbital shaking (200 rpm, 24 h). Astrocytes were trypsinised (0.05% trypsin-EDTA, 3 min) and re-seeded onto poly-D-lysine (PDL, 10 \u0026micro;g/ml)-coated plates, used within two passages.\u003c/p\u003e \u003cp\u003ePrimary neurons were isolated from E16\u0026ndash;18 C57BL/6 J mouse or SD rat embryos. Hippocampi were processed as above, digested with 0.125% trypsin-EDTA (5 min), filtered (40 \u0026micro;m), and plated at 4\u0026ndash;6 \u0026times; 10⁴ cells/cm\u0026sup2; on PDL-coated plates in Neurobasal-A\u0026thinsp;+\u0026thinsp;2% B-27\u0026thinsp;+\u0026thinsp;1% GlutaMAX\u0026thinsp;+\u0026thinsp;1% antibiotic\u0026ndash;antimycotic. Half-medium changes were performed every 3 days.\u003c/p\u003e \u003cp\u003eTAK-242 (TLR4 antagonist) was dissolved in 0.1% DMSO (250 \u0026micro;M) and diluted to 100 \u0026micro;M in culture medium. Astrocytic cultures (C8-D1A or primary) were pre-treated with 100 \u0026micro;M TAK-242 for 8 h, followed by 5 mM NH₄Cl for 48 h. Vehicle controls received 0.1% DMSO. Supernatants from treated astrocytes were diluted 1:1 with phenol-red-free Neurobasal-A\u0026thinsp;+\u0026thinsp;2% B-27 and applied to HT22 or primary neurons for 24 h. For cytokine challenge, neurons were treated with 10 ng/ml IL-1β, IL-6, TNF-α, or vehicle for 24 h. All conditions were tested in triplicate.\u003c/p\u003e\n\u003ch3\u003eEstablishment of HE animal models\u003c/h3\u003e\n\u003cp\u003eHE models were established by bile duct ligation (BDL) in mice or intraperitoneal (i.p.) injection of 150 mg/kg thioacetamide (TAA) in rats. For BDL: mice were anaesthetised with sevoflurane (3\u0026ndash;4% in 1 L/min O₂), a mid-line laparotomy was performed, and the common bile duct was ligated with 4\u0026thinsp;\u0026minus;\u0026thinsp;0 silk (proximally and at the pancreatic border) without transection. Sham-operated mice underwent identical manipulation without ligation. In accordance with the experimental protocol, WT C57BL/6 J mice received intraperitoneal injections of TAK-242 (3 mg/kg every other day) or its vehicle. In parallel, \u003cem\u003eGFAP-Cre\u003c/em\u003e; \u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice or \u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice were treated for 4 weeks starting post-surgery: daily gavage with vehicle (PBS with 0.2% SDS), rifaximin (50 mg/kg), or daily intraperitoneal injection of simvastatin (0.2 mg\u0026middot;kg⁻\u0026sup1;), as well as the corresponding combined treatment. For TAA model: rats received TAA (in sterile 0.9% saline) once daily for 3 consecutive days; controls received saline. 5% glucose in 0.9% saline was administered orally (25 ml/kg) 12 h after the first TAA injection. Rats were pre-treated with TAK-242 (3 mg/kg i.p.) or vehicle every other day for 3 injections before TAA administration.\u003c/p\u003e\n\u003ch3\u003eH\u0026E staining\u003c/h3\u003e\n\u003cp\u003eAnimals were euthanized by cervical dislocation after sodium pentobarbital anaesthesia (100 mg/kg i.p.). Livers were fixed in 4% neutral-buffered formalin, dehydrated, cleared in xylene, and embedded in paraffin. 4-\u0026micro;m sections were stained with hematoxylin and eosin, examined under a bright-field microscope (Olympus, Japan).\u003c/p\u003e\n\u003ch3\u003eNissl staining\u003c/h3\u003e\n\u003cp\u003e After behavioural tests, mice/rats (n\u0026thinsp;=\u0026thinsp;5 per group) were transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4). Brains were post-fixed in 4% PFA (4\u0026deg;C, 24 h), dehydrated, cleared, and embedded in paraffin. 5-\u0026micro;m coronal sections were deparaffinized, rehydrated, stained with 0.1% cresyl violet (Servicebio, G1036) for 8 min, differentiated in 95% ethanol, cleared in xylene, and coverslipped. Hippocampal CA1 neurons were imaged (\u0026times;40 objective) and counted using Fiji ImageJ v1.53.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGolgi staining\u003c/h2\u003e \u003cp\u003eBrains were dissected and immersed in Golgi-Cox OptimStain\u0026trade; Kit solutions A\u0026thinsp;+\u0026thinsp;B (Hitobiotec, HTKNS1125) in the dark (RT, 14 days), then transferred to solution C (72 h, RT). 100-\u0026micro;m coronal sections were cut, air-dried, developed with solutions D:E:ddH₂O (1:1:2), dehydrated, cleared, and coverslipped. Neurons were imaged (200\u0026times; for Sholl analysis, 1000\u0026times; for spine density) with Fiji ImageJ (experimenter-blinded).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNeuN immunohistochemistry\u003c/h3\u003e\n\u003cp\u003eSections were prepared as for Nissl staining. Endogenous peroxidase was blocked with 3% H₂O₂ in methanol (15 min, RT). Antigen retrieval was performed in 10 mM sodium citrate (pH 6.0, 95\u0026deg;C, 10 min). Sections were blocked with 5% normal goat serum\u0026thinsp;+\u0026thinsp;0.3% Triton X-100 (1 h, RT), incubated with rabbit anti-NeuN (4\u0026deg;C, overnight), followed by biotinylated goat anti-rabbit IgG (1 h, RT) and VECTASTAIN Elite ABC-HRP kit (Vector PK-6100). Immunoreactivity was visualized with 0.05% DAB\u0026thinsp;+\u0026thinsp;0.01% H₂O₂. Sections were counter-stained with hematoxylin, dehydrated, and coverslipped. NeuN-positive cells were counted (\u0026times;40 objective) using Fiji ImageJ.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eParaffin sections were deparaffinized, rehydrated, fixed in 10% neutral buffered formalin (10 min), and antigen-retrieved (citrate buffer, pH 6.0, microwave). Sections were permeabilized with 0.3% Triton X-100 (30 min), blocked with 5% BSA (1 h, RT), and incubated with primary antibody pairs (4\u0026deg;C, overnight): rabbit anti-NeuN (Thermo Fisher Scientific, PA5-78499) + rabbit anti-cleaved Caspase-3 (Cell Signaling Technology, 9661); mouse anti-GFAP (ServiceBio, GB12096) + rabbit anti-TLR4 (Thermo Fisher Scientific, PA5-23124), p-P65 (Cell Signaling Technology, 3033) or NLRP3 (ServiceBio, GB114320). After washing, species-matched Alexa-Fluor-conjugated secondary antibodies (Goat Anti-Mouse IgG H\u0026amp;L (Alexa Fluor\u0026reg; 488), ab150113; Goat Anti-Rabbit IgG H\u0026amp;L (Alexa Fluor\u0026reg; 594), ab150080) were applied (1 h, RT, dark). Sections were counter-stained with DAPI (1 \u0026micro;g/ml, 10 min) and mounted. Images were acquired (Olympus BX53) and analysed (Fiji ImageJ, blinded).\u003c/p\u003e \u003cp\u003eFor six-colour multiplex immunofluorescence, 5-\u0026micro;m sections were stained with NeuN (Thermo Fisher Scientific, PA5-78499), cleaved Caspase 3 (Cell Signaling Technology, 9661), GFAP (ServiceBio, GB12096), C3 (Thermo Fisher Scientific, PA5-21349), S100A10 (Thermo Fisher Scientific, PA5-95505) and DAPI using the TG TSA Multiplex IHC Assay Kit (TissueGnostics, TGFP550) and imaged on a TissueFAXS Cytometry platform.