Sodium valproate induces pancreatic injury by disruption of one carbon metabolism

Sodium valproate induces pancreatic injury by disruption of one carbon metabolism | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL British Journal of Pharmacology This is a preprint and has not been peer reviewed. Data may be preliminary. 9 January 2026 V1 Latest version Share on Sodium valproate induces pancreatic injury by disruption of one carbon metabolism Authors : Wenhao Cai , Di Wu , Yuying Li , Michael Chvanov , Mohammad Awais , Shiva Seyed Forootan , Xiaoli Meng 0000-0002-7774-2075 , … Show All … , Diane Latawiec , Joseph Brown P , Ziyu Li , Wenhua He , Anthony Evans , R. Mukherjee , Peter Szatmary , David Criddle N , Christopher Goldring , Wei Huang , Qing Xia , and Robert Sutton [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.176797098.85981401/v1 Published British Journal of Pharmacology Version of record Peer review timeline 332 views 126 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Objective: Valproate medications are a leading cause of drug-associated acute pancreatitis (DAAP), yet the underlying mechanisms remain unclear. This study aimed to determine how sodium valproate (Na-VPA) induces pancreatic injury and contributes to acute pancreatitis (AP). Methods: Freshly isolated murine pancreatic acinar cells (PACs) were treated with Na-VPA or major VPA metabolites, and cytotoxicity was assessed by spectrofluorometry and confocal imaging. In vivo , C57BL/6 mice were administered Na-VPA with or without caerulein-induced pancreatitis (CER-AP), and pancreatic injury was evaluated by biochemical and histological analyses. Pancreatic mRNA-sequencing and pseudo-targeted metabolomics were conducted to identify dysregulated pathways. Intracellular methionine-cycle metabolites were quantified by LC-MS/MS, while expression of one-carbon metabolism enzymes was determined by RT-qPCR and western blotting. AutoDock Vina was employed to predict binding affinities of VPA to key metabolic enzymes. The therapeutic effect of S-adenosylmethionine (SAM) supplementation on Na-VPA-exacerbated CER-AP was also tested. Results: Na-VPA and its metabolites induced concentration- and time-dependent PAC death, independent of toxic calcium signaling. In vivo , Na-VPA aggravated CER-AP, increasing pancreatic histology scores and biochemical parameters. Transcriptomic and metabolomic analyses showed dysregulated one-carbon metabolism, validated by altered mRNA and protein expression analysis of key rate-limiting enzymes. Molecular docking indicated direct interactions between VPA and several metabolic enzymes. Na-VPA also activated the pancreatic endoplasmic reticulum (ER) stress pathway. In PACs, Na-VPA reduced methionine and SAM levels, while supplementation with methionine or SAM markedly attenuated cell injury; conversely, ethionine exacerbated it. Moreover, SAM supplementation in vivo significantly ameliorated pancreatic damage and biochemical alterations in Na-VPA–exacerbated CER-AP. Conclusion: Na-VPA disrupts one-carbon metabolism, triggering ER stress and acinar cell injury and exacerbating experimental pancreatitis. These findings provide new mechanistic insight into valproate-associated AP and identify metabolic targets for potential prevention or therapy. \articletype Original Articles Original Article Sodium valproate induces pancreatic injury by disruption of one carbon metabolism Wenhao Cai 1,2# , Di Wu 3# , Yuying Li 1# , Michael Chvanov 2 , Muhammad Awais 2 , Shiva Seyed Forootan 4 , Xiaoli Meng 4 , Diane Latawiec 2 , Joseph P Brown 4 , Ziyu Li 1 , Wenhua He 5 , Anthony Evans 6 , Rajarshi Mukherjee 2 , Peter Szatmary 2 , David N Criddle 2 , Chris Goldring 4 , Wei Huang 1,2 *, Qing Xia 1 *, Robert Sutton 2 * \articletype Original Articles 1 West China Centre of Excellence for Pancreatitis, Institute of Integrated Traditional Chinese and Western Medicine, West China-Liverpool Biomedical Research Centre, West China Hospital, Sichuan University, Chengdu, Sichuan, China 2 Liverpool Pancreatitis Research Group, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, UK \articletype Original Articles 3 Pancreas Center, First Affiliated Hospital of Nanjing Medical University, Nanjing Medical University, Nanjing, Jiangsu, China \articletype Original Articles 4 MRC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, University of Liverpool, UK \articletype Original Articles 5 Department of Gastroenterology, Jiangxi Provincial Key Laboratory of Digestive Diseases, Jiangxi Clinical Research Center for Gastroenterology, Digestive Disease Hospital, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi, China \articletype Original Articles 6 Computational Biology Facility, University of Liverpool, Liverpool, UK *Correspondence to: Wei Huang, West China Centre of Pancreatitis for Excellence, Institute of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, Translational Medicine Comprehensive Building, No. 5 Dianxin Road, Chengdu 610041, China. E-mail: [email protected] Qing Xia, West China Centre of Excellence for Pancreatitis, Institute of Integrated Traditional Chinese and Western Medicine, West China-Liverpool Biomedical Research Centre, West China Hospital, Sichuan University, Chengdu 610041, China. E-mail: [email protected] Robert Sutton, Liverpool Pancreatitis Research Group, Institute of Systems, Molecular and Integrative Biology, University of Liverpool and Liverpool University Hospitals NHS Foundation Trust, Liverpool L69 3GE, UK. E-mail: [email protected] \articletype Original Articles # Wenhao Cai, Di Wu, and Yuying Li contributed equally to this work. \articletype Original Articles Objective: Valproate medications are a leading cause of drug-associated acute pancreatitis (DAAP), yet the underlying mechanisms remain unclear. This study aimed to determine how sodium valproate (Na-VPA) induces pancreatic injury and contributes to acute pancreatitis (AP). \articletype Original Articles Methods: Freshly isolated murine pancreatic acinar cells (PACs) were treated with Na-VPA or major VPA metabolites, and cytotoxicity was assessed by spectrofluorometry and confocal imaging. In vivo , C57BL/6 mice were administered Na-VPA with or without caerulein-induced pancreatitis (CER-AP), and pancreatic injury was evaluated by biochemical and histological analyses. Pancreatic mRNA-sequencing and pseudo-targeted metabolomics were conducted to identify dysregulated pathways. Intracellular methionine-cycle metabolites were quantified by LC-MS/MS, while expression of one-carbon metabolism enzymes was determined by RT-qPCR and western blotting. AutoDock Vina was employed to predict binding affinities of VPA to key metabolic enzymes. The therapeutic effect of S-adenosylmethionine (SAM) supplementation on Na-VPA-exacerbated CER-AP was also tested. Results: Na-VPA and its metabolites induced concentration- and time-dependent PAC death, independent of toxic calcium signaling. In vivo , Na-VPA aggravated CER-AP, increasing pancreatic histology scores and biochemical parameters. Transcriptomic and metabolomic analyses showed dysregulated one-carbon metabolism, validated by altered mRNA and protein expression analysis of key rate-limiting enzymes. Molecular docking indicated direct interactions between VPA and several metabolic enzymes. Na-VPA also activated the pancreatic endoplasmic reticulum (ER) stress pathway. In PACs, Na-VPA