Functional and Transcriptional Effects of a Hydrogen Sulfide Donor on the Intestinal Epithelial Barrier

Functional and Transcriptional Effects of a Hydrogen Sulfide Donor on the Intestinal Epithelial Barrier | 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 Functional and Transcriptional Effects of a Hydrogen Sulfide Donor on the Intestinal Epithelial Barrier Janaíne Prata Oliveira, Matthias Sligtenhorst, Cansu Akkaya, Astrid Verbiest, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7324506/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted 15 You are reading this latest preprint version Abstract The intestinal epithelial barrier is essential for protecting against pathogens and toxins while permitting nutrient and water absorption. Barrier dysfunction is a hallmark of inflammatory diseases affecting the gastrointestinal (GI) tract and beyond. Hydrogen sulfide (H₂S) has emerged as a critical regulator of intestinal homeostasis. This study examines the effects of the H₂S-releasing compound 4-hydroxithiobenzamide (TBZ) on epithelial barrier integrity. While TBZ did not prevent interferon-γ and tumor necrosis factor-α (IFN/TNF)-induced epithelial cell death, it reversed cytokine-induced increases in transepithelial permeability. Interestingly, TBZ alone elevated paracellular permeability, yet normalized it under inflammatory conditions, indicating a context-dependent effect. H₂S-producing enzymes localized apically in intestinal epithelial cells, suggesting spatial regulation. Transcriptomic analysis implicated oxidative phosphorylation as a pathway mediating TBZ’s effects. These findings advance our understanding of H₂S in intestinal barrier regulation and support TBZ as a candidate therapeutic agent for conditions marked by barrier dysfunction in an inflammatory context. Biological sciences/Cell biology Health sciences/Diseases Health sciences/Gastroenterology Biological sciences/Immunology Biological sciences/Molecular biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The intestinal epithelial cell (IEC) monolayer is crucial for maintaining the intestinal barrier, which protects against xenobiotics and pathogens while regulating nutrient absorption and permeability 1 , 2 . Disruption of this barrier is associated with a range of gastrointestinal (GI) diseases and is increasingly recognized as a central mechanism driving systemic conditions 3 , 4 . Such disruption often involves increased paracellular permeability due to the breakdown of junctional complexes or epithelial cell death 5 . Hydrogen sulfide (H₂S) is a pleiotropic gasotransmitter that exhibits protective effects at physiological levels through its anti-inflammatory and antioxidant properties but becomes toxic at high concentrations 6 , 7 . In humans, H 2 S is endogenously produced by enzymes such as cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CTH, or CSE), and 3-mercapto-sulfurtransferase (MPST, or 3-MST) 8 , 9 . Impaired biosynthesis of endogenous H 2 S worsens intestinal barrier integrity and promotes conditions like colitis 10 , 11 . In contrast, exogenous H 2 S delivery restores barrier function and reduces intestinal inflammation in colitis models 12 . Interestingly, evidence suggests that sulfur-linked compounds, such as 4-hydroxithiobenzamide (TBZ) 13 , have the potential to release protective H₂S. This effect has been observed in association with both steroidal 14 and non-steroidal anti-inflammatory drugs 15 . For example, coupling of TBZ to ketoprofen (ATB-352) enhances analgesic efficacy while eliminating harmful gastric effects 16 . Similarly, TBZ coupled to naproxen (ATB-346) prevents intestinal damage and inflammation 17 . These findings underscore the clinical potential of the H 2 S donor TBZ, although its effects in isolation or the molecular mechanisms underlying its protective effects remain unclear. Most studies on H 2 S donors have employed in vivo models, making it difficult to pinpoint its individual contributions specifically to epithelial permeability and cell death during intestinal barrier failure. Moreover, insights into the involved molecular mechanisms largely stem from targeted approaches investigating a limited number of genes or signaling pathways. This study seeks to evaluate the protective effects of TBZ on IECs, assessing cellular viability and intestinal barrier integrity under both normal and inflammatory conditions. Additionally, we mapped the distribution of endogenous H 2 S-synthesizing enzymes in the healthy human colorectal mucosa. To gain unbiased insights into the involved mechanisms, we employed bulk RNA-sequencing of cells treated with TBZ under normal and inflammatory conditions. Results Effect of H 2 S donors on IEC viability. Before starting the experimental design (Fig. 1 A), we determined the cytotoxicity of TBZ at different concentrations (5, 50 and 500 µM) in Caco-2/TC7 cells. Here we found that only 500 µM caused any significant cytotoxic effects (Fig. 1 B), as determined by the relative reduction in luminescence, indicating reduced reductive capacity of cells; a proxy for the number of live cells in culture. Afterwards, we assessed cell viability of Caco-2/TC7 cells using the same assay under normal conditions or after stimulation with IFN/TNF, followed by treatment with vehicle or 50 µM TBZ. Accordingly, TBZ treatment did not affect cell viability compared to vehicle-treated cells, however, stimulation of cells with IFN/TNF significantly reduced cell viability compared with vehicle (Fig. 1 C). This was not reversed by treatment with 50 µM TBZ. Similar results were obtained in intestinal organoids after treatment of control or IFN/TNF stimulated organoids (Fig. S1 A). These results were further confirmed in Caco-2/TC7 and HIEC-6 cells by quantifying intracellular ATP levels, which again can be used as a proxy for cell viability. In both cell lines, there was no cytotoxic effect of 50 µM TBZ observed in the absence of inflammatory stimuli (Fig. 1 D-E). Stimulation of cells with IFN/TNF significantly reduced cell viability compared with vehicle, which was again not rescued by 50 µM TBZ. Effect of the H 2 S donor on intestinal epithelial barrier integrity As HIEC-6 cells cannot reach full confluence, we employed Caco-2/TC7 cell monolayers in a trans-well system as well as intestinal organoids to investigate the effect of H 2 S donors on epithelial barrier integrity (Fig. 2 A). In the absence of inflammatory stimuli, treatment with TBZ for 72 h did not change trans-epithelial resistance (TEER) of Caco-2/TC7 cell monolayers when compared to vehicle. Conversely, stimulation with IFN/TNF reduced TEER, whereas treatment with TBZ partially reversed this effect (Fig. 2 B). We further investigated the effects of the H 2 S donor on epithelial barrier function by assessing paracellular permeability of FITC-Dextran in both Caco-2/TC7 monolayers and intestinal organoids. We observed that treatment with TBZ for 72 hours had contrasting effects under control vs. inflamed contexts. When alone, 50 µM TBZ increased paracellular permeability to FITC-Dextran in both Caco-2/TC7 cells and organoids. Stimulation with IFN/TNF also resulted in increased paracellular permeability. In contrast though, 50 µM TBZ reversed this disruptive effect of IFN/TNF and restored FITC-dextran influx to levels observed