\u003c/p\u003e \u003cp\u003eFor cell immunofluorescence, primary astrocytes and neurons on coverslips were fixed with 4% PFA (15 min, RT), permeabilized with 0.3% Triton X-100, blocked with 5% BSA (1 h), and incubated with primary antibody pairs (4\u0026deg;C, overnight): mouse anti-GFAP (ServiceBio, GB12096) + rabbit anti-TLR4 (Thermo Fisher Scientific, PA5-23124), p-P65 (Cell Signaling Technology, 3033) or NLRP3 (ServiceBio, GB114320); mouse anti-β3-Tubulin (Cell Signaling Technology, 4466), rabbit anti-cleaved Caspase-3 (Cell Signaling Technology, 9661). Secondary antibodies were applied (1 h, RT), nuclei counter-stained with DAPI, and images acquired on a Leica TCS SP8 confocal microscope (\u0026times;40 oil-immersion objective).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy\u003c/h2\u003e \u003cp\u003eHippocampal blocks (1 mm\u0026sup3;) were fixed in 2.5% glutaraldehyde (4\u0026deg;C, overnight), post-fixed in 1% OsO₄ (1 h), dehydrated, and embedded in Epon-812. 60-nm ultrathin sections were stained with 3% uranyl acetate and lead citrate, observed under a JEM-1200EX transmission electron microscope (JEOL, Japan) at 8000\u0026ndash;15000\u0026times;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eHippocampal tissues, cells, and organoids were lysed in RIPA buffer (Yeasen, 20101ES60) supplemented with 1 mM PMSF (ServiceBio, G2008) and 1\u0026times; phosphatase inhibitor cocktail (Millipore Sigma, 4906837001) (100:1:1). Lysates were rotated (4\u0026deg;C, 30 min), centrifuged (12000\u0026times;g, 30 min, 4\u0026deg;C), and protein concentration determined by BCA assay (Beyotime, P0010). 30 \u0026micro;g protein was separated by 10%/12% SDS-PAGE, transferred to 0.45 \u0026micro;m PVDF membranes (Merck Millipore, USA), blocked with 5% non-fat dry milk (1 h, RT), and incubated with primary antibodies (4\u0026deg;C, overnight): cleaved Caspase-3 (Cell Signaling Technology, 9661), Caspase-3 (Cell Signaling Technology, 9662), BAX (ServiceBio, GB15690), BCL2 (Cohesion Biosciences, CPA1095), α-Tubulin (Abclonal, A6830), TNF-α (ServiceBio, GB11188), IL-6 (Affinity Biosciences, DF6087), IL-10 (Abclonal, A2171), IL-1β (Abcam, ab283818), C3 (Thermo Fisher Scientific, PA5-21349), S100A10 (Thermo Fisher Scientific, PA5-95505), TLR4 (Thermo Fisher Scientific, PA5-23124), NF-κB p65 (Cell Signaling Technology, 8242), Phospho-NF-κB p65 (Cell Signaling Technology, 3033), NLRP3 (ServiceBio, GB114320). After washing, HRP-conjugated secondary antibodies (goat anti-mouse or goat anti-rabbit, 1:10000) were applied (1 h, RT). Bands were visualized with ECL reagent (Servicebio, G2014) and quantified with Fiji ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLiver function detection\u003c/h2\u003e \u003cp\u003eOrbital venous blood was collected after anaesthesia, kept on ice (4\u0026deg;C), clotted, and centrifuged (3000\u0026times;g, 15 min, 4\u0026deg;C). Serum ALT (C009-2-1), AST (C010-2-1), TBIL (C019-1-1), and DBIL (C019-2-1) levels were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute) per manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAmmonia level determination\u003c/h2\u003e \u003cp\u003eBlood and hippocampal homogenates (1:9 w/v in ice-cold saline) were centrifuged (3500\u0026times;g, 15 min, 4\u0026deg;C). Ammonia levels in supernatants were assayed with the Ammonia/Ammonium Microplate Assay Kit (Absin, abs580164) per manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eHippocampal tissues were homogenised in PBS\u0026thinsp;+\u0026thinsp;protease inhibitor cocktail, centrifuged (12000\u0026times;g, 30 min, 4\u0026deg;C). Supernatant IL-1β, IL-6, IL-10, and TNF-α levels were measured using commercial ELISA kits (Mouse IL-1 beta: Thermo Fisher Scientific, BMS6002-2; Mouse IL-6: Thermo Fisher Scientific, BMS603-2HS; Mouse IL-10: Thermo Fisher Scientific, BMS614; Mouse TNF alpha: Thermo Fisher Scientific, BMS607-3; Rat IL-1β: Elabscience, E-EL-R0012; Rat IL-6: Elabscience, E-EL-R0015; Rat IL-10: Elabscience, E-EL-R0016; Rat TNF-α: Elabscience, E-EL-R2856) per manufacturer\u0026rsquo;s instructions, normalized to total protein content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eY-Maze test\u003c/h2\u003e \u003cp\u003eY-maze test assessed cognitive function and spatial reference memory in HE models. The 120\u0026deg; three-arm maze included three phases: 30-min individual habituation, training (blocked arm; 10 min/rats or 8 min/mice exploration of accessible arms, 1-h interval), and testing (novel arm opened; 5-min exploration of all three arms from the original starting arm). Arm entry times were recorded and analyzed with a Smart Video Tracking System (Stoelting Co., USA) for memory evaluation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eNovel object recognition test\u003c/h2\u003e \u003cp\u003eNovel Object Recognition test (exploiting rodents' novelty preference) assessed recognition memory and cognitive function. Setup included an open-field chamber and HD camera linked to EthoVision XT 17.0 (Noldus, Netherlands). After 3 days of 5-min habituation, animals underwent 5-min familiarization (two identical objects, A\u0026thinsp;+\u0026thinsp;A), followed by 1-h delay and 5-min test (one object replaced with a novel square, A\u0026thinsp;+\u0026thinsp;B). Exploration time/tracks were recorded via EthoVision. Recognition memory was quantified by recognition index [(Novel time/Total exploration time) \u0026times;100%] and discrimination index [(Novel\u0026minus;Familiar time)/Total exploration time\u0026times;100%], with higher values indicating better memory.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFlow-cytometric apoptosis assay\u003c/h2\u003e \u003cp\u003eHT22 neurons (2 \u0026times; 10⁵ cells/well) were treated, harvested, centrifuged (300\u0026times;g, 5 min, 4\u0026deg;C), and resuspended in 1\u0026times; binding buffer (1 \u0026times; 10⁶ cells/ml). 100 \u0026micro;l cell suspension was incubated with 5 \u0026micro;l APC-conjugated Annexin-V and 10 \u0026micro;l 7-AAD (Annexin V-APC/7-AAD Apoptosis Analysis Kit, Absin, abs50008) (15 min, RT, dark). 400 \u0026micro;l binding buffer was added, and samples analysed on a BD FACSCanto II flow cytometer. Apoptotic fractions (Annexin-V⁺/7-AAD⁻ + Annexin-V⁺/7-AAD⁺) were quantified with FlowJo v10.