reduced methionine and SAM levels, while supplementation with methionine or SAM markedly attenuated cell injury; conversely, ethionine exacerbated it. Moreover, SAM supplementation in vivo significantly ameliorated pancreatic damage and biochemical alterations in Na-VPA–exacerbated CER-AP. \articletype Original Articles Conclusion: Na-VPA disrupts one-carbon metabolism, triggering ER stress and acinar cell injury and exacerbating experimental pancreatitis. These findings provide new mechanistic insight into valproate-associated AP and identify metabolic targets for potential prevention or therapy. \articletype Original Articles Key words: \articletype Original Articles Drug-associated acute pancreatitis, sodium valproate, pancreatic acinar cell, one carbon metabolism. \articletype Original Articles AHCY S-adenosylhomocysteine hydrolase ALT Alanine aminotransferase AP Acute pancreatitis AST Aspartate aminotransferase BiP Binding immunoglobulin protein CBS Cystathionine β-synthase CER Caerulein CER-AP Caerulein-induced acute pancreatitis CHOP C/EBP homologous protein Ct Cycle threshold DAAP Drug-associated acute pancreatitis Ddit3 DNA damage-inducible transcript 3 DE Differential expression DEG Differentially expressed gene DEM Differentially expressed metabolite Eif2s1 Eukaryotic translation initiation factor 2 subunit 1 ER Endoplasmic reticulum Ern1 Endoplasmic reticulum to nucleus signaling 1 GABA γ-aminobutyric acid GNMT Glycine N-methyltransferase GSEA Gene set enrichment analysis HCA Hierarchical clustering analysis HDAC Histone deacetylase Hmox1 Haeme oxygenase 1 Hspa5 Heat shock protein family A member 5 IL-1β Interleukin-1 beta IL-6 Interleukin-6 i.p. Intraperitoneal KEGG Kyoto Encyclopedia of Genes and Genomes Kd Dissociation constant LC-MS/MS Liquid chromatography–tandem mass spectrometry MAT1A Methionine adenosyltransferase 1A MAT2A Methionine adenosyltransferase 2A MPO Myeloperoxidase MTHFR Methylenetetrahydrofolate reductase MTR Methionine synthase Na-VPA Sodium valproate NES Normalized enrichment score ORA Over-representation analysis PAC Pancreatic acinar cell PCA Principal component analysis PI Propidium iodide QC Quality control RNA-seq RNA sequencing RT-qPCR Real-time quantitative PCR SAH S-adenosylhomocysteine SAM S-adenosylmethionine SEM Standard error of the mean sXBP1 Spliced X-box-binding protein 1 uXBP1 Unspliced X-box-binding protein 1 VPA Valproic acid WB Western blotting \articletype Original Articles 1. Introduction Acute pancreatitis (AP) is a common inflammatory disease of the exocrine pancreas that causes severe abdominal pain and multiple organ dysfunction that may result in pancreatic necrosis and persistent organ failure, with an overall mortality of 1-5% (Petrov & Yadav, 2019; Szatmary et al., 2022). The aetiology of AP is varied, with several risk factors identified as primary causes, including gallstones, excess alcohol consumption, hypertriglyceridaemia, and drugs (Szatmary et al., 2022). Drugs are estimated to account for 2-5% of all AP cases (Nitsche, Maertin, Scheiber, Ritter, Lerch & Mayerle, 2012; Vinklerova, Prochazka, Prochazka & Urbanek, 2010), with higher proportions in paediatric patients at over 20% (Uc & Husain, 2019). Although more than 500 drugs have been linked with AP, fewer than 50 have a definite association with AP from well documented re-challenge data (Bellocchi, Campagnola & Frulloni, 2015; Wolfe et al., 2020). Despite the significance of drug-associated AP (DAAP), however, the mechanisms have been largely unexplored. Sodium valproate (Na-VPA) and other valproate medications (valproic acid (VPA) and valproate semisodium) are widely used to treat epilepsy, bipolar disorder, neuropathic pain, and migraine. Valproate medications increase aminobutyric acid (GABA) levels in the central nervous system though inhibition of degradative enzymes and/or neuronal reuptake, block voltage-gated sodium channels, and inhibit histone deacetylases (HDACs) (Johannessen, 2000; Löscher, 1999). The most recent systematic reviews of DAAP indicate valproate medications are associated with the highest number of case reports of DAAP (Bischof et al., 2023; Meczker et al., 2020; Wolfe et al., 2020). Few experimental studies (Eisses et al., 2015; Ghoneim, Alrefai, Elsamanoudy, Abo El-Khair & Khalaf, 2020; Walker, Smith, Barsoum & Macallum, 1990; Xu, Guo, Liang, Li & Chen, 2019) have investigated the effects of valproate medications on the exocrine pancreas, and none have sought to determine mechanisms by which they induce AP. In 1990, Walker and colleagues reported on a preclinical toxicological investigation to explore the safety of calcium VPA as an alternative valproate medication, demonstrating that 125, 250, and 500 mg/kg/day calcium VPA administered to Wistar rats for 1 year but not for 13 weeks caused a dose-dependent increase in the incidence and severity of atrophic pancreatitis, accompanied by modest acinar vacuolisation and inflammatory infiltrates (Walker, Smith, Barsoum & Macallum, 1990). A recent study sought to develop biomarkers of hepatotoxicity administering 250 and 500 mg/kg/day VPA to male Kunming mice for 31 days and simultaneously found no obvious pancreatic histopathological damage (Xu, Guo, Liang, Li & Chen, 2019). Eisses et al. combined 125 mg/kg VPA at 12-hourly intervals either 4 days before or 7 days after induction of AP in Swiss Webster mice with the classical caerulein hyperstimulation AP model (CER-AP), to investigate the effects of HDAC inhibition by VPA on pancreatic recovery (Eisses et al., 2015). VPA was found to retard pancreatic recovery from AP through inhibition of HDACs. Although short-term VPA alone did not induce identifiable pancreatic injury, VPA administered before CER-AP induced net increases in pancreatic inflammation and vacuolisation, as well as reduced acinar content over that in CER-AP alone, indicating potential for this model in investigation of the effects of valproate medications on the pancreas. \articletype Original Articles Here we have investigated mechanisms by which Na-VPA and its major metabolites injure the pancreas and may induce AP. We have used freshly isolated pancreatic acinar cells (PACs) and in vivo models for experiments by plate reader, confocal microscopy, liquid chromatography with tandem mass spectrometry (LC-MS/MS), next generation sequencing, pseudo-targeted metabolomics, western blotting, and RT-qPCR experiments, together with molecular docking analysis. We have identified disrupted one carbon metabolism as a key mechanism by which Na-VPA induces pancreatic injury. 2. Material and methods 2.1. Animals Wild type CD-1 and C57BL/6J mice were purchased from Charles River UK Ltd (Margate, UK) and housed in the state-of-the-art animal unit of the University of Liverpool. Generation of cyclophilin D knockout mice ( Ppif -/- ) mice were previously described (Armstrong et al., 2018; Mukherjee et al., 2016). For in vitro experiments, PACs were isolated from 10 to 12-week-old CD-1 male mice when using wild type cells alone, or C57BL/6 male mice when comparing to Ppif -/- mice. For in vivo experiments, male C57BL/6J 10 to 12-week-old mice were used. All animals were allowed to acclimatize for one week under temperature-controlled conditions with a 12-hour light/dark cycle, with free access to water and standard laboratory chow. \articletype Original Articles 2.2. Plate reader cell death assay and NAD(P)H/FAD autofluorescence assay Mouse PACs were freshly isolated following procedures provided in supplemental methods. Time-course experiments were conducted at 37°C using a POLARstar Omega fluorescence microplate reader (BMG Labtech, Ortenberg, Germany). The cells were seeded in flat-bottomed 96-well microplates at a density of 200,000/well. Necrosis was detected using propidium iodide (P3566, Invitrogen™, Loughborough, UK) at a final concentration of 10 μg/ml, using a 520 nm excitation and a 590 nm emission wavelength. Autofluorescence assays for NAD(P)H and FAD involved excitation/emission wavelengths of 340/440 nm and 430/520 nm respectively. Fluorescence intensity was normalised to the initial readings, with duplicates for each condition. 