in vehicle-treated non stimulated cells and organoids (Fig. 2 C-E). Moreover, these effects were concentration-dependent in organoids (Fig. S1 B). Effect of TBZ on gene expression To gain insights into the mechanisms underlying the protective effects of the H 2 S donor on IECs stimulated with IFN/TNF, we performed an exploratory RNA-seq analysis. Stimulation of control Caco-2/TC7 cells with TBZ resulted in the highest number of differentially expressed genes (DEGs; logFC > 2 or < -2). In total, 494 genes showed altered expression, with 335 upregulated and 159 downregulated. In contrast, stimulation with TNF/IFN exhibited a less pronounced transcriptional effect, altering the expression of 76 genes. Comparative analysis between IFN/TNF-stimulated cells treated or untreated with TBZ revealed 46 differentially expressed genes. When comparing TBZ-treated cells under control vs. inflamed (IFN/TNF-stimulated) conditions, only five genes were differentially expressed (Fig. 3 A, Data S1-4). These findings suggest that TBZ exerts a strong regulatory effect on transcription. Moreover, the limited number of differentially expressed genes between cell groups (control vs. inflamed) treated with TBZ indicates that this H 2 S donor has dominant transcriptional activity in relation to this inflammatory stimulation. Principal component analysis (PCA) further supports these conclusions (Fig. 3 B). Clustered heatmap analysis revealed the regulation of four major gene modules. Gene module S1 was broadly regulated by stimulation of Caco-2/TC7 cells with IFN/TNF and/or TBZ. Notably, the pro-inflammatory gene LYPD6B was strongly upregulated. Members of the LY6 superfamily are known to be upregulated in the intestinal epithelium in inflammatory bowel disease (IBD), and their cross-linking promotes chemokine production 18 . Genes in module S2 were specifically upregulated by TBZ in both control and IFN/TNF-treated cells. Among these, NDUFA4 and COX5A , which are involved in oxidative phosphorylation in mitochondria were noticeably upregulated. In contrast, gene module S3 was downregulated by TBZ, while module S4 was downregulated by both TBZ and/or IFN/TNF stimulation (Fig. 3 C). Hence, we performed geneset enrichment analysis using the HALLMARK collection. With a threshold of logFC > 0.5, we identified oxidative phosphorylation-related genes as differentially enriched in control Caco-2/TC7 cells treated with TBZ (logFC = 0.55) (Fig. 3 D, E). This enrichment was driven by the upregulation of genes such as COX5A , FH , COX6C , NDUFA4 , and others (Data S5). In additional comparisons, the logFC threshold was lowered to > 0.2. In IFN/TNF-stimulated cells, we observed the enrichment of three genesets: Interferon Response, G2M Checkpoint, and Apical Surface (logFC = 0.24, 0.24, and 0.20, respectively). Among the genes associated with the interferon response, IFIH1 , IFITM2 , CASP1 , and CASP8 are key examples of upregulated genes (Fig. 3 F, G; Data S6). Similar to the findings in control cells, TBZ treatment of IFN/TNF-stimulated cells also enriched the Oxidative Phosphorylation pathway (logFC = 0.35). This enrichment was driven by the upregulation of genes such as COX5A , COX6C , NDUFA4 , and members of the solute carrier family ( SLC25A3-5 ) (Fig. 3 H, I; Data S7). Finally, a comparison between control and IFN/TNF-treated cells exposed to TBZ revealed only mild differences in geneset enrichment (Fig. 3 J, L; Data S8). Changes in gene expression were further confirmed with RT-qPCR, where the expression of LYPD6B, NDUFA4, and COX6C were assessed (Fig. 3 M-O). Immunoreactivity pattern of HS-synthesizing enzymes We assessed protein expression of the H 2 S-synthetizing enzymes CSE and 3-MST by immunostaining colorectal tissue slices prepared from mucosal biopsies obtained from healthy volunteers. Overall, the expression of CSE and 3-MST was more pronounced in the human ascending colon (Fig. 4 A) than in the rectum (Fig. 4 B). In the ascending colon, 3-MST staining was highly enriched in epithelial cells (co-localized with the pan epithelial marker EPCAM). Conversely, CSE expression was present in epithelial cells as well as in cells within the lamina propria. Of note, both CSE and 3-MST displayed a similar subcellular distribution pattern in colonic epithelial cells, with a marked enrichment in the apical pole. Discussion Improving the intestinal barrier is a promising therapeutic strategy for treatment of chronic intestinal diseases, disorders affecting the gut-liver axis and other distant organ systems rooted in an impaired intestinal barrier 19 . Previous studies using hybrid H 2 S-releasing NSAIDs have shown protective effects on the gastrointestinal system 17 , 20 , 21 . However, most of these studies were conducted in vivo , and the underlying cellular mechanisms remain unclear. In this in vitro study, we aimed to investigate whether the effect of H 2 S on the intestinal barrier is associated with a regulatory role over cell death and/or epithelial permeability. Our findings show that TBZ did not influence cell death induced by IFN/TNF in both Caco-2/TC7 and HIEC-6 cells. These observations suggest that TBZ does not counteract the pro-apoptotic effects of IFN/TNF, potentially mediated by caspases 1 and 8, which are both upregulated in cells stimulated with these cytokines 22 , 23 . The intrinsic apoptosis pathway involves the release of pro-apoptotic proteins from the mitochondrial intermembrane space into the cytosol, while the extrinsic pathway, triggered by death receptors of the TNF superfamily, recruits cytoplasmic adaptor proteins and activates downstream caspase cascades 24 . While H₂S may protect against cell death induced by the intrinsic or mitochondrial pathway through upregulation of oxidative phosphorylation genes, it appears to have no effect on cell death mediated by the extrinsic pathway, which is the context of our study design. In addition to inducing apoptosis, the combination of IFN and TNF disrupts the intestinal epithelial barrier by increasing myosin light chain kinase (MLCK1) activity, leading to endocytosis of the tight junction protein occludin and increased paracellular permeability 25 . In our study, TBZ alone had no effect on transcellular permeability. However, while IFN/TNF treatment reduced transepithelial electrical resistance (TEER), TBZ effectively restored this barrier function. Notably, TBZ exhibited divergent effects on paracellular permeability: it increased FITC-dextran influx in untreated Caco-2/TC7 cells and organoids but restored epithelial barrier integrity in cells stimulated with IFN/TNF. These findings align with previous reports highlighting the pleiotropic effects of H₂S, which can influence cellular homeostasis, inflammation, and mitochondrial function in a concentration- and context-dependent manner 26 , 27 . Thus, while our data reinforce prior evidence supporting the protective role of H₂S against inflammation-induced intestinal barrier disruption 12 , 28 , further research is needed to determine the precise contexts in which H₂S could be clinically beneficial. To gain deeper insights into the molecular mechanisms involved in epithelial barrier protection by H 2 S in an inflammatory context, we performed an exploratory RNA-seq analysis followed by cross-validation by RT-qPCR. As mentioned, treatment