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eshRNA-mediated TLR4 knockdown\u003c/h2\u003e \u003cp\u003eThree shRNAs targeting mouse TLR4 (NM_021297) were designed: shTLR4-1 (5\u0026prime;-CCTGTAAGTTACCTGCATATT-3\u0026prime;), shTLR4-2 (5\u0026prime;-CCCTCCATAGACTTCAATTAT-3\u0026prime;), shTLR4#3 (5\u0026prime;-TAGAGGTAGTTCCTAATATTA-3\u0026prime;) and a non-targeting control shNC (5\u0026prime;-CCTAAGGTTAAGTCGCCCTCG-3\u0026prime;). Lentiviruses were produced for the two most effective shRNAs (shTLR4#1 and shTLR4#2) and shNC. C8-D1A astrocytes were transduced (MOI\u0026thinsp;\u0026asymp;\u0026thinsp;5, 8 \u0026micro;g/ml polybrene, 12 h) and selected with 0.5 \u0026micro;g/ml puromycin (7 days). Knockdown efficiency was verified by Western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eBulk RNA-Seq and analysis\u003c/h2\u003e \u003cp\u003eHippocampal tissues from Sham/BDL mice and Ctrl/TAA rats (n\u0026thinsp;=\u0026thinsp;5 per group) were collected. Total RNA (RIN\u0026thinsp;\u0026gt;\u0026thinsp;7.0, 28S:18S\u0026thinsp;\u0026gt;\u0026thinsp;1.8) was isolated with TRIzol (Invitrogen). Poly(A) mRNA was enriched with NEB Next Poly(A) mRNA Magnetic Isolation Module, fragmented, and reverse transcribed. Libraries were constructed with NEB Next Ultra RNA Library Prep Kit for Illumina and sequenced on an Illumina NovaSeq 6000 (2 \u0026times; 150 bp). Reads were trimmed with Trimmomatic v0.36, aligned to genomes (GRCm38/Rnor_6.0) with STAR, quantified by featureCounts, and filtered (average TPM\u0026thinsp;\u0026lt;\u0026thinsp;0.1). Differentially expressed genes (DEGs) were identified with edgeR (|logFC| \u0026gt; 0.585, FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and analysed by Metascape and GSEA (clusterProfiler).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003escRNA/snRNA-seq and analysis\u003c/h2\u003e \u003cp\u003eHippocampal single-cell/single-nucleus suspensions were prepared for 10\u0026times; Genomics Chromium capture. Libraries were constructed with Chromium Single Cell 3\u0026prime; Reagent Kits v3.1 and sequenced on Illumina NovaSeq 6000. Data were processed with Seurat: genes expressed in \u0026ge;\u0026thinsp;3 cells and cells with \u0026ge;\u0026thinsp;500 features were retained; doublets removed with DoubletFinder; batches aligned with Harmony. PCA, graph-based clustering, and UMAP embedding were performed. Cluster markers were identified with FindAllMarkers, and GSEA was conducted with clusterProfiler.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eHuman brain transcriptome analysis\u003c/h2\u003e \u003cp\u003eGSE41919 and GSE57193 datasets were downloaded from the Gene Expression Omnibus (GEO). GSE41919 initially contained 8 post-mortem brain samples from HE patients and 8 non-cirrhosis controls (GPL14550). Applying the sample-exclusion criteria of Hsu et al., 6 specimens that crossed the phenotypic boundary in hierarchical clustering and PCA (GSM1027454, GSM1027458, GSM1027462, GSM1027465, GSM1027467, GSM1027468) were removed, leaving 6 HE and 4 control samples. GSE57193 included 4 HE and 4 control brains (same platform) and was analysed separately without additional sample exclusion. DEGs were identified with limma (adjusted P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, | log₂foldchange | \u0026gt; 0.585). GSEA was performed with hallmark gene sets. Neuroinflammatory and apoptotic signatures were quantified by GSVA v1.46, and Pearson correlation was computed.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eOrganoid culture and differentiation\u003c/h2\u003e \u003cp\u003ehPSCs were differentiated into cerebral organoids. EBs were formed in 96-well round-bottom plates (9,000 cells/well) with EB differentiation medium\u0026thinsp;+\u0026thinsp;10 \u0026micro;M Y-27632. Neuroectodermal induction was performed on day 5, EBs embedded in Matrigel (Corning, 354253) on days 7\u0026ndash;10, and maturation initiated on day 10 with orbital shaking (65 rpm). Mature organoids (day 40) were exposed to 5 mM NH₄Cl or 5 \u0026micro;M simvastatin. Organoids were released with Cell Recovery Solution (Corning, 354253) for protein extraction.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eDrug prediction, docking and molecular dynamics\u003c/h2\u003e \u003cp\u003eTLR4 was uploaded to DSigDB via Enrichr to identify candidate drugs (FDA/EMA-approved, blood-brain-barrier permeable, adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01). TLR4 structure (PDB) and compound structures (PubChem) were downloaded. Docking was performed with AutoDock Vina and compounds with a binding energy \u0026lt; -5 kcal/mol were selected for subsequent molecular dynamics simulations. Molecular dynamics of TLR4-candesartan/simvastatin complexes were evaluated with GROMACS 2020.6 (100 ns production run). Complex stability was analysed with VMD and PyMOL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eQuantitative data were analysed with GraphPad Prism 10, presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Two-group comparisons used unpaired \u003cem\u003et\u003c/em\u003e-test; multi-group comparisons used one-way \u003cem\u003eANOVA\u003c/em\u003e followed by \u003cem\u003eDunnett\u0026rsquo;s or Holm\u0026ndash;Š\u0026iacute;d\u0026aacute;k\u003c/em\u003e post-test. Significance levels are indicated in figure legends. Sample sizes were determined by independent experiments with multiple replicates.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eConvergent Evidence from BDL and TAA Models Identifies Hippocampal Neuronal Apoptosis as a Hallmark of HE\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe established a classic bile duct ligation (BDL) mouse model of HE in C57BL/6J mice, in which chronic hyperammonemia induced by surgical bile duct ligation led to HE after four weeks. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Histological analysis of BDL model livers revealed severe structural injury, including extensive necrosis, steatosis, and inflammatory infiltrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Consistent with this structural damage, hepatic function was significantly compromised, as indicated by marked increases in AST, ALT, TBil, and DBil (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Furthermore, the systemic and central effects of the model were confirmed by elevated ammonia levels in both the blood and the brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Cognitive impairment is a well-established hallmark of HE. To assess this in our model, we subjected BDL mice to Y-maze and novel-object recognition (NOR) tests. The BDL mice exhibited significant spatial working memory deficits in the Y-maze, as evidenced by reduced entries into and less time spent in the novel arm, indicating a decreased preference for novelty (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Consistently, in the NOR test, the recognition and discrimination indices were significantly lower in BDL mice, confirming a general recognition memory deficit (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These results collectively demonstrate that BDL mice develop robust cognitive