2.3. Time-lapse cell death measurement using confocal microscopy Isolated PACs were loaded in a four-compartment 35 mm glass bottom dish from Greiner Bio-One (627871, Stonehouse, UK) at a density of 1 million per compartment with a range of concentrations of tested compounds and propidium iodide (1 µM), measured at excitation 488 nm, emission 557-645 nm. Cells were imaged every 20 mins for 18 h using a time-lapse scanning mode in Zeiss LSM710 system (Zeiss, Heidelberg, Germany). \articletype Original Articles 2.4. Confocal fluorescence microscopy for cell function experiments Isolated PACs loaded in a poly-D-lysine coated 35 mm dish (P35GC-1.5-14-C, MATTEK, Ashland, US) were viewed on a Zeiss LSM710 with a 63x C-Apochromat water immersion objective with constant perfusion of challenged compound or control solutions. Fluo 4-AM (F14201, Invitrogen™, Loughborough, UK) was used to assess cytosolic calcium signalling with a final concentration of 5 µM at excitation 488 nm and emission 505 nm. 2.5. LC-MS/MS \articletype Original Articles The detailed descriptions of the methods applied for LC-MS/MS were provided in the supplemental methods. LC gradient conditions and multiple reaction monitoring transitions and fragmentation parameters are presented in Table S1 and Table S2, respectively. \articletype Original Articles 2.6. Hyperstimulation-induced AP and Na-VPA administration Hyperstimulation CER-AP was induced by seven intraperitoneal (i.p.) injections of 50 μg/kg caerulein at one h intervals. Mice were humanely sacrificed 8 h after the first injection. Control mice received four i.p. injections of saline of the same volume. Na-VPA at 500 mg/kg was dissolved in 100 μl saline and administered once by i.p. injection daily for 8 days, and mice were humanely sacrificed on the 8 th day, either after CER-AP induced as described, or without CER-AP. To determine the therapeutic effects of SAM on Na-VPA plus CER-AP model, SAM was administered once i.p. daily (20 mg/kg) for 8 days, with or without concurrent Na-VPA administration, given at a 1-hour interval on the same day. Severity was determined through biochemical parameters and pancreatic histology ( supplemental methods ). \articletype Original Articles 2.7. RNA extraction and RT-qPCR Cells were lysed using the QIAshredder cell-lysate homogenizers (79656, Qiagen, Manchester, UK) and then processed for total RNA extraction using a RNeasy Mini kit (74104, Qiagen, Manchester, UK). The reverse transcription of RNA to cDNA was conducted using LunaScript™ RT SuperMix Kit (E3010, NEB, Salisbury, UK) on a Mastercycler® nexus gradient thermocycler (Eppendorf, Hamburg, Germany). Real time quantitative PCR was performed using SYBR Green Luna® Universal qPCR Master Mix (M3003, NEB, Salisbery, UK) on an Applied Biosystems ViiA-7 system (Thermo Fisher Scientific, Oxford, UK). A total of 100 ng cDNA was added to each well for PCR reactions. For each sample, the average cycle threshold (Ct) value was normalized to 18S ribosomal RNA and the relevant control sample, using the formula 2 −∆∆Ct . Primers used are provided in Table S3 . \articletype Original Articles 2.8. mRNA sequencing Total RNA extracted in our laboratory was submitted to TAmiRNA (Vienna, Austria) for sequencing. Read counts for each library were normalized, and differential expression (DE) analysis was performed using the DESeq2 package. Principal component analysis (PCA) was conducted on variance-stabilized transformed (vst) counts. Genes with p < 0.05 were considered differentially expressed (DEGs). DEGs and all expressed background genes were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment using over-representation analysis (ORA) and gene set enrichment analysis (GSEA) via the clusterProfiler package. Gene fold changes were calculated for two contrasts: Na-VPA vs. control and Na-VPA + CER-AP vs. CER-AP. Ranked gene lists were used for GSEA with KEGG mouse gene sets, and significantly enriched metabolic pathways ( P < 0.05) were extracted for visualization. \articletype Original Articles 2.9. Pseudo-targeted metabolomics Pseudo-targeted metabolomics of pancreatic tissue was performed using LC–MS on a high-sensitivity SCIEX QTRAP® 6500+ platform. Twenty-four samples (four groups, n = 6 per group) were analyzed. The workflow included sample preparation, metabolite extraction, LC–MS acquisition, and data processing. Raw LC–MS files (.wiff) were processed in SCIEX OS v1.4 for peak detection, integration, and alignment. Peak areas were used as relative quantification values, normalized to total ion intensity and quality-control (QC) samples to ensure data consistency. Multivariate analysis was performed using the limma package, applying linear mixed models to evaluate the effects of Na-VPA and CER-AP. Statistically significant differentially expressed metabolites (DEMs) were identified after log transformation ( P < 0.05). PCA, hierarchical clustering (HCA), and metabolite–metabolite correlation analyses were conducted to explore metabolic patterns and relationships with transcriptomic features. KEGG-based pathway enrichment of DEMs was performed using enrichr, with all detected metabolites as background, to identify dysregulated metabolic pathways under experimental conditions. \articletype Original Articles 2.10. Western blotting Total protein from pancreatic tissue was extracted using RIPA buffer (89901, Thermo Fisher Scientific, UK) supplemented with PMSF (1 mM) and cOmplete™ Mini Protease Inhibitor (Roche, Switzerland). Protein concentration was determined using the Pierce™ BCA assay kit (Thermo Fisher Scientific, UK). Lysates were mixed with 4× Laemmli buffer (β-mercaptoethanol added 1:10), boiled at 95 °C for 10 min, and stored at −20 °C or used immediately. Equal amounts of protein (30 µg) were separated on Any kD™ Mini-PROTEAN® TGX™ precast gels (Bio-Rad) and transferred to PVDF membranes using the Trans-Blot® Turbo system (Bio-Rad). Membranes were blocked with 5% non-fat milk in TBST for 2 h, incubated overnight at 4 °C with primary antibodies, and then with HRP-conjugated secondary antibodies for 1 h at room temperature. Bands were visualized using ECL substrate (Bio-Rad) and imaged with the ChemiDoc™ system. Densitometry was performed using Image Lab software (Bio-Rad). β-actin or vinculin served as loading controls, and hepatocyte lysates were used as positive controls for one-carbon metabolism enzymes. Antibody details are listed in Table S4 . \articletype Original Articles 2.11. Molecular docking A molecular docking strategy was used to predict the binding affinity between VPA and target proteins. Three-dimensional structures of the proteins