with TBZ displayed the strongest transcriptional effect, surpassing the effect of IFN/TNF. It is unclear though if TBZ has a more dominant effect on transcription over IFN/TNF, since stimulation with these cytokines took place 24 h before cells were exposed to TBZ and further cultured for additional 72 h. Nevertheless, our findings do indicate that H 2 S has a regulatory action on gene expression that has not been reported before. Additionally, geneset enrichment analysis highlighted regulation of oxidative phosphorylation in both control and IFN/TNF-treated cells exposed to TBZ, indicating that H 2 S may serve as a master regulator of mitochondrial homeostasis through transcriptional regulation. This enrichment was driven by upregulation of multiple genes, including COX5A , COX6C , NDUFA4 , and SLC25A3-5 . The subunits COX5A and COX6C are components of cytochrome c oxidase (Complex IV), which plays a crucial role in the electron transport chain and ATP synthesis. COX5A modulates Complex IV activity, while COX6C maintains its structural integrity 29 . NDUFA4 , another component of Complex IV, contributes to electron transport and proton gradient formation, and its dysfunction is linked to mitochondrial disorders 30 . Furthermore, SLC25A3-5 are mitochondrial transporters that facilitate the exchange of essential metabolites like phosphate and ADP/ATP, thus ensuring efficient ATP production and cellular energy balance 31 . Although no direct evidence in the existing literature links these specific genes to tight junction regulation or intestinal permeability, mitochondrial function, oxidative stress, and cellular energy balance are key regulators of epithelial barrier integrity 25 , 32 , 33 . Indeed, maintenance of this barrier is an energy-intensive process that relies on the coordination of tight junction proteins, cytoskeletal components, and other junctional elements 34 . Therefore, disruptions in mitochondrial activity can compromise tight junctions and increase permeability. A recent study demonstrated that STARD7, which mediates phosphatidylcholine transfer from the endoplasmic reticulum to mitochondria for membrane stabilization and respiration, is downregulated in the epithelium of patients with IBD. Moreover, its deficiency impairs tight junctions, the intestinal barrier, and worsens experimental colitis 35 . Several studies have assessed the regulation of H 2 S-synthesizing enzymes in IBD and in pre-clinical models of colitis, and although there are conflicting findings, the expression of H 2 S-synthesizing enzymes has been reported to be altered in these conditions, particularly in those sites exhibiting active inflammation 10 , 36 – 38 . For instance, a recent study reported that 3-MST expression was markedly decreased in colonic samples collected from both patients with IBD and DSS-treated mice, while 3-MST deficiency led to increased colitis severity 39 . In order to get better insights into the cellular and subcellular distribution of H 2 S-synthesizing enzymes in the human intestinal epithelium, we performed immunofluorescence on human colorectal tissue sections from healthy volunteers. One key finding in these experiments was the observation that 3-MST and CSE immunoreactivities were overall enriched in the apical pole of colonic epithelial cells, therefore, coincidently with the localization of junctional complexes. This subcellular enrichment in colonic epithelial cells might have interesting functional purposes in regulating mitochondrial homeostasis and junctional complex integrity mediated through H 2 S synthesis. In other polarized cells, such as neurons, there is growing evidence pointing to mitochondrial morphology and functional diversity within subcellular domains 40 . Nevertheless, this aspect remains uncharted in intestinal epithelial cells. Our study underpins cellular and molecular mechanisms underlying the protective effect of TBZ towards intestinal epithelial barrier integrity in inflammation. Of note, we provide evidence indicating that H 2 S may represent a master regulator of genes involved in oxidative phosphorylation and mitochondrial homeostasis. These findings highlight promising therapeutic prospects for H 2 S-releasing compounds to treat conditions rooted in mitochondrial and intestinal epithelial barrier dysfunction. However, a deeper understanding of H₂S’s dual effects is essential to harness its benefits while mitigating potential risks. Material and Methods IEC culture The human colon adenocarcinoma cell line Caco-2/TC7 (Sigma Aldrich, Germany; cat. #SCC209), and the normal human small intestinal epithelial cell line HIEC-6 (ATCC, United States; cat. #CRL-3266) were genotyped by the commercial suppliers through STR analysis to verify the unique identity of the cell lines. The cells were mycoplasma free and were grown under hypoxia (5% O 2 , 95% CO 2 , 37ºC and 90% humidity, CBF260; Binder, GmbH). Caco-2/TC7 cells were grown in DMEM/F12 medium, supplemented with 1% Glutamax, 10% FBS, and 1% penicillin/streptomycin. HIEC-6 cells were grown in Opti-MEM®I supplemented with 1% Glutamax, 10% Fetal Bovine Serum (FBS), and 1% penicillin/streptomycin. Experimental design Caco-2/TC7 and HIEC-6 cells were grown to 80–90% confluency before starting experiments. Cells were treated with TBZ (50 µM) or vehicle (DMSO 0.1%) for 72 hours. For the inflammatory conditions, cells were initially exposed to IFNγ (2.5 ng/mL) for 3 hours to induce expression of the receptor for TNFα, followed by treatment with TNFα (10 ng/mL) or vehicle (culture medium) for a total period of 24 hours. After which cells were treated with TBZ or vehicle for an additional 72 hours. Cellular viability assessment Th1 cytokines such as IFNγ and TNFα are key mediators in gastrointestinal lesions and have been reported to cause IEC apoptosis in a synergistic way 41 – 43 . Therefore, we assessed the effect of the H 2 S donor on IEC viability in normal and inflammatory conditions. Firstly, we employed the RealTime-Glo™ MT Cell Viability Assay (Promega; Cat. G9711) with Caco-2/TC7 cells, which measures the reductive capacity of cells, and is a proxy for cell viability. Secondly, we employed the CellTiter-Glo® Luminescent Cell Viability Assay (Promega; Cat. G7570) with both Caco-2/TC7 and HIEC-6 cells, which measures ATP levels, and is also a measure of cell viability. In brief, Caco-2/TC7 and HIEC-6 cells were plated in 96-well plates, containing 5x10 4 cells per well. After 1 week of growth, the experimental design aforementioned was followed. Afterwards, the cells were treated for 30 minutes with either RealTime-Glo™ MT Cell Viability reagent to measure the reductive capacity of cells, or with CellTiter-Glo® reagent to measure the amount of ATP, after which luminescence was measured using a luminometer (FLUOstar Omega, BMG Labtech, Hepatology Research Unit, KU Leuven). RealTime-Glo™ MT Cell viability assay readings were plotted against baseline. Raw CellTiter-Glo® assay readings were interpolated against an ATP standard curve (0.001–10 µM). Experiments were repeated independently 3 times. Evaluation of epithelial barrier integrity The non-tumoral cell line HIEC-6 expresses certain tight junction proteins, but lacks occludin, an important component of junctional complexes, and a major target of TNFα-induced barrier