impairment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe hippocampus serves as a central hub for cognitive processing, and damage to hippocampal neurons is a critical factor in the development of cognitive impairment(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Nissl staining of our BDL model hippocampus showed neuronal loss and damage features, including neuronal shrinkage, darkened staining, and irregular contours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), as well as the similar phenotype was observed in NeuN immunohistochemistry (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Structural integrity was further compromised at the single neuron level, as Golgi staining revealed simplified dendritic arbors and decreased spine density (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK), collectively pointing to a profound disruption of neuronal connectivity that underlies the observed cognitive deficits. Ultrastructural evidence of neuronal apoptosis was observed via transmission electron microscopy (TEM), as BDL hippocampal neurons displayed classic morphological changes, including electron-dense cytoplasm, clumped chromatin margination, and cellular shrinkage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). Consistent with these morphological findings, we demonstrated a robust activation of the intrinsic apoptotic pathway, characterized by increased abundance of cleaved Caspase-3 and BAX, and a decrease in BCL2 in BDL hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM). Further supporting this, immunofluorescence revealed a significant decrease in NeuN signal intensity concomitant with an increase in cleaved Caspase-3 intensity within the hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN). Our results indicated ongoing apoptosis and neuronal loss in BDL mice.\u003c/p\u003e \u003cp\u003eTo extend these findings, we generated another complementary HE model in SD rats via intraperitoneal injection of thioacetamide (TAA) (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). This paradigm induced severe hepatocellular injury, as shown by extensive necrosis and inflammatory infiltration (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB), elevated serum markers of liver damage (AST, ALT, TBil, DBil), and increased hippocampal ammonia (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC and S1D). TAA-exposed rats recapitulated the cognitive deficits observed in BDL mice, exhibiting impaired performance in both Y-maze and NOR tests (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE and S1F). Mirroring the hippocampal pathology, NeuN immunostaining confirmed neuronal loss in the hippocampus of TAA‑treated rats (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG). Ultrastructural analysis by TEM further revealed apoptotic morphology in hippocampal neurons, including electron-dense cytoplasm and chromatin condensation (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eH). Consistently, immunofluorescence demonstrated decreased NeuN signal alongside increased cleaved Caspase-3 intensity (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eI). Together, the convergent findings from BDL and TAA models robustly establish hippocampal neuronal apoptosis as a conserved pathological hallmark in HE.\u003c/p\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003eA1 Reactive Astrocytes Drive Hippocampal Neuronal Apoptosis in HE\u003c/h2\u003e \u003cp\u003eTo identify key pathways driving neuronal apoptosis in HE, we conducted bulk RNA-seq on hippocampi from mice model. The enrichment pathways of differentially expressed genes highlighted significant involvement of the acute inflammatory response, neuronal apoptosis regulation, and interleukin-6-family signaling in BDL model (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Notably, gene set enrichment analysis (GSEA) revealed significant up-regulation of inflammatory and neuroinflammation gene sets, alongside down-regulation of neuron development and projection signatures in BDL hippocampi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), indicating prominent neuroinflammation. Consistent with this transcriptomic profile, western blot and ELISA confirmed elevated pro-inflammatory cytokines IL-1β, IL-6, TNF-α and reduced anti-inflammatory IL-10 in BDL hippocampi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), underscoring a neuroinflammatory microenvironment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAstrocytes, the most abundant glial cells in the central nervous system, rapidly undergo molecular and structural remodeling when the brain microenvironment is disturbed, a process known as reactive astrogliosis(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). These reactive astrocytes secrete a spectrum of factors such as IL-1β, IL-6, TNF-α, \u003cem\u003eetc.\u003c/em\u003e, and severely reactive astrocytes are capable of triggering neuronal apoptosis and cognitive decline in acute ischemic and hemorrhagic stroke(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). To investigate the role of this process in HE, we employed single-cell/nucleus RNA sequencing on hippocampal tissue from mice model (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Our results revealed a marked expansion of the reactive astrocyte cluster alongside an increased proportion of apoptotic neurons in the BDL group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). In addition, TEM of hippocampal astrocytes revealed the characteristic profile of reactive astrogliosis in BDL mice, as shown by swollen cell bodies and mitochondrial swelling with fragmented cristae (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). These hypertrophic astrocytes frequently surrounded neurons with condensed nuclei and shrunken cytoplasm, indicating that reactive astrocytes spatially associate with apoptotic neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). These results indicated that astrocyte activation is a pivotal driver of neuroinflammation and neuronal apoptosis in BDL mice. Reactive astrocytes in mammals are broadly categorized into two types: A1 astrocytes secrete complement C3 and pro-inflammatory cytokines, exerting neurotoxic effects that impair synapses and promote neuronal death. In contrast, A2 astrocytes release trophic factors such as BDNF and S100A10, along with anti-inflammatory mediators, thereby supporting neuronal survival and repair(\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). As expected, immunofluorescence staining showed markedly more cleaved Caspase-3 positive neurons in BDL hippocampi compared to sham groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). In addition, a striking spatial relationship was observed: cleaved Caspase-3 positive neurons in BDL mice were closely surrounded by A1 astrocytes, whereas neurons in sham mice were associated with resting or A2 astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). Collectively, these observations suggest that the shift of astrocytes toward a pro-inflammatory A1 phenotype creates a deleterious microenvironment that promotes hippocampal neuronal apoptosis in the BDL model.