were obtained from the UniProt database (https://www.uniprot.org/) and downloaded in PDB format. Homologous multimeric proteins underwent processing and repair using Maestro software. Water molecules and ligands were removed using Pymol. Hydrogen atoms and charges were added using Autodock Tools, and the processed proteins saved in PDBQT format. The small molecule ligand was obtained from the ZINC database in SDF format. Free energy of the molecule was minimized using Chem3D, and the file subsequently converted to MOL2 format. Subsequently, the affinities (binding free energy) between the small molecule and protein targets were calculated with the AutoDock Vina docking model. The dissociation constant ( K d ) was calculated based on the binding affinity: Kd = e ΔG bind /RT (where ΔG bind is the binding free energy, R is the gas constant ≈ 1.987 cal/(mol·K), T is the temperature in Kelvin set as room temperature = 298 K). PyMOL software was applied to visualise the results. \articletype Original Articles 2.12. Chemicals All the chemicals used in the study were purchased from Sigma Aldrich (Merck, Gillingham, UK), with exceptions listed as below. 2-propyl-4-Pentenoic Acid (4-ene-VPA, item No. 23090), 2-propyl-2-Pentenoic Acid (2-ene-VPA, item No. 19591), and S-adenosylhomocysteine-d4 (Item No. 9000372) were from Cayman Chemical (Ann Arbor, Michigan, US). 3-keto-valproic acid sodium salt (3-keto-VPA, item No. sc-216476) was from Santa Cruz (Santa Cruz Biotechnology, Dallas, TX, US). \articletype Original Articles 2.13. Statistical analysis Data were presented as mean ± standard error of the mean (SEM) for parametric data. Two-tailed Student’s t-test (two groups) and one-way ANOVA (more than two groups) were performed for parametric data. Two-way ANOVA was performed for grouped analyses. Tukey test or Dunnett’s test were used for corrections for multiple comparisons. Mann-Whitney test (two groups) and Kruskal-Wallis test (more than two groups) were conducted for non-parametric data with Dunn’s test used for multiple comparisons. P values < 0.05 were considered significant. Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, La Jolla, CA, US). \articletype Original Articles 3. Results 3.1. Na-VPA and its metabolites caused PAC death independent of toxic calcium signals Na-VPA at 1-20 mM caused significantly PAC necrosis in a concentration- and time-dependent manner ( Fig. 1A ). Confocal time-lapse imaging confirmed that 20 mM Na-VPA induced significantly more cell death than solvent control from 7 h ( Fig. 1B ). The parent drug VPA caused significantly cell death at 10 mM ( Fig. 1C ). Major Na-VPA metabolites including 2-ene-VPA, 3-keto-VPA, and 4-ene-VPA, also caused markedly more cell death at 5 or 10 mM ( Fig 1D-F ). However, low concentrations of pancreatitis toxins did not enhance Na-VPA toxicity ( Fig. S1A-B ). Confocal calcium signalling showed no significant abnormal cytosolic calcium signals with exposure to 5 or 20 mM Na-VPA in various conditions ( Fig. 1G-L ). Moreover, calcium exclusion or inositol 1,4,5-trisphosphate receptor inhibition by caffeine did not protect against Na-VPA induced cell death ( Fig. S1C-D ). Additionally, Na-VPA, 3-keto-VPA, and 4-ene-VPA significantly reduced intracellular NADH autofluorescence ( Fig. S1E-G ), indicating decreased oxidative phosphorylation. Cyclophilin D depletion (constitutive knockout Ppif -/- ) did not significantly mitigate cell death ( Fig. S1H-J ). \articletype Original Articles 3.2 Na-VPA exacerbated pancreatic injury in mild CER-AP and induces transcriptomic alterations linked to one-carbon metabolism A one-week Na-VPA pretreatment was combined with a mild CER-AP model ( Fig. 2A ), adapted from Eisses et al .(Eisses et al., 2015) Na-VPA alone reduced the weight of mice from day two, an effect maintained throughout the study ( Fig. 2B ). Combined Na-VPA with CER-AP significantly increased pancreatic inflammation, necrosis, and total injury scores compared to CER-AP alone ( Fig. 2C-D ). Significant increases in serum amylase, serum lipase, pancreatic trypsin and MPO activities, IL-6, and serum ALT were found in the combined Na-VPA with CER-AP group over CER-AP alone ( Fig. 2E-J ). Moreover, serum IL-6 levels were significantly higher in Na-VPA group versus controls ( Fig. 2H ). Serum AST was not significantly increased ( Fig. 2K ). Transcriptomic profiling of pancreatic tissue revealed distinct molecular signatures ( Fig. S2 ). PCA showed a clear separation between CER-AP and control samples along PC1 (87 percent of variance) and an additional separation induced by Na-VPA on PC2 (6 percent variance) ( Fig. 2L ). Differential gene expression analysis identified 104 consistently upregulated and 46 consistently downregulated genes shared between Na-VPA vs. control and Na-VPA + CER-AP vs. CER-AP comparisons ( Fig. 2M-O ). To further interpret pathway-level alterations, GSEA indicated that Na-VPA monotherapy suppressed “valine, leucine and isoleucine degradation” and activated “glycosphingolipid biosynthesis” compared with controls ( Fig. 2P ). Notably, Na-VPA + CER-AP significantly inhibited metabolism-related pathways, particularly “one-carbon pool by folate,” suggesting a key role for impaired one-carbon metabolism in Na-VPA-mediated exacerbation of AP ( Fig. 2Q ). 3.3. Pancreatic metabolomics reveals disruption of one carbon metabolism pathway To elucidate metabolic alterations associated with Na-VPA treatment in the presence or absence of CER stimulation, pseudo-targeted metabolomics was performed, covering 16 major chemical classes and more than 800 metabolites, predominantly belonging to lipids and amino acid derivatives ( Fig. S3A&B ). PCA revealed distinct separations corresponding to CER-AP induction and Na-VPA exposure, consistent with the patterns observed in transcriptomics ( Fig. 3A ). A total of 123, 284, and 59 metabolites were significantly increased, and 65, 102, and 76 metabolites were significantly decreased in the Na-VPA vs. control, CER-AP vs. control, and Na-VPA + CER-AP vs. CER-AP comparisons, respectively ( Fig. 3B ). Notably, 10 metabolites, including S-adenosylmethionine (SAM), were consistently downregulated in both Na-VPA vs. control and Na-VPA + CER-AP vs. CER-AP contrasts ( Fig. 3C ). Pathway enrichment analysis highlighted “one-carbon pool by folate” and “cysteine and methionine metabolism” as the top perturbed metabolic pathways in these groups ( Fig. 3D ). Focusing on one-carbon metabolism-related metabolites, SAM, methionine, 5-methyl-THF, and several others showed marked decreases following Na-VPA exposure ( Fig. 3E ). Correlation analysis further demonstrated that SAM and methionine displayed strong associations with one-carbon metabolism-related genes as well as multiple metabolite clusters, suggesting their central role in mediating Na-VPA-induced impairment of the methionine cycle ( Fig. 3F ). \articletype Original Articles 3.4. Na-VPA disrupts expression of rate-limiting enzymes in one carbon metabolism As transcriptomic and metabolomic analyses indicated dysregulation of one-carbon metabolism, we quantified temporal changes in the mRNA expression of key rate-limiting enzymes in PACs. mRNA levels of methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), and methionine adenosyltransferase 2A (MAT2A) were significantly reduced by Na-VPA, whereas cystathionine β-synthase (CBS) was upregulated ( Fig. 4A ). Protein expression of these enzymes in pancreatic tissue showed consistent trends: MTR protein was significantly decreased by Na-VPA; MAT2A protein was elevated in CER-AP but markedly reduced when Na-VPA was added; and CBS protein was significantly increased following Na-VPA exposure ( Fig. 4B&C ). In contrast, MAT1A, glycine N-methyltransferase (GNMT), and S-adenosylhomocysteine hydrolase (AHCY) showed no significant mRNA or protein changes. To assess whether Na-VPA could directly inhibit these enzymes, molecular docking analysis was performed. As shown in Fig. 4D-I , Na-VPA bound within the active sites of all five enzymes, with the strongest affinity observed for MAT2A. These binding affinities were comparable to those previously reported for VPA interactions with its neurological therapeutic targets(Piplani, Verma & Kumar, 2016). 