dysfunction 44 . Therefore, the assessment of the effects of the H 2 S donor on transcellular permeability was done with Caco-TC7 cells, while paracellular permeability was assessed in this cell line as well as in human intestinal organoids. In brief, 5x10 5 Caco-2/TC7 cells were seeded on a Costar 6.5 mm 2 Transwell system with polyester membrane inserts with 0.4 µm pores (StemCell Technologies) and differentiated for 21 days. 750 µL and 250 µL media were added to the basal and apical compartment of each well, respectively, and changed every 2 days. A well only containing culture medium was used as a blank. After 21 days, stimuli and treatment of intestinal epithelial were conducted, as described in the experimental design. Transepithelial electrical resistance (TEER) was measured with the EVOM device (EVM-MT-03-01, WPI, GmbH). The values were measured in Ω*cm 2 and blank-corrected. Moreover, paracellular permeability was measured by the influx of FITC-dextran (MW: 3 kDa) from the apical to the basal compartment. Briefly, after the last TEER measurement, wells were washed twice with DMEM/F12 without phenol red. FITC-dextran (250 µL; 100 µM in medium without phenol red) was added to the apical compartment and then 750 µL of culture medium was added to the basal compartment. Aliquots (100 µL) of the basal compartment were collected, and fluorescence intensity was measured using a microplate reader (FLUOstar Omega, BMG Labtec) with excitation at 496 nm, emission at 524 nm, and a cutoff wavelength at 515 nm. Additionally, intestinal organoids were used to assess paracellular epithelial barrier integrity. In short, organoids from diverse intestinal regions were cultured based on previously described methods 45 . For experimental setups, organoids were digested into single cells, 500 of which were plated in BME drops. Organoids were treated according to the experimental design after four days of growth. 72 hours after initiation of TBZ treatment, organoids were overlaid with advanced DMEM/F12 containing 100 µM FITC-dextran (MW: 3kDa). One hour afterwards, organoids were washed 3 times with fresh advanced DMEM/F12 and treated with DAPI to assess cell death. Imaging was performed using a Perkin Elmer Operetta CLS microscope (VIB BioImaging Core Leuven), after which the mean FITC-dextran and DAPI signal per organoid was analyzed using ImageJ. Bulk RNA-sequencing (RNA-seq) Caco-2/TC7 cells were treated according to the aforementioned experimental design. Three biological replicates per condition were pooled and subjected to total RNA extraction using the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Since biological replicates were pooled during RNA extraction, the RNA-seq analysis is exploratory (FDR = 1). RNA-seq was performed by the Genomics Core KU Leuven. Briefly, cDNA libraries were synthesized with the QuantSeq 3’ mRNA Seq Kit (Lexogen, Inc.) and sequenced on an Illumina HiSeq4000 system. The raw sequencing reads were initially checked for quality using FastQC, and adapter sequences and low-quality bases were trimmed using Trimmomatic. Reads were then aligned to the reference genome (GRCh38) using Hisat2. Following normalization, counts and sample tables were uploaded into Omics Playground v3 (BigOmics Analytics, Switzerland) for further assessment of quality control, differential gene expression, and geneset enrichment. A heatmap was generated using the ComplexHeatmap R/Bioconductor package 46 on scaled log-expression values (z-score) using Euclidean distance and Ward linkage using the fastcluster R package. The available methods to select the top features are sd (standard deviation) - features with the highest standard deviation across all the samples, marker - features that are overexpressed in each phenotype class compared to the rest, or by PCA - principal component analysis (performed using the irlba R package). Geneset enrichment was performed using CAMERA 47 , GSEA 48 , ssGSEA 49 , fGSEA, GSVA 50 , and fry 51 . The q-values yielded by the different methods were then combined into a meta-q value, where the meta value corresponds to the maximum. Hence, geneset enrichment analysis focused on the HALLMARK collection 52 . Immunofluorescence Human colorectal mucosa samples were collected from healthy volunteers. Tissue sections (10 µm thickness) were fixed with 4% paraformaldehyde (PFA) for 20 min at RT. The sections were then permeabilized with 0.03% Triton X-100 in PBS at room temperature for 5 min and then incubated with a blocking solution containing 3% BSA in PBS for 60 min at room temperature. The sections were subsequently incubated (overnight at 4°C) with primary antibodies: mouse anti-cystathionine γ-lyase (CSE; Invitrogen, cat. #MA5-25423,1:50), rabbit anti-3-mercaptopyruvate sulfurtransferase (3MST; Invitrogen, cat. #PA5-51548, 1:50) and goat anti-epithelial cell adhesion molecule (EPCAM; R&D Systems, cat. #AF960, 1:100). After primary incubation, the sections were incubated with secondary antibodies conjugated to the fluorophores AlexaFluor 488, 594, and 647 (Invitrogen) for 1.5 h at room temperature, and then stained for 5 minutes with DAPI. Cover slides were mounted using mounting medium (ProLong Gold, Thermo Fisher). Photomicrographs were captured using a confocal microscopy (Zeiss LSM 880) equipped with Zen software. Images were analyzed using Fiji Image J software (1.53 t, NIH, Baltimore, MD, USA, https://imagej.nih.gov/ij/ ). Statistical analysis Data are expressed as mean ± standard error of the mean (S.E.M). With the aid of GraphPad Prism v10 software, mean group differences were analyzed by either 1- or 2-way ANOVA, followed by post-tests with correction for multiple comparisons by controlling the false discovery rate. Values of p < 0.05 were considered significant. Declarations Funding: This study was supported by the KU Leuven startup grant STG/22/023 and C1 grant C14/23/135. JPO received a scholarship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (CNPq, 312514/2019-0) and from the Coordenação de Aperfeiçoamento de Pessoal e Nível Superior - Brazil (CAPES; 88887.694612/2022-00). We would like to acknowledge the support from FAPESP (processes 2016/06146-3 and 2019/14051-2). MNM and SKPC are recipients of scientific productivity scholarships from the Brazilian National Council for Scientific and Technological Development (CNPq; processes 306294/2019-2 and 312514/2019-0, respectively). Acknowledgments: Images were recorded on a Zeiss LSM 880 – Airyscan (Cell and Tissue Imaging Cluster (CIC), supported by Hercules AKUL/15/37_GOH1816N and FWO G.0929.15 to Pieter Vanden Berghe, University of Leuven. Figures 1A and 2A and the graphical abstract were created with BioRender.com. We thank Prof. Dr. Guy Boeckxstaens for providing rectal tissue samples from healthy volunteers and Dr. Hannelie Korf for reviewing the manuscript. Marc Ferrante is a Senior Clinical Investigator of the Research Foundation - Flanders (FWO), Belgium. Author contributions: JPO, MvS, SC and ADS: study conceptualization and design. JPO, MvS, MW, VFA and ADS: contributed to data acquisition. JPO, ADS, MvS, SC and CA: involved in data analysis and/or interpretation. JLW, AV, TV: resources. JPO and ADS: drafted the manuscript. ADS, JPO, MvS, CA, MNM, SvdM, MF, JLW and SC: Reviewing, Editing, Resources. All authors read and approved