\u003c/p\u003e \u003cp\u003eInterrogating the TAA rat model, hippocampal bulk RNA-seq revealed a pro-inflammatory gene signature\u0026mdash;encompassing apoptosis, inflammatory response, TNF-α/NF-κB, and IL-6/JAK-STAT3 pathways\u0026mdash;that closely mirrored the BDL profile (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA and Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). This transcriptional conservation was reinforced at the protein level, with marked increases in IL-1β, IL-6, and TNF-α and a decrease in IL-10 (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB and S2C). In addition, our immunofluorescence demonstrated that apoptotic neurons in TAA rats were flanked by A1-type astrocytes, while control neurons neighbored resting or A2-type cells (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD). These convergent results from both models establish that HE-induced astrocytic activation and A1-mediated neuroinflammation are key drivers of hippocampal neuronal apoptosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eAmmonia Drives Astrocyte Reactivation and Neuroinflammation to Induce Neuronal Apoptosis\u003c/h2\u003e \u003cp\u003eTo further confirm that ammonia stimulates astrocyte reactivation, leading to the release of pro-inflammatory factors and subsequent hippocampal neuronal apoptosis, we triggered primary mouse astrocytes with 5 mM NH\u003csub\u003e4\u003c/sub\u003eCl for 48 h to mimic HE-associated hyperammonemia, which induced a clear pro-inflammatory shift. Western blot analysis showed upregulated levels of C3, IL-1β, IL-6, and TNF-α concomitant with downregulation of IL-10 and S100A10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Consistent with this, immunofluorescence staining demonstrated enhanced GFAP and C3 expression but reduced S100A10 in ammonia-treated astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), corroborating their activation toward a neurotoxic phenotype. In addition, we treated primary hippocampal neurons with conditioned medium from NH₄Cl-exposed astrocytes (AC-CM). AC-CM, compared to control medium (Ctrl-CM), increased cleaved Caspase-3 expression and induced neuronal injury characterized by somal disruption and axonal breakage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). This neurotoxicity was directly replicated by treating neurons with IL-1β, IL-6, or TNF-α individually (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), pinpointing these cytokines as key mediators.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next sought to validate these findings in a rat model. Ammonia exposure (5 mM NH₄Cl, 48 h) induced a consistent pro-inflammatory phenotype in rat primary astrocytes, evidenced by upregulated pro-inflammatory markers (C3, IL-1β, IL-6, TNF-α) and downregulated anti-inflammatory markers (IL-10, S100A10) on western blot (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA) and immunofluorescence (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB and S3C). Conditioned medium from these rat astrocytes (AC-CM) was potently neurotoxic, elevating cleaved Caspase-3 and causing somal disruption and axonal fragmentation in rat hippocampal neurons (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eD and S3E). This neurotoxicity was directly attributable to specific cytokines, as individual application of IL-1β, IL-6, or TNF-α to neurons replicated the apoptotic damage (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eF and S3G), confirming the conserved role of these astrocyte-derived factors.\u003c/p\u003e \u003cp\u003eTo ensure experimental consistency and further validate our findings in a defined system, we employed the C8-D1A astrocyte cell line. Treatment with 5 mM NH₄Cl for 48 h induced a pro-inflammatory shift in these cells, marked by increased C3, IL-1β, IL-6, and TNF-α and decreased IL-10 and S100A10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Conditioned medium from these cells (AC-CM) triggered significant apoptosis in neuronal HT22 cells, shown by a higher apoptotic cell proportion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI) and elevated pro-apoptotic protein levels (BAX, cleaved Caspase-3) with reduced BCL2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). Direct treatment with IL-1β, IL-6, or TNF-α reproduced this apoptotic effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK), confirming that astrocyte-derived cytokines are sufficient to induce neuronal apoptosis across experimental models.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eAstrocytic TLR4/NF-κB/NLRP3 Signaling Drives Neuroinflammation and Neuronal Apoptosis in HE\u003c/h2\u003e \u003cp\u003ePattern-recognition receptors on astrocytes enable rapid detection of tissue damage(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). TLR4, a key member of this family, amplifies neuroinflammation by disrupting the blood-brain barrier and boosting pro-inflammatory factors(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Although microglia are the primary source of TLR4 in the healthy brain(\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), whether hyperammonemia induces TLR4 upregulation and activation in astrocytes is not known. Previous work in LPS and trauma models shows that astrocytic TLR4 signaling activates NF-κB, upregulates NLRP3, and increases IL-1β, IL-6, and TNF-α production(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). We proposed a mechanism that hyperammonemia engages the TLR4/NF-κB/NLRP3 cascade in astrocytes, driving astrocytic reactivation and subsequent neuronal apoptosis in HE (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Immunofluorescence of hippocampal tissue in BDL mice revealed that increased co-expression of GFAP with TLR4, NF-κB, and NLRP3, indicating activation of this pathway \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). This was recapitulated \u003cem\u003ein vitro\u003c/em\u003e, treating primary murine astrocytes with 5 mM NH₄Cl for 48 hours significantly increased protein levels of TLR4, NF-κB, and NLRP3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Critically, pretreatment with the TLR4 inhibitor TAK-242 abolished these increases and reversed the inflammatory profile, reducing