3.5. Na-VPA causes endoplasmic reticulum stress in PACs and in vivo In addition, we examined markers of endoplasmic reticulum (ER) stress in PACs and in vivo . In PACs treated with 10 mM Na-VPA, mRNA levels of heat shock protein family A member 5 (Hspa5, encoding BIP protein), DNA damage inducible transcript 3 (Ddit3, encoding CHOP protein), and ER to nucleus signalling 1 (Ern1) were significantly upregulated after 4-12 h compared to controls ( Fig. 5A ). Although spliced X-Box binding protein 1 (sXBP1) and unspliced XBP1 (uXBP1) were unaffected, sXBP1/uXBP1 ratio was significantly increased at 12 h ( Fig. 5A ). Eukaryotic translation initiation factor 2 subunit 1 (Eif2s1) mRNA levels remained unchanged ( Fig. 5A ). Similar findings were made in pancreatic tissue from Na-VPA-treated mice. Hspa5 mRNA was significantly elevated in Na-VPA group versus controls ( Fig. 5B ). Eif2s1 mRNA levels was increased in CER-AP alone versus controls ( Fig. 5B ). Notably, Hspa5, Ddit3, sXBP1, Eif2s1, and Ern1 mRNAs were significantly higher in the Na-VPA with CER-AP group compared to CER-AP alone ( Fig. 5B ). Western blotting showed an increase of BiP in the Na-VPA alone group compared to controls, although no further differences of BiP protein were seen in the Na-VPA with CER-AP or CER-AP alone groups ( Fig. 5C ). CHOP protein expression was significantly higher in CER-AP alone compared to controls and was markedly increased in the Na-VPA with CER-AP group ( Fig. 5C ). Additionally, pancreatic mRNA levels of the oxidative stress marker haeme oxygenase 1 ( Fig. 5D ) and the pro-inflammatory markers IL-1β and TNF-α ( Fig. 5E ) were significantly higher in the Na-VPA with CER-AP group compared to CER-AP alone. \articletype Original Articles 3.6. Effects of Na-VPA on metabolite profiles of the methionine cycle in vitro and the effects of key metabolites on Na-VPA induced PAC death Given that metabolomics and enzyme analyses highlighted the importance of the methionine cycle, we quantified intracellular levels of methionine, SAM, S-adenosylhomocysteine (SAH), and homocysteine in PACs exposed to 10 or 20 mM Na-VPA. Representative chromatograms and quantitative data are shown in Fig. 6A&B . Na-VPA significantly reduced intracellular methionine and SAM at 12 h and 18 h, and markedly decreased SAH at 18 h. Homocysteine showed a slight, non-significant reduction at 18 h. Functionally, methionine supplementation (0.5–1 mM) significantly protected PACs from Na-VPA-induced cell death ( Fig. 6C ), whereas non-toxic concentrations of ethionine (5–10 mM) exacerbated injury ( Fig. 6D ). SAM supplementation (0.5–1 mM) also markedly attenuated Na-VPA-induced cell death ( Fig. 6E ). In contrast, homocysteine or folic acid supplementation did not modify Na-VPA toxicity ( Fig. S4 ). \articletype Original Articles 3.7. SAM supplementation alleviated Na-VPA exacerbated CER-AP in vivo In the Na-VPA exacerbated CER-AP model, SAM was co-administered with Na-VPA at 15 mg/kg/d ( Fig. 7A ), based on dosing previously used in the choline deficient, ethionine supplemented diet-induced AP (CDE-AP) model (Lu et al., 2003). SAM alone had no adverse effects on the pancreas. In contrast, SAM supplementation markedly improved pancreatic injury in Na-VPA+CER-AP mice, significantly reducing inflammatory infiltration, necrosis, and total scores ( Fig. 7B&C ). Moreover, SAM supplementation significantly reduced serum amylase, lipase, pancreatic trypsin activity, pancreatic MPO activity, serum IL-6, serum ALT and serum ALT ( Fig. 7D-J ). A schematic summary of the proposed mechanism of Na-VPA pancreatic toxicity is shown in Fig. 7K . \articletype Original Articles 4. Discussion We found Na-VPA and its metabolites induce pancreatic injury by disruption of one carbon metabolism, the principal components of which are the methionine and folate cycles, transsulfuration, and glutathione pathways (Ducker & Rabinowitz, 2017). Na-VPA caused depletion of PAC methionine, SAM and SAH, and dose-dependent PAC necrosis that was significantly alleviated by administration of supplementary methionine, SAM, but not homocysteine or folate. In vivo SAM supplementation significantly protected against Na-VPA exacerbated CER-AP. These findings identify a critical vulnerability in the methionine cycle, crucial to protein and nucleotide synthesis essential to pancreatic digestive enzyme production. PACs, with protein turnover among the highest of all cells (Rooman et al., 2013; Saluja, Dudeja, Dawra & Sah, 2019), depend on methionine for initiating protein synthesis, as methionine’s codon (AUG) starts most protein translations (Danchin, Sekowska & You, 2020). SAM is a methyl donor to histones, influencing transcription (Mentch et al., 2015) and epigenetic modifications (Clare, Brassington, Kwong & Sinclair, 2019), and regulates the nutrient sensitive target of rapamycin for translation and adenosine monophosphate protein kinase for ATP restoration (González, Hall, Lin & Hardie, 2020; Martínez-Chantar et al., 2006). SAH, a precursor of glutathione through the transsulfuration pathway, reduces peroxides and free radicals generated by cell stress (Ducker & Rabinowitz, 2017). Consistent with disordered transcription, translation and cell protection consequent upon insufficient supply of methionine cycle substrates, ER stress, which contributes to AP (Habtezion, Gukovskaya & Pandol, 2019), was evident in PACs and in the pancreata of mice exposed to Na-VPA. Na-VPA treatment prior to mild CER-AP induction significantly increased ER stress and activation of immune response genes. The contrary effects to supplementary methionine, which stimulates mitochondrial respiration, of the amino acid ethionine, a methionine antagonist that competes for adenosyl groups from ATP, underlines the importance of one carbon metabolism in PACs and its potential disruption in CDE-AP (Niederau, Lüthen, Niederau, Grendell & Ferrell, 1992; Rao, Tuma & Lombardi, 1976). Together with our identification of the independence of Na-VPA pancreatic injury from toxic calcium signals, these findings support distinction of an amino acid-dependent pathway (Zhang et al., 2018) from a calcium-dependent pathway in the pathogenesis of AP, pathways that may nevertheless interact. Valproate medications have a narrow therapeutic index and high pharmacokinetic variability, with potential toxicities above the therapeutic range of 350-700 µM for epilepsy and up to 870 µM for bipolar disorder, despite ~90% plasma protein binding in humans (Klotz & Antonin, 1977; Lin, Cao, Au & Dahri, 2022). We