the manuscript. Data availability statement: The RNA-seq datasets generated and analyzed during the present study are publicly available in the European Genome-Phenome Archive (EGA) under accession number EGAS50000001237. Any additional original data reported in this study are available from the corresponding author upon reasonable request. Ethics statement: The study protocol was approved by the Ethics Committee of the University Hospital Leuven (Ethics protocols S62059 and S68855). The study was performed in accordance with the ethical standards established in the Declaration of Helsinki and written informed consent was obtained from all participants prior to the study. Conflict of interest disclosure: Marc Ferrante received research grants from AbbVie, EG Pharma, Janssen, Pfizer, Takeda and Viatris; consultancy fees from AbbVie, AgomAb Therapeutics, Boehringer Ingelheim, Celgene, Celltrion, Eli Lilly, Janssen-Cilag, Merck Sharp and Dohme, MRM Health, Pfizer, Takeda and ThermoFisher; and speakers’ fees from AbbVie, Biogen, Boehringer Ingelheim, Dr Falk Pharma, Ferring, Janssen-Cilag, Merck Sharp and Dohme, Pfizer, Takeda, Truvion Healthcare and Viatris. John L. Wallace is a co-founder of Antibe Therapeutics Inc. References Soderholm, A.T., and Pedicord, V.A. (2019). Intestinal epithelial cells: at the interface of the microbiota and mucosal immunity. Immunology 158 , 267-280. 10.1111/imm.13117. Odenwald, M.A., and Turner, J.R. (2017). The intestinal epithelial barrier: a therapeutic target? Nat Rev Gastroenterol Hepatol 14 , 9-21. 10.1038/nrgastro.2016.169. 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Marc Ferrante received research grants from AbbVie, EG Pharma, Janssen, Pfizer, Takeda and Viatris; consultancy fees from AbbVie, AgomAb Therapeutics, Boehringer Ingelheim, Celgene, Celltrion, Eli Lilly, Janssen-Cilag, Merck Sharp and Dohme, MRM Health, Pfizer, Takeda and ThermoFisher; and speakers’ fees from AbbVie, Biogen, Boehringer Ingelheim, Dr Falk Pharma, Ferring, Janssen-Cilag, Merck Sharp and Dohme, Pfizer, Takeda, Truvion Healthcare and Viatris. John L. Wallace is a co-founder of Antibe Therapeutics Inc. Supplementary Files DataS1DEGVehTBZxVeh.xlsx Suplementary files: Data S1. DEG Veh+TBZ x Veh DataS2DEGIFNTNFVehxVeh.xlsx Data S2. DEG IFN+TNF+Veh x Veh DataS3DEGIFNTNFTBZxIFNTNFVeh.xlsx Data S3. DEG IFN+TNF+TBZ x IFN+TNF+Veh DataS4DEGIFNTNFTBZxVehTBZ.xlsx Data S4. DEG IFN+TNF+TBZ x Veh+TBZ DataS5GenesetEnrichmentVehTBZxVeh.xlsx Data S5. Geneset enrichment Veh+TBZ x Veh DataS6GenesetEnrichmentIFNTNFVehxVeh.xlsx Data S6. Geneset enrichment IFN+TNF+Veh x Veh DataS7GenesetEnrichmentIFNTNFTBZxIFNTNFVeh.xlsx Data S7. Geneset enrichment IFN+TNF+TBZ x IFN+TNF+Veh DataS8GenesetEnrichmentIFNTNFTBZxVehTBZ.xlsx Data S8. Geneset enrichment IFN+TNF+TBZ x Veh+TBZ FigS1.png Figure S1: Effects of TBZ on viability and barrier integrity of intestinal organoids A. Assessment of mean DAPI signal intensity (% of vehicle) per live intestinal organoid after treatment of control or IFN/TNF treated organoids with vehicle or TBZ. Higher signal intensity indicates more cell death. B. Assessment of FITC-dextran influx (% of vehicle) in intestinal organoids after treatment of control IFN/TNF treated organoids with 5 µM or 50 µM TBZ. 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17:54:27","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":136900,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7324506/v1/5350542124314aab91899dc0.html"},{"id":92325790,"identity":"01a92cdb-f73d-415a-ab66-5bc6aaff442b","added_by":"auto","created_at":"2025-09-27 17:54:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":142810,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of H2S donor on intestinal epithelial cell viability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of TBZ on IEC viability. A. \u003c/strong\u003eRepresentative scheme for the cellular viability assays employed with intestinal epithelial cell lines Caco-2/TC7 and HIEC-6. \u003cstrong\u003eB.\u003c/strong\u003e Luminescence proportional to the number of live cells (% of vehicle) of Caco-2/TC7 cells treated with 5µM, 50µM, or 500µM TBZ for 72 hours. \u003cstrong\u003eC.\u003c/strong\u003e Alterations in luminescence proportional to the number of live cells (% of vehicle) in Caco-2/TC7 cells treated with 50µM TBZ for 72 hours under control or inflammatory conditions. \u003cstrong\u003eD.\u003c/strong\u003eAlterations in intracellular ATP (% of vehicle) in Caco-2/TC7 cells treated with 50µM TBZ for 72 hours under control or inflammatory conditions. \u003cstrong\u003eE.\u003c/strong\u003eAlterations in intracellular ATP in HIEC-6 cells treated with TBZ under control or inflammatory conditions. Data are represented as Mean ± SEM from 3 independent experiments. Ordinary one-way ANOVA test, with Dunnett’s multiple comparisons test (B) or Tukey’s multiple comparisons test (C-E).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7324506/v1/5613d674487466c4c4dcff91.png"},{"id":92325262,"identity":"78370f30-9115-4fc1-89f5-7a5acd7b3024","added_by":"auto","created_at":"2025-09-27 17:46:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":175502,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of the H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eS donor TBZ on intestinal epithelial permeability. A.\u003c/strong\u003e Representative scheme of the epithelial permeability \u003cem\u003ein vitro\u003c/em\u003e models. \u003cstrong\u003eB\u003c/strong\u003e. TEER measurements of Caco-2/TC7 cells treated with 50µM TBZ for 72 hours under control or inflammatory conditions. \u003cstrong\u003eC\u003c/strong\u003e. Fluorescence (% of vehicle) after FITC-dextran influx in Caco-2/TC7 cells treated with 50µM TBZ for 72 hours under control or inflammatory conditions. \u003cstrong\u003eD.\u003c/strong\u003e Representative images of FITC-dextran influx in intestinal organoids, with relative FITC fluorescence (% of vehicle). Scale bar = 100 µm. \u003cstrong\u003eE.\u003c/strong\u003e Quantification of fluorescence (% of vehicle) after FITC-dextran influx in intestinal organoids. Single organoid measurements from three biological replicates were pooled. Data are represented as Mean ± SEM from 3 independent experiments. Ordinary one-way ANOVA with Tukey’s multiple comparisons test.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7324506/v1/7dfa9a9aaa9457b3d39d558d.png"},{"id":92325260,"identity":"e4a16a6d-6c42-4959-8e5b-7d1250e175cc","added_by":"auto","created_at":"2025-09-27 17:46:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":116571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of the H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eS donor TBZ on gene expression in IEC.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e Summary table indicating the number of DEG (Up, upregulated or Down, downregulated) in each comparison. \u003cstrong\u003eB.\u003c/strong\u003e PCA plot indicating the proximity/distance of transcriptional profiles among experimental groups. \u003cstrong\u003eC.