NF-κB, NLRP3, C3, IL-1β, IL-6, and TNF-α while restoring IL-10 and S100A10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHaving validated this pathway \u003cem\u003ein vitro\u003c/em\u003e, we next investigated its role in established HE models. In BDL model treated with TAK-242 (3 mg kg⁻\u0026sup1; every 48 h for four weeks), hippocampal astrocytes showed significant decrease fluorescence intensity of GFAP, NF-κB, and NLRP3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Western blot and ELISA analyses of hippocampal tissue confirmed parallel decreases in IL-1β, IL-6, and TNF-α, alongside a recovery of IL-10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). These data establish astrocytic TLR4/NF-κB/NLRP3 signaling as a key driver of neuroinflammation in the BDL model. As expected, preemptive administration of TAK-242 similarly reduced hippocampal levels of NF-κB, NLRP3, and GFAP (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA and S4B), and decreased IL-1β, IL-6, and TNF-α while increasing IL-10 in TAA rat model (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eC). Collectively, these results demonstrate that pharmacological TLR4 inhibition suppresses the astrocytic NF-κB/NLRP3 axis and attenuates neuroinflammation in both murine and rat models of HE.\u003c/p\u003e \u003cp\u003eTo determine whether blocking the astrocytic TLR4 pathway protects neurons, we exposed mouse primary neurons to conditioned medium from astrocytes. When astrocytes were pretreated with the TLR4 inhibitor TAK-242 before NH\u003csub\u003e4\u003c/sub\u003eCl treatment, the resulting medium failed to increase cleaved Caspase-3 in neurons, unlike medium from vehicle-pretreated astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). Similarly, conditioned medium from C8-D1A astrocytes with stable TLR4 shRNA knockdown, under the same ammonium exposure, caused a marked reduction in HT22 neuronal apoptosis compared to control shRNA, as shown by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL). These data demonstrate that inhibiting astrocytic TLR4 signaling effectively attenuates neuronal apoptosis \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDual Modulation of Astrocytic TLR4 Alleviates Neuroinflammation and Cognitive Deficits in HE\u003c/h3\u003e\n\u003cp\u003eTo determine whether pharmacological blockade of astrocytic TLR4 protects neurons \u003cem\u003ein vivo\u003c/em\u003e, we administered TAK-242 (3 mg kg⁻\u0026sup1;, i.p. every 48 h) or vehicle to BDL mice for four weeks. Administration of TAK-242 to BDL mice significantly reduced hippocampal neuronal loss and apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These changes were coincided with decreased GFAP and C3 together with elevated S100A10 fluorescence, indicating a shift in astrocytes from a pro-inflammatory A1 to a protective A2 phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This was accompanied by an anti-apoptotic molecular profile including decreased BAX/cleaved Caspase-3 and increased BCL2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Crucially, these improvements extended to cognitive function, with treated mice performing better in Y-maze and novel object recognition tests (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Identical treatment in TAA rats yielded equivalent protection across histopathological, molecular, and behavioral readouts (Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eA-S5F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo complement the pharmacological approach, we genetically ablated TLR4 specifically in astrocytes using \u003cem\u003eGFAP\u003c/em\u003e-\u003cem\u003eCre\u003c/em\u003e; \u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice. After BDL surgery, these mice showed preserved hippocampal neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG) and attenuated neuroinflammation (reduced NLRP3, NF-κB, IL-1β, IL-6, TNF-α; increased IL-10) and apoptosis (reduced cleaved Caspase-3, BAX) at the molecular level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). The finding supported by immunofluorescence and TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). Paralleling the TAK-242 treatment, cognitive deficits were also rescued in the knockout mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM). Together, these data demonstrate that targeting astrocytic TLR4\u0026mdash;either pharmacologically or genetically\u0026mdash;effectively dampens neuroinflammation, prevents neuronal apoptosis, and restores cognitive function in HE.\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eDrug Repurposing of Simvastatin via Astrocytic TLR4 Inhibition Rescues Cognition in HE\u003c/h2\u003e \u003cp\u003eWhile our results demonstrated that ammonia elicits pro-inflammatory cytokine release from astrocytes, leading to neuronal injury, its relevance to human HE remained unclear. To translate these findings, we analyzed public transcriptomic data from post-mortem HE patient brains (GSE57193 and GSE41919). GSEA of GSE57193 showed marked enrichment of gene sets related to apoptosis, inflammatory response, and key signaling pathways such as IL-6\u0026ndash;JAK\u0026ndash;STAT3, TNF-α via NF-κB and complement (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Furthermore, a positive correlation was observed between neuroinflammatory and neuronal apoptosis gene signatures across patient samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), confirming concomitant neuroinflammation and apoptosis in human HE. Our results indicated that the TLR4/NF-κB/NLRP3 axis may play a critical role in neuroinflammation in patients with HE.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBuilding on the identification of TLR4 as a potential therapeutic target, we aimed to repurpose clinically approved drugs that target TLR4 to alleviate HE-related cognitive impairment. We first performed molecular docking of compounds from several drug classes (e.g., statins, ARBs, NSAIDs, Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Four compounds\u0026mdash;candesartan, simvastatin, dexamethasone, and methylprednisolone\u0026mdash;showed promising binding affinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Based on potential hepatotoxicity, we focused on candesartan and simvastatin for further analysis. Molecular dynamics simulations revealed that simvastatin had a more stable binding mode with TLR4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). We then experimentally validated these candidates, in ammonia-treated C8-D1A astrocytes, only simvastatin pretreatment effectively reduced markers of reactive astrogliosis and pro-inflammatory cytokine expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Notably, in human iPSC-derived cerebral organoids, simvastatin treatment suppressed ammonia-induced upregulation of key inflammatory mediators-C3, NF-κB, NLRP3, IL-1β, IL-6, TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Thus, our integrated screening identifies simvastatin as a TLR4-targeting agent capable of dampening astrocyte-driven neuroinflammation, positioning it for therapeutic repurposing in HE.