found Na-VPA induced significant necrosis of PACs within 24 hours at concentrations as low as 1 mM, perhaps partly because of increased vulnerability consequent on the cell isolation procedure in HEPES buffer, which lacks extracellular protein to bind Na-VPA. The acute injury of PACs resulted in decreased mRNA expression of the majority of one carbon metabolism enzymes integral to normal cellular function, including MTHFR, MTR, and MAT2A, changes consistent with the observed depletion of methionine and SAM in PACs. Additionally, Na-VPA upregulated CBS, suggesting activation of the transsulfuration pathway and further diversion of substrates away from methionine regeneration. In mice, plasma protein binding of VPA is far lower at Boughattas, 2013) and despite 500 mg/kg/day for 8 days, which from pharmacokinetic investigations (Ben-Cherif, Dridi, Aouam, Ben-Attia, Reinberg & Boughattas, 2013) can be estimated to result in plasma concentrations of 3-6 mM, Na-VPA did not induce AP, consistent with previous murine studies (Eisses et al., 2015; Xu, Guo, Liang, Li & Chen, 2019). In our model, Na-VPA exposure in vivo downregulated MTR and MAT2A and upregulated CBS in either Na-VPA versus control or Na-VPA+CER-AP versus CER-AP comparisons, mirroring the metabolomic evidence of reduced methionine and SAM availability. Combined with molecular docking showing high binding affinity of VPA for MAT2A and MTR, these findings support a mechanism in which Na-VPA directly inhibits these enzymes, thereby limiting methionine and SAM formation, while CBS upregulation further disrupts methionine-cycle homeostasis. However, in view of the relative absence of AP following medium-term Na-VPA administration in murine species (Eisses et al., 2015; Ghoneim, Alrefai, Elsamanoudy, Abo El-Khair & Khalaf, 2020; Walker, Smith, Barsoum & Macallum, 1990; Xu, Guo, Liang, Li & Chen, 2019) and most patients (Bischof et al., 2023), despite the frequency of valproate medication-induced AP (Bischof et al., 2023; Meczker et al., 2020; Wolfe et al., 2020), other factors such as genetic, epigenetic, and environmental factors likely contribute to valproate-associated AP (Barreto et al., 2021). Therapeutic effects of valproate medications are attributed to inhibition of GABA-clearing enzymes, voltage-gated sodium channels, and HDACs (Johannessen, 2000; Löscher, 1999), but not enzymes regulating one carbon metabolism, disruption of which is an off-target effect. Molecular docking analyses of on-target effects have calculated binding free energies of VPA with enzymes regulating GABA at between -3.21 to -6.21 Kcal/mol (Piplani, Verma & Kumar, 2016) (K i 34.68 mM-28.29 µM; GABA transaminase -4.99 Kcal/mol and 218.28 µM; succinate semialdehyde dehydrogenase –5.33 Kcal/mol and 124.44 µM), and with NavMs and Nav1.2 voltage-gated sodium channels at -3.3 to -6.2 Kcal/mol (Zanatta et al., 2019). If valproate medications’ therapeutic effects were resulted from binding to a single target, these values would indicate relatively weak affinity (Tonge, 2018); valproate medications, however, appear promiscuous in their interactions, which may be due to their small, branched chain fatty acid structure. Islet cell GABA receptors and voltage-gated sodium channels contribute to the regulation of insulin secretion but are not present in PACs (Babic & Travagli, 2014; Petersen & Findlay, 1987), secretion from which is only indirectly modulated by GABA via the dorsal motor nucleus of the vagus (Babic & Travagli, 2014). Off-target, VPA binding to glutathione reductase, which maintains the supply of reduced glutathione to protect cells from reactive oxygen species, has been calculated to have binding free energy of -5.07 Kcal/mol and K i 190.91 µM (Alam et al., 2023). Here, we found binding free energies between Na-VPA and enzymes regulating the methionine and folate cycles from -4.8 to -5.6 Kcal/mol (K d 301-78 µM; lowest values were for MAT2A), of a similar order to those described for on-target effects, although these parameters describe thermodynamic not kinetic stability (Tonge, 2018). Nevertheless, this order of binding with multiple enzymes regulating one carbon metabolism implicates direct interaction of Na-VPA and its metabolites with these enzymes in the toxic effects of valproate medications on the pancreas. One carbon metabolism has been increasingly implicated as a pathogenetic contributor to AP (Huang et al., 2022; Lu et al., 2003; Rius-Perez et al., 2020). Building on the finding of decreased hepatic SAM in CDE-AP, Lu et al administered 15 mg/kg SAM i.p. every 24 h before and during CDE-AP induction, which not only corrected pancreatic SAM deficiency but also prevented AP (Lu et al., 2003). Our study has demonstrated similar effects of in vivo and in vitro administration of SAM on alleviating Na-VPA associated cell injury and pancreatic injury. In CER-AP induced by 7 injections of caerulein, Rius-Pérez et al found marked depletion of pancreatic methionine, SAM and glutathione but not SAH or homocysteine within 3 h of the first caerulein injection (Rius-Perez et al., 2020), as similar in our findings. However, SAM administration exacerbated CER-AP, attributed to transsulfuration blockade caused by inducible nitric oxide synthase-mediated tyrosine-nitration of CBS. It was not in line with the findings from our study and the previous CDE-AP study, maybe due to the difference of model, dosage, and dosing frequencies. A metabolomics study of CER-AP showed similar changes in pancreatic methionine, SAM, and glutathione levels at the same time points, but these levels rose significantly at 12 and 24 h, suggesting either compensation and/or blockade (Huang et al., 2022). Unlike in our work that showed 4-ene-VPA and other VPA metabolites to be toxic to PACs, these studies did not examine the impact of VPA metabolites. Nevertheless, as we show for the pancreas, these metabolites are implicated in injury of various tissues (Ghodke-Puranik et al., 2013; Kassahun, Farrell & Abbott, 1991; Rettie, Rettenmeier, Howald & Baillie, 1987; Tang & Abbott, 1996), notably liver where VPA toxicity can be severe (Ghodke-Puranik et al., 2013; Zhu et al., 2024): there 4-ene-VPA is generated by cytochrome P-450, and VPA and its metabolites inhibit mitochondrial β-oxidation, compromising ATP production and NAD(P)H generation (Ezhilarasan & Mani, 2022). In PACs we found Na-VPA, 3-keto-VPA, and 4-ene-VPA cause mitochondrial impairment independent of cyclophilin D, consistent with impairment of β-oxidation that is not improved or may be exacerbated by deletion of cyclophilin D (Tavecchio, Lisanti, Bennett, Languino & Altieri, 2015). Unlike with longer term administration of valproate medications (Eisses et al., 2015; Ghoneim, Alrefai, Elsamanoudy, Abo El-Khair & Khalaf, 2020; Walker, Smith, Barsoum & Macallum, 1990; Xu, Guo, Liang, Li & Chen, 2019), our experiments in PACs were of necessity short-term using high concentrations of Na-VPA and VPA metabolites, since PACs dedifferentiate and do not survive more than 24-48 h in culture (Logsdon & Williams, 1983). Despite this, injury emerged at concentrations as low as 1 mM, which could be approached if not reached in vivo by the combined concentrations of Na-VPA and its metabolites over the medium- or long-term, when other variables may come into play e.g. neurohormonal stimulation (Ben-Cherif, Dridi, Aouam, Ben-Attia, Reinberg & Boughattas, 