\u003c/strong\u003e Heatmap indicating upregulated (red) and downregulated (blue) genes within 4-gene modules (S1-4) across experimental conditions. \u003cstrong\u003eD, F, H, J\u003c/strong\u003e. Plots showing the top enriched\u0026nbsp;or selected gene set for the chosen comparison between groups. Black vertical bars indicate the rank of\u0026nbsp;genes in the\u0026nbsp;gene\u0026nbsp;set in the sorted list metric. The green curve corresponds to the 'running statistics' of the enrichment score (ES). The more the green ES curve is shifted to the upper left of the graph, the more the\u0026nbsp;gene\u0026nbsp;set is enriched in the first group. Conversely, a shift of the ES curve to the lower right, corresponds to more enrichment in the second group. \u003cstrong\u003eE, G, I, L.\u003c/strong\u003e Enrichment barplot of selected comparisons for the gene set that is selected on the Enrichment analysis table. \u003cstrong\u003eM, N, O.\u003c/strong\u003e RT-qPCR quantification of differentially expressed genes LYPD6B, NDUFA4, and COX6C.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7324506/v1/b1f9461db1fafab82c18edd0.png"},{"id":92325794,"identity":"cb4e3723-cd95-4f77-aa8e-d8b714bc56b6","added_by":"auto","created_at":"2025-09-27 17:54:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":452636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunoreactivity pattern of H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eS-synthesizing enzymes in the healthy human colorectal mucosa\u003c/strong\u003e. \u003cstrong\u003eA and B\u003c/strong\u003e. Expression pattern of Epithelial cell adhesion molecule (EPCAM, pan epithelial cell marker, gray), and H\u003csub\u003e2\u003c/sub\u003eS-synthesizing enzymes CSE (green) and 3-MST (magenta) from human colon (A) and rectum (B) biopsies. Nuclei were counterstained with DAPI. Immunofluorescence staining was performed three times, independently. Scale bar, 50 µm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7324506/v1/f4058fbe49eb699a05c36bd3.png"},{"id":103765542,"identity":"c0fdcd75-23de-4d76-9259-e55254c2dc6f","added_by":"auto","created_at":"2026-03-02 16:03:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1696160,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7324506/v1/983f6a0b-891d-43e9-9679-82b551a326f0.pdf"},{"id":92325261,"identity":"ba90cf12-bf4a-49a7-b1c1-6f29e119ad1e","added_by":"auto","created_at":"2025-09-27 17:46:27","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":46022,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuplementary files:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData S1. DEG Veh+TBZ x Veh\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"DataS1DEGVehTBZxVeh.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7324506/v1/584fa4b4f913d7321646e98d.xlsx"},{"id":92325791,"identity":"3d9e4db2-0a22-4afa-a749-5c37b567ea8c","added_by":"auto","created_at":"2025-09-27 17:54:27","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14395,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eData S2. DEG IFN+TNF+Veh x Veh\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"DataS2DEGIFNTNFVehxVeh.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7324506/v1/de99e479d42ae6530e67ffaf.xlsx"},{"id":92325274,"identity":"23c6f8b5-2573-4f63-a54e-8674716f63db","added_by":"auto","created_at":"2025-09-27 17:46:27","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":12059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eData S3. DEG IFN+TNF+TBZ x IFN+TNF+Veh\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"DataS3DEGIFNTNFTBZxIFNTNFVeh.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7324506/v1/55921be3e0b5449e66f27e90.xlsx"},{"id":92325277,"identity":"b1b17c76-1bf3-4e1e-9d27-d15abdde716f","added_by":"auto","created_at":"2025-09-27 17:46:27","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":7884,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eData S4. DEG IFN+TNF+TBZ x Veh+TBZ\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"DataS4DEGIFNTNFTBZxVehTBZ.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7324506/v1/9cf0619e3a47d12aeac16345.xlsx"},{"id":92325796,"identity":"44e72d41-6c1d-4268-afea-d12e5497ae34","added_by":"auto","created_at":"2025-09-27 17:54:27","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":15976,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eData S5. Geneset enrichment Veh+TBZ x Veh\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"DataS5GenesetEnrichmentVehTBZxVeh.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7324506/v1/35b90ec32f9cfcc5d32500f2.xlsx"},{"id":92325283,"identity":"f4fb95f1-e4bf-46e4-919b-a8884212943e","added_by":"auto","created_at":"2025-09-27 17:46:28","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":12118,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eData S6. Geneset enrichment IFN+TNF+Veh x Veh\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"DataS6GenesetEnrichmentIFNTNFVehxVeh.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7324506/v1/20918d534e6ffb49e0f57013.xlsx"},{"id":92325279,"identity":"1e63375a-3c33-4527-b2d2-e611d3977bd4","added_by":"auto","created_at":"2025-09-27 17:46:27","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":15976,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eData S7. Geneset enrichment IFN+TNF+TBZ x IFN+TNF+Veh\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"DataS7GenesetEnrichmentIFNTNFTBZxIFNTNFVeh.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7324506/v1/58139acc5085c84e9d2777a2.xlsx"},{"id":92325276,"identity":"fe3e6b50-fc44-4707-b4ff-7e08ad0a21f3","added_by":"auto","created_at":"2025-09-27 17:46:27","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":15853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eData S8. Geneset enrichment IFN+TNF+TBZ x Veh+TBZ\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"DataS8GenesetEnrichmentIFNTNFTBZxVehTBZ.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7324506/v1/dc61b63f6b566f2d7a249cac.xlsx"},{"id":92325268,"identity":"19ac19f0-7a67-44b6-8ef0-c7dc243980fd","added_by":"auto","created_at":"2025-09-27 17:46:27","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":101216,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1: Effects of TBZ on viability and barrier integrity of intestinal organoids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eAssessment of mean DAPI signal intensity (% of vehicle) per live intestinal organoid after treatment of control or IFN/TNF treated organoids with vehicle or TBZ. Higher signal intensity indicates more cell death. \u003cstrong\u003eB.\u003c/strong\u003e Assessment of FITC-dextran influx (% of vehicle) in intestinal organoids after treatment of control IFN/TNF treated organoids with 5 µM or 50 µM TBZ.