\u003c/p\u003e \u003cp\u003eTo validate the therapeutic potential of simvastatin \u003cem\u003ein vivo\u003c/em\u003e, we administered it every other day for four weeks to our BDL model. Simvastatin treatment significantly improved cognitive function, increasing novel arm entries in the Y-maze and the recognition index in the novel object recognition test (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Its efficacy was comparable to rifaximin, the classical HE-therapy drug (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Interestingly, rifaximin showed a more pronounced effect in \u003cem\u003eGFAP\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eTLR4\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice, prompting us to test a combination therapy. Co-administration of simvastatin and rifaximin resulted in superior amelioration of cognitive impairment compared to either drug alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Collectively, these results establish​ astrocytic TLR4 as a validated​ therapeutic target for HE. They further nominate​ simvastatin, either as monotherapy or in synergistic combination with rifaximin, as a ready-to-repurpose​ treatment strategy to alleviate cognitive impairment.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHepatic encephalopathy, a severe neuropsychiatric complication of liver disease, manifests as progressive cognitive impairment. Existing treatments focusing on ammonia lowering offer only modest and unsustained efficacy, underscoring the critical need for novel central nervous system-targeted interventions. Astrocytes, owing to their unique ammonia metabolism, are critically involved in HE pathogenesis. Yet, how hyperammonemia precisely triggers these cells to propagate neuroinflammation and subsequent neuronal damage is unclear, representing a key knowledge gap. In this study, we define a pathogenic cascade wherein hyperammonemia activates the TLR4/NF-κB/NLRP3 axis in astrocytes, leading to neuroinflammation, neuronal apoptosis, and cognitive dysfunction.​ Furthermore, we repurpose the lipid-lowering agent simvastatin as a TLR4 antagonist that effectively disrupts this cascade and rescues cognition, offering a readily translatable therapeutic strategy (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003eA key remaining question is how hyperammonemia initiates the astrocytic TLR4/NF-κB/NLRP3 cascade. Prior work in endothelial cells established that ammonia rapidly elevates TLR4 levels via a Ca\u003csup\u003e2+\u003c/sup\u003e-dependent ROS burst(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). The conservation of this rapid Ca\u003csup\u003e2+\u003c/sup\u003e-oxidant response in astrocytes suggests a similar pathway may upregulate TLR4(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Mitochondrial damage provides a potential source of ligands to engage TLR4. Specifically, damage-associated molecular patterns (mtDAMPs) released from mitochondria can act as potent agonists for TLR4, including mtDNA, cardiolipin and cytochrome C(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Accumulating evidence has demonstrated that hyperammonemia induces mitochondrial injury in astrocytes during the pathogenesis of HE(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Consistent with this, the TME showed that astrocytic mitochondria exhibited marked injury in our BDL model, characterized by swelling and cristae fragmentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Notably, each of these mtDAMP components has been identified as an TLR4 ligand(\u003cspan additionalcitationids=\"CR43 CR44\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), with the capacity to initiate downstream NF-κB and NLRP3 signaling cascades. Moreover, mtDAMPs released upon mitochondrial damage can act on recipient cells via autocrine, paracrine, or vesicle-mediated secretory pathways(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e)\u003csup\u003e,\u003c/sup\u003e(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). We propose a mechanism for HE: ammonia may first enhance astrocytic TLR4 expression via Ca\u003csup\u003e2+\u003c/sup\u003e-ROS signaling, and the receptor is then activated by mtDAMPs released from damaged mitochondria, triggering the TLR4/NF-κB/NLRP3 axis and astrocytic type A1 reactivity.\u003c/p\u003e \u003cp\u003eSimvastatin, a widely used lipid-lowering agent with known certain neuroprotective properties and established clinical application in cerebrovascular accidents(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), emerged from our drug screening as a promising TLR4 modulator. Additionally, simvastatin can scavenge superoxide anions and suppress NADH and NADPH oxidase-mediated reactive oxygen species generation, thereby reducing lipid peroxidation in liver and brain tissues and indirectly alleviating oxidative stress-related exacerbation of HE(\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Beyond these metabolic and antioxidant effects, accumulating evidence indicates that statins confer broad anti-inflammatory actions in the brain by down-regulating TLR4(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). In models of intracerebral hemorrhage, simvastatin lowers TLR4 expression, blunts NF-κB activation, and decreases subsequent production of IL-1β and TNF-α, resulting in attenuated neuronal damage and improved neurological outcome(\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Given its established clinical safety profile and low cost, simvastatin is uniquely positioned for rapid therapeutic repurposing. Our data support the progression to dose-finding and proof-of-concept trials in HE patients. Notably, simvastatin co-administered with rifaximin yielded synergistic cognitive benefits, likely through complementary actions on the gut-liver-brain axis. This combination strategy, simultaneously reducing peripheral toxin load and central neuroinflammation, presents a promising translational avenue for alleviating HE-related cognitive impairment.