2013), higher demands for β-oxidation (Ezhilarasan & Mani, 2022), and/or increased amino acid loading of the pancreas (Danchin, Sekowska & You, 2020; Rooman et al., 2013; Saluja, Dudeja, Dawra & Sah, 2019), on a background of differing predisposing risks (Barreto et al., 2021). \articletype Original Articles 5. Conclusions In summary, our findings identify disruption of one-carbon metabolism as a central mechanism by which Na-VPA and its metabolites injure pancreatic acinar cells and exacerbate experimental pancreatitis. By depleting methionine-cycle intermediates, directly interacting with key metabolic enzymes, and activating ER stress pathways, Na-VPA compromises essential metabolic and proteostatic processes required for acinar cell integrity. These metabolic disturbances occurred independently of pathological calcium signaling, delineating a distinct injury pathway from classical mechanisms of AP. Importantly, restoration of methionine-cycle metabolites, particularly through SAM supplementation, significantly mitigated Na-VPA–induced acinar cell injury in vitro and pancreatic damage in vivo , highlighting the therapeutic potential of targeting metabolic vulnerability in valproate-associated AP. Collectively, these results provide mechanistic insight into DAAP caused by valproate medications and suggest that metabolic support of one-carbon pathways may represent a promising strategy for prevention or intervention. \articletype Original Articles Acknowledgements \articletype Original Articles The authors thank the staff in the Biomedical Service Unit of University of Liverpool for their assistance in animal management. \articletype Original Articles Author Contributions WC: Conceptualisation; methodology; validation; formal analysis; visualization, investigation; writing—original draft; writing—review and editing. DW and YL: methodology; software, visualization, investigation; writing—review and editing. MC: Conceptualisation; methodology. MA, SSF, and XM: Methodology; validation. DL: Data curation. JPB and ZL: Methodology. AE: Formal analysis. WHe, RM, PS, DNC: Writing-review and editing. CG: Funding acquisition, writing—review and editing. QX and WHuang: Funding acquisition, resources, writing—review and editing. RS: Conceptualisation; methodology; funding acquisition, resources, project administration, writing—original draft; writing—review and editing. \articletype Original Articles Availability of data and materials The data that support the findings of this study are available from the corresponding author upon reasonable request. The code for the RNA-seq and pseudo-targeted metabolomics analysis were in the Github: https://github.com/Pancreatologist/Sodium-valproate-induces-pancreatic-injury. \articletype Original Articles Ethics approval All animal studies were ethically reviewed and conducted according to UK Animals (Scientific Procedures) Act 1986 under a project license approved by UK Home Office. \articletype Original Articles Funding \articletype Original Articles This work was supported by the China Scholarship Council (201806240274 to WC), West China-Liverpool Clinician Scientist Development Award (to WC), National Institute for Health and Care Research Senior Investigator Award (to RS), TransBioLine Consortium from the Innovative Medicines Initiative 2 Joint Undertaking (821283 to CG and RS). Conflict of Interest Statement RS has consulted for AbbVie, CalciMedica, Cypralis, Eagle Pharmaceuticals, GlaxoSmithKline (GSK), Graybug, Novartis, Oppenheimer, Perceptive Advisors, Quark Ventures, Recro Pharma, Soleus Capital, Takeda, and Vical; and has received research funding from EA Pharma, GSK, Lilly, Merck/MSD, Pfizer as well as multiple public sources in the last five years; and has received drug supply of Remicade® (infliximab) for the RAPID-I trial ( NCT03684278 funded by UK NIHR/MRC EME Programme) from Merck/MSD. RS is collaborating in the IMI2 TransBioLine Consortium with Janssen (J&J), Lilly, Merck/MSD, Novartis, Pfizer, Genentech (Roche), and Sanofi-Aventis. All funds received have been paid to the University of Liverpool and/or Liverpool University Hospitals NHS Foundation Trust. \articletype Original Articles REFERENCES Alam MN, Singh L, Khan NA, Asiri YI, Hassan MZ, Afzal O , et al. (2023). 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Mechanisms of Pancreatic Injury Induced by Basic Amino Acids Differ Between L-Arginine, L-Ornithine, and L-Histidine. Front Physiol 9 : 1922.Zhu J, Wang Z, Sun X, Wang D, Xu X, Yang L , et al. (2024). Associations between one-carbon metabolism and valproic acid-induced liver dysfunction in epileptic patients. Front Pharmacol 15 : 1358262. \articletype Original Articles Figure legends Fig . 1. In vitro , Na-VPA and its metabolites caused dose and time dependent PAC cell death independent of toxic calcium signaling. (A) 1-20 mM Na-VPA caused time and dose dependent cell death; data were presented as mean ± SEM with 17 cell preparations for each group. (B) Confocal time-lapse imaging confirmed the cell death caused by 20 mM Na-VPA. (C-F) The parent drug valproic acid and metabolites 2-ene-VPA, 3-keto-Na-VPA, and 4-ene-VPA at concentrations ranging from 1-10 mM also caused PAC cell death; data were presented as mean ± SEM with ≥ 3 cell preparations for each group. (G-L) Na-VPA toxicity is independent of calcium overload, representative calcium signals in the presence of Na-VPA at various conditions were displayed, 30-50 cells were recorded from ≥ 3 cell preparations. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Na-VPA, sodium valproate; VPA, valproic acid; TPG, thapsigargin; PAC, pancreatic acinar cell; SEM, standard error of the mean. Fig. 2. In vivo , Na-VPA pretreatment exacerbated pancreatic damage of a mild CER-AP mouse model. (A) Experimental design: mice received Na-VPA (500 mg/kg/day, i.p.) for 8 days. On day 8, the Na-VPA+CER-AP group additionally received seven injections of caerulein (50 μg/kg, 1-h intervals). The Na-VPA group received Na-VPA alone; the CER-AP group received caerulein alone; controls received saline. Each group contained 8 mice. (B) Body weight changes during Na-VPA treatment. (C) Representative H&E-stained pancreatic sections. (D) Histological scoring of oedema, inflammatory infiltration, necrosis, and total injury. (E–F) Serum amylase and lipase. (G–H) Pancreatic trypsin and MPO activities. (I–K) Serum IL-6, ALT, and AST levels. (L) PCA of all pancreatic transcriptomic samples. (M) Volcano plot of DEGs (Na-VPA vs. control), highlighting upregulated (red) and downregulated (blue) genes. (N) Volcano plot of DEGs (Na-VPA+CER-AP vs. CER-AP). (O) Venn diagram illustrating overlap of DEMs among the two contrasts. (P) GSEA of DEGs (Na-VPA vs. control) showing the top five activated and suppressed pathways ranked by NES. (Q) GSEA of DEGs (Na-VPA+CER-AP vs. CER-AP), with top five activated and suppressed pathways by NES. Na-VPA, sodium valproate; i.p., intraperitoneal; CER-AP, caerulein-induced acute pancreatitis; H&E, haematoxylin and eosin; MPO, myeloperoxidase; IL-6, interleukin-6; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, principal component analysis; DEG, differentially expressed gene; DEM, differentially expressed metabolite; GSEA, gene set enrichment analysis; NES, normalized enrichment score. Fig. 3. Pseudo-targeted metabolomics analysis of pancreatic tissue. (A) PCA of variance-stabilized