\u003c/p\u003e","description":"","filename":"FigS1.png","url":"https://assets-eu.researchsquare.com/files/rs-7324506/v1/a67dad5da9b695b382f4f131.png"}],"financialInterests":"Competing interest reported. Marc Ferrante received research grants from AbbVie, EG Pharma, Janssen, Pfizer, Takeda and Viatris; consultancy fees from AbbVie, AgomAb Therapeutics, Boehringer Ingelheim, Celgene, Celltrion, Eli Lilly, Janssen-Cilag, Merck Sharp and Dohme, MRM Health, Pfizer, Takeda and ThermoFisher; and speakers’ fees from AbbVie, Biogen, Boehringer Ingelheim, Dr Falk Pharma, Ferring, Janssen-Cilag, Merck Sharp and Dohme, Pfizer, Takeda, Truvion Healthcare and Viatris. John L. Wallace is a co-founder of Antibe Therapeutics Inc.","formattedTitle":"Functional and Transcriptional Effects of a Hydrogen Sulfide Donor on the Intestinal Epithelial Barrier","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe intestinal epithelial cell (IEC) monolayer is crucial for maintaining the intestinal barrier, which protects against xenobiotics and pathogens while regulating nutrient absorption and permeability \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Disruption of this barrier is associated with a range of gastrointestinal (GI) diseases and is increasingly recognized as a central mechanism driving systemic conditions \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Such disruption often involves increased paracellular permeability due to the breakdown of junctional complexes or epithelial cell death \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHydrogen sulfide (H₂S) is a pleiotropic gasotransmitter that exhibits protective effects at physiological levels through its anti-inflammatory and antioxidant properties but becomes toxic at high concentrations \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In humans, H\u003csub\u003e2\u003c/sub\u003eS is endogenously produced by enzymes such as cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CTH, or CSE), and 3-mercapto-sulfurtransferase (MPST, or 3-MST) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Impaired biosynthesis of endogenous H\u003csub\u003e2\u003c/sub\u003eS worsens intestinal barrier integrity and promotes conditions like colitis \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In contrast, exogenous H\u003csub\u003e2\u003c/sub\u003eS delivery restores barrier function and reduces intestinal inflammation in colitis models \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eInterestingly, evidence suggests that sulfur-linked compounds, such as 4-hydroxithiobenzamide (TBZ) \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, have the potential to release protective H₂S. This effect has been observed in association with both steroidal \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e and non-steroidal anti-inflammatory drugs \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. For example, coupling of TBZ to ketoprofen (ATB-352) enhances analgesic efficacy while eliminating harmful gastric effects \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Similarly, TBZ coupled to naproxen (ATB-346) prevents intestinal damage and inflammation \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. These findings underscore the clinical potential of the H\u003csub\u003e2\u003c/sub\u003eS donor TBZ, although its effects in isolation or the molecular mechanisms underlying its protective effects remain unclear.\u003c/p\u003e\u003cp\u003eMost studies on H\u003csub\u003e2\u003c/sub\u003eS donors have employed \u003cem\u003ein vivo\u003c/em\u003e models, making it difficult to pinpoint its individual contributions specifically to epithelial permeability and cell death during intestinal barrier failure. Moreover, insights into the involved molecular mechanisms largely stem from targeted approaches investigating a limited number of genes or signaling pathways. This study seeks to evaluate the protective effects of TBZ on IECs, assessing cellular viability and intestinal barrier integrity under both normal and inflammatory conditions. Additionally, we mapped the distribution of endogenous H\u003csub\u003e2\u003c/sub\u003eS-synthesizing enzymes in the healthy human colorectal mucosa. To gain unbiased insights into the involved mechanisms, we employed bulk RNA-sequencing of cells treated with TBZ under normal and inflammatory conditions.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eEffect of H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eS donors on IEC viability.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBefore starting the experimental design (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), we determined the cytotoxicity of TBZ at different concentrations (5, 50 and 500 \u0026micro;M) in Caco-2/TC7 cells. Here we found that only 500 \u0026micro;M caused any significant cytotoxic effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), as determined by the relative reduction in luminescence, indicating reduced reductive capacity of cells; a proxy for the number of live cells in culture.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfterwards, we assessed cell viability of Caco-2/TC7 cells using the same assay under normal conditions or after stimulation with IFN/TNF, followed by treatment with vehicle or 50 \u0026micro;M TBZ. Accordingly, TBZ treatment did not affect cell viability compared to vehicle-treated cells, however, stimulation of cells with IFN/TNF significantly reduced cell viability compared with vehicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). This was not reversed by treatment with 50 \u0026micro;M TBZ. Similar results were obtained in intestinal organoids after treatment of control or IFN/TNF stimulated organoids (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese results were further confirmed in Caco-2/TC7 and HIEC-6 cells by quantifying intracellular ATP levels, which again can be used as a proxy for cell viability. In both cell lines, there was no cytotoxic effect of 50 \u0026micro;M TBZ observed in the absence of inflammatory stimuli (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E). Stimulation of cells with IFN/TNF significantly reduced cell viability compared with vehicle, which was again not rescued by 50 \u0026micro;M TBZ.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eEffect of the H\u003csub\u003e2\u003c/sub\u003eS donor on intestinal epithelial barrier integrity\u003c/h2\u003e\u003cp\u003eAs HIEC-6 cells cannot reach full confluence, we employed Caco-2/TC7 cell monolayers in a trans-well system as well as intestinal organoids to investigate the effect of H\u003csub\u003e2\u003c/sub\u003eS donors on epithelial barrier integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In the absence of inflammatory stimuli, treatment with TBZ for 72 h did not change trans-epithelial resistance (TEER) of Caco-2/TC7 cell monolayers when compared to vehicle. Conversely, stimulation with IFN/TNF reduced TEER, whereas treatment with TBZ partially reversed this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe further investigated the effects of the H\u003csub\u003e2\u003c/sub\u003eS donor on epithelial barrier function by assessing paracellular permeability of FITC-Dextran in both Caco-2/TC7 monolayers and intestinal organoids. We observed that treatment with TBZ for 72 hours had contrasting effects under control vs. inflamed contexts. When alone, 50 \u0026micro;M TBZ increased paracellular permeability to FITC-Dextran in both Caco-2/TC7 cells and organoids. Stimulation with IFN/TNF also resulted in increased paracellular permeability. In contrast though, 50 \u0026micro;M TBZ reversed this disruptive effect of IFN/TNF and restored FITC-dextran influx to levels observed in vehicle-treated non stimulated cells and organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E). Moreover, these effects were concentration-dependent in organoids (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEffect of TBZ on gene expression\u003c/h3\u003e\n\u003cp\u003eTo gain insights into the mechanisms underlying the protective effects of the H\u003csub\u003e2\u003c/sub\u003eS donor on IECs stimulated with IFN/TNF, we performed an exploratory RNA-seq analysis. Stimulation of control Caco-2/TC7 cells with TBZ resulted in the highest number of differentially expressed genes (DEGs; logFC\u0026thinsp;\u0026gt;\u0026thinsp;2 or \u0026lt; -2). In total, 494 genes showed altered expression, with 335 upregulated and 159 downregulated. In contrast, stimulation with TNF/IFN exhibited a less pronounced transcriptional effect, altering the expression of 76 genes. Comparative analysis between IFN/TNF-stimulated cells treated or untreated with TBZ revealed 46 differentially expressed genes. When comparing TBZ-treated cells under control vs. inflamed (IFN/TNF-stimulated) conditions, only five genes were differentially expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Data S1-4).