\u003c/p\u003e \u003cp\u003eSeveral limitations warrant consideration. A key limitation is the indirect evidence for astrocyte-specific ammonia handling; direct assessment of NH₄⁺ flux across neural cell types is required. In addition, the precise molecular steps connecting ammonia to astrocytic TLR4 activation remain incompletely resolved. Specifically, the roles of the Ca\u0026sup2;⁺-ROS cascade in receptor upregulation and of mtDAMPs as TLR4 ligands are plausible yet unverified; targeted experiments in astrocytes would help clarify these mechanisms. Third, the specific role of astrocyte-derived factors requires further delineation. Although astrocytic cytokines were associated with neuronal apoptosis, a comprehensive profiling of the astrocyte secretome was not performed, and we did not disrupt potential pro-inflammatory crosstalk from microglia(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Consequently, the individual contributions of astrocyte-versus microglia-mediated neurotoxicity remain unresolved. Furthermore, all efficacy data for simvastatin are derived from animal studies. Its clinical potential must be confirmed through subsequent dose-finding, pharmacokinetic, and long-term safety trials in HE patients. In summary, our study defines a mechanism in hepatic encephalopathy where hyperammonemia elicits astrocyte reactivity via the TLR4/NF-κB/NLRP3 pathway, driving neuroinflammation, neuronal apoptosis, and cognitive decline. Targeting this pathway with simvastatin reverses these deficits, offering a directly repurposable therapy for patients with HE.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Chongqing Medical Leading Talent Program [grant YXLJ202521 to L.Z.];the Discipline Excellence Talent Program [grant 2023XKRC005 to L.Z.]; the Chongqing Technology Innovation and Application Development General Program [grant CSTB2023TIAD-GPX0013 to L.Z.];the National Natural Science Foundation of China [grant 8257116374 to X.X.]; the Chongqing Natural Science Foundation [grant CSTB2024NSCQ-KJFZZDX0016 to X.X.];and the National Natural Science Foundation of China [grant 32500501 to R.T.].\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eS.Z., P.N., R.T., X.X., and L.Z. designed the study; L.Z., R.T., and S.Z. wrote the manuscript and prepared the figures; S.Z. performed most experiments; P.N. assisted with animal models and primary cell isolation; Y.T. conducted bioinformatics analysis and drug screening; Q.L. performed flow cytometry-based apoptosis detection; C.H., S.Z., and X.J. assisted with behavioral tests and animal drug administration; J.Y., X.H. and Z.W. provided support for immunofluorescence staining experiments; W.L., Y.H. and N.Y. performed the evaluation of clinical public datasets related to HE; all authors reviewed and edited the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe gratefully acknowledge Drs. Chaoqun Wang, Ruihao Yang, Senlin Li, Bin Han, Zhengfei Bi and Cyagen Biosciences, Beijing Vital River Laboratory Animal Technology, Yeasen biotechnology for inspiring discussions or reagents. The graphical illustrations in this work were created using the BioGDP platform (BioGDP.com), developed as the Generic Diagramming Platform (GDP) (Jiang S et al., Nucleic Acids Res, 2025).\u003c/p\u003e\u003ch2\u003eAvailability of Data and Materials\u003c/h2\u003e \u003cp\u003eSequencing data in this study have been deposited in the OMIX (National Genomics Data Center, China) under the accession numbers OMIX014552 (mouse bulk RNA-seq), OMIX014554 (rat bulk RNA-seq), and OMIX014555 (mouse scRNA-seq and snRNA-seq). The datasets are currently under restricted access and a permanent public access will be enabled via \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngdc.cncb.ac.cn/omix\u003c/span\u003e\u003cspan address=\"https://ngdc.cncb.ac.cn/omix\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e after publication. Human bulk RNA-sequencing data were obtained from public repositories (GEO: GSE57193 and GEO: GSE41919).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTsai CY, Wu JCC, Wu CJ, Chan SHH. Protective role of VEGF/VEGFR2 signaling against high fatality associated with hepatic encephalopathy via sustaining mitochondrial bioenergetics functions. J Biomed Sci. 2022;29(1):47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEngelmann C, Claria J, Szabo G, Bosch J, Bernardi M. 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Cell Death Dis. 2023;14(4):285.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9288348/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9288348/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCognitive impairment remains a significant neuropsychiatric challenge in hepatic encephalopathy (HE). Characterizing how astrocytes mediate ammonia-driven neuroinflammation may identify therapeutic targets for HE-related cognitive decline. We demonstrate that hippocampal neuronal apoptosis in HE is driven by astrocyte-mediated neuroinflammation through the TLR4/NF-κB/NLRP3 signaling axis. Both genetic deletion and pharmacological blockade of astrocytic TLR4 attenuated neuroinflammation and neuronal apoptosis. Notably, we identified the drug simvastatin as a potent inhibitor of this pathway, which curbed astrocyte reactivity and rescued cognitive function. These findings establish astrocytic TLR4/NF-κB/NLRP3 signaling as a key driver of HE-related cognitive decline and nominate simvastatin as a readily translatable inhibitor of this pathogenic cascade\u003c/p\u003e","manuscriptTitle":"Astrocyte-Dependent Neuroinflammation Triggers Hippocampal Neuronal Apoptosis through the TLR4/NF-κB/NLRP3 Axis in Hepatic Encephalopathy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-30 09:57:52","doi":"10.21203/rs.3.rs-9288348/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-06T11:11:37+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-22T15:42:22+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-22T12:23:12+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-04-22T06:40:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-02T15:04:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-01T07:15:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2026-04-01T07:15:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"89c70487-96e5-49fb-a8d9-0ab8b3043577","owner":[],"postedDate":"April 30th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-06T11:11:37+00:00","index":1,"fulltext":"This content is not available."}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66785836,"name":"Biological sciences/Neuroscience/Cognitive neuroscience/Cognitive control"},{"id":66785837,"name":"Health sciences/Medical research/Drug development"}],"tags":[],"updatedAt":"2026-04-30T09:57:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-30 09:57:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9288348","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9288348","identity":"rs-9288348","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}