metabolite expression, with each point representing an individual sample and colors indicating experimental groups. (B) Volcano plots showing differentially expressed metabolites (DEMs) for three contrasts: Na-VPA vs. control, CER-AP vs. control, and Na-VPA+CER-AP vs. CER-AP. Increased metabolites are shown in red and decreased metabolites in blue. (C) Venn diagram illustrating overlap of DEMs among the three contrasts. (D) KEGG pathway enrichment of DEMs for Na-VPA vs. control (left) and Na-VPA+CER-AP vs. CER-AP (right). (E) Box plots of key metabolites involved in the one-carbon metabolism pathway. (F) Correlation of SAM and methionine levels with expression of one-carbon metabolism-related genes (left), and classification of all detected metabolites (right). Na-VPA, sodium valproate; CER-AP, caerulein-induced acute pancreatitis; SAM, S-adenosylmethionine; SAH, S-adenosyl-homocysteine; THF, tetrahydrofolate; PCA, principal component analysis; DEM, differentially expressed metabolite; KEGG, Kyoto Encyclopedia of Genes and Genomes. Fig. 4. Na-VPA disrupts expression of rate-limiting enzymes in one carbon metabolism and molecular docking analysis. (A) In vitro , Na-VPA perturbates mRNA expressions of rate-limiting enzymes in one carbon metabolism of PACs from 1h to 18h, n ≥ 3 for each group. (B-C) In vivo , Na-VPA dysregulates protein expressions of rate-limiting enzymes in one carbon metabolism in pancreas. (D) Binding affinities between VPA and one carbon metabolism enzymes. (E-I) Graphical representation of modeling interactions formed by VPA and five enzymes including MTHFR, MTR, MAT2A, GNMT, and AHCY. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; MAT1A, methionine adenosyltransferase 1A; MAT2A, methionine adenosyltransferase 2A; GNMT, glycine N-methyltransferase; AHCY, S-adenosylhomocysteine hydrolase; CBS, cystathionine β-synthase; Na-VPA, sodium valproate. Fig. 5. Na-VPA causes ER stress in PACs in vitro and pancreas in vivo . (A) mRNA expressions of ER stress markers in vitro. (B) mRNA expressions of ER stress markers in vivo . (C) Protein expressions of ER stress markers in vivo . (D) mRNA expression of oxidative stress marker Hmox1. (E) mRNA expressions of inflammation markers. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. BiP, binding immunoglobulin protein; CHOP, c/ebp homologous protein; Ddit3, DNA damage inducible transcript 3; ER, endoplasmic reticulum; Ern1, endoplasmic reticulum to nucleus signaling 1; Eif2s1, eukaryotic initiation factor 2 subunit alpha; Hmox1, heme oxygenase 1; Hspa5, heat shock protein family a member 5; IL-1β, interleukin 1 beta; sXBP1, spliced x-box binding protein 1; uXBP1, TNF-α, tumor necrosis factor alpha; unspliced x-box binding protein 1. Fig. 6. In vitro , Na-VPA caused disturbance of one carbon metabolism in PACs, supplementation of metabolites protected against Na-VPA induced PAC death. (A) Representative base peak intensity chromatograms of LC-MS/MS showing the target metabolites including methionine, SAM, SAH, and homocysteine were well-acquired. (B) Changes of the levels of major metabolites in methionine cycle in PACs after Na-VPA exposure, SAM and SAH levels were significantly reduced; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; n = 3 for each group. (C) Methionine at 0.5 mM and 1.0 mM significantly reduced Na-VPA caused cell death, n = 3 for each group. (D) Ethionine (antagonist of methionine) at 5 mM and 10 mM significantly exacerbated cell death, n = 3 for each group. (E) SAM at 0.5 mM and 1.0 mM significantly reduced cell death, n = 3 for each group. * P < 0.05, # P < 0.01, & P < 0.001, Na-VPA plus metabolite vs Na-VPA alone at the same concentration of Na-VPA at each time point. Na-VPA, sodium valproate; PAC, pancreatic acinar cell; SAM, S-adenosylmethionine; SAH, S-adenosyl-homocysteine. Fig. 7. SAM supplementation alleviated Na-VPA exacerbated CER-AP in vivo . (A) Experimental design: mice received Na-VPA (500 mg/kg/day, i.p.) and SAM (15 mg/kg/day, i.p.) for 8 days; on day 8, the Na-VPA+CER-AP group additionally received seven injections of caerulein (50 μg/kg, 1-h intervals); the SAM group received SAM alone; each group contained 8 mice. (B) Representative H&E-stained pancreatic sections. (C) Histological scoring of oedema, inflammatory infiltration, necrosis, and total injury. (D–E) Serum amylase and lipase. (F–G) Pancreatic trypsin and MPO activities. (H-J) Serum IL-6, ALT, and AST levels. (K) Schematic illustration summarizing the proposed mechanism of Na-VPA–induced pancreatic toxicity. Information & Authors Information Version history V1 Version 1 09 January 2026 Peer review timeline Published British Journal of Pharmacology Version of Record 21 Apr 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection British Journal of Pharmacology Authors Affiliations Wenhao Cai West China Hospital of Sichuan University View all articles by this author Di Wu The First Affiliated Hospital With Nanjing Medical University View all articles by this author Yuying Li West China Hospital of Sichuan University View all articles by this author Michael Chvanov University of Liverpool Institute of Systems Molecular and Integrative Biology View all articles by this author Mohammad Awais University of Liverpool Institute of Systems Molecular and Integrative Biology View all articles by this author Shiva Seyed Forootan University of Liverpool Department of Pharmacology and Therapeutics View all articles by this author Xiaoli Meng 0000-0002-7774-2075 University of Liverpool Department of Pharmacology and Therapeutics View all articles by this author Diane Latawiec University of Liverpool Institute of Systems Molecular and Integrative Biology View all articles by this author Joseph Brown P University of Liverpool Department of Pharmacology and Therapeutics View all articles by this author Ziyu Li West China Hospital of Sichuan University View all articles by this author Wenhua He First Affiliated Hospital of Nanchang University Jiangxi Department of Gastroenterology View all articles by this author Anthony Evans University of Liverpool Institute of Systems Molecular and Integrative Biology View all articles by this author R. Mukherjee University of Liverpool Institute of Systems Molecular and Integrative Biology View all articles by this author Peter Szatmary University of Liverpool Institute of Systems Molecular and Integrative Biology View all articles by this author David Criddle N University of Liverpool Institute of Systems Molecular and Integrative Biology View all articles by this author Christopher Goldring University of Liverpool Department of Pharmacology and Therapeutics View all articles by this author Wei Huang West China Hospital of Sichuan University View all articles by this author Qing Xia West China Hospital of Sichuan University View all articles by this author Robert Sutton [email protected] University of Liverpool Institute of Systems Molecular and Integrative Biology View all articles by this author Metrics & Citations Metrics Article Usage 332 views 126 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Wenhao Cai, Di Wu, Yuying Li, et al. Sodium valproate induces pancreatic injury by disruption of one carbon metabolism. Authorea . 09 January 2026. DOI: https://doi.org/10.22541/au.176797098.85981401/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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