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese findings suggest that TBZ exerts a strong regulatory effect on transcription. Moreover, the limited number of differentially expressed genes between cell groups (control vs. inflamed) treated with TBZ indicates that this H\u003csub\u003e2\u003c/sub\u003eS donor has dominant transcriptional activity in relation to this inflammatory stimulation. Principal component analysis (PCA) further supports these conclusions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eClustered heatmap analysis revealed the regulation of four major gene modules. Gene module S1 was broadly regulated by stimulation of Caco-2/TC7 cells with IFN/TNF and/or TBZ. Notably, the pro-inflammatory gene \u003cem\u003eLYPD6B\u003c/em\u003e was strongly upregulated. Members of the LY6 superfamily are known to be upregulated in the intestinal epithelium in inflammatory bowel disease (IBD), and their cross-linking promotes chemokine production \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eGenes in module S2 were specifically upregulated by TBZ in both control and IFN/TNF-treated cells. Among these, \u003cem\u003eNDUFA4\u003c/em\u003e and \u003cem\u003eCOX5A\u003c/em\u003e, which are involved in oxidative phosphorylation in mitochondria were noticeably upregulated. In contrast, gene module S3 was downregulated by TBZ, while module S4 was downregulated by both TBZ and/or IFN/TNF stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eHence, we performed geneset enrichment analysis using the HALLMARK collection. With a threshold of logFC\u0026thinsp;\u0026gt;\u0026thinsp;0.5, we identified oxidative phosphorylation-related genes as differentially enriched in control Caco-2/TC7 cells treated with TBZ (logFC\u0026thinsp;=\u0026thinsp;0.55) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). This enrichment was driven by the upregulation of genes such as \u003cem\u003eCOX5A\u003c/em\u003e, \u003cem\u003eFH\u003c/em\u003e, \u003cem\u003eCOX6C\u003c/em\u003e, \u003cem\u003eNDUFA4\u003c/em\u003e, and others (Data S5).\u003c/p\u003e\u003cp\u003eIn additional comparisons, the logFC threshold was lowered to \u0026gt;\u0026thinsp;0.2. In IFN/TNF-stimulated cells, we observed the enrichment of three genesets: Interferon Response, G2M Checkpoint, and Apical Surface (logFC\u0026thinsp;=\u0026thinsp;0.24, 0.24, and 0.20, respectively). Among the genes associated with the interferon response, \u003cem\u003eIFIH1\u003c/em\u003e, \u003cem\u003eIFITM2\u003c/em\u003e, \u003cem\u003eCASP1\u003c/em\u003e, and \u003cem\u003eCASP8\u003c/em\u003e are key examples of upregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G; Data S6).\u003c/p\u003e\u003cp\u003eSimilar to the findings in control cells, TBZ treatment of IFN/TNF-stimulated cells also enriched the Oxidative Phosphorylation pathway (logFC\u0026thinsp;=\u0026thinsp;0.35). This enrichment was driven by the upregulation of genes such as \u003cem\u003eCOX5A\u003c/em\u003e, \u003cem\u003eCOX6C\u003c/em\u003e, \u003cem\u003eNDUFA4\u003c/em\u003e, and members of the solute carrier family (\u003cem\u003eSLC25A3-5\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, I; Data S7). Finally, a comparison between control and IFN/TNF-treated cells exposed to TBZ revealed only mild differences in geneset enrichment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ, L; Data S8).\u003c/p\u003e\u003cp\u003eChanges in gene expression were further confirmed with RT-qPCR, where the expression of LYPD6B, NDUFA4, and COX6C were assessed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eM-O).\u003c/p\u003e\n\u003ch3\u003eImmunoreactivity pattern of HS-synthesizing enzymes\u003c/h3\u003e\n\u003cp\u003eWe assessed protein expression of the H\u003csub\u003e2\u003c/sub\u003eS-synthetizing enzymes CSE and 3-MST by immunostaining colorectal tissue slices prepared from mucosal biopsies obtained from healthy volunteers. Overall, the expression of CSE and 3-MST was more pronounced in the human ascending colon (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) than in the rectum (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In the ascending colon, 3-MST staining was highly enriched in epithelial cells (co-localized with the pan epithelial marker EPCAM). Conversely, CSE expression was present in epithelial cells as well as in cells within the lamina propria. Of note, both CSE and 3-MST displayed a similar subcellular distribution pattern in colonic epithelial cells, with a marked enrichment in the apical pole.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eImproving the intestinal barrier is a promising therapeutic strategy for treatment of chronic intestinal diseases, disorders affecting the gut-liver axis and other distant organ systems rooted in an impaired intestinal barrier \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Previous studies using hybrid H\u003csub\u003e2\u003c/sub\u003eS-releasing NSAIDs have shown protective effects on the gastrointestinal system \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, most of these studies were conducted \u003cem\u003ein vivo\u003c/em\u003e, and the underlying cellular mechanisms remain unclear. In this \u003cem\u003ein vitro\u003c/em\u003e study, we aimed to investigate whether the effect of H\u003csub\u003e2\u003c/sub\u003eS on the intestinal barrier is associated with a regulatory role over cell death and/or epithelial permeability.\u003c/p\u003e\u003cp\u003eOur findings show that TBZ did not influence cell death induced by IFN/TNF in both Caco-2/TC7 and HIEC-6 cells. These observations suggest that TBZ does not counteract the pro-apoptotic effects of IFN/TNF, potentially mediated by caspases 1 and 8, which are both upregulated in cells stimulated with these cytokines \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The intrinsic apoptosis pathway involves the release of pro-apoptotic proteins from the mitochondrial intermembrane space into the cytosol, while the extrinsic pathway, triggered by death receptors of the TNF superfamily, recruits cytoplasmic adaptor proteins and activates downstream caspase cascades \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. While H₂S may protect against cell death induced by the intrinsic or mitochondrial pathway through upregulation of oxidative phosphorylation genes, it appears to have no effect on cell death mediated by the extrinsic pathway, which is the context of our study design.\u003c/p\u003e\u003cp\u003eIn addition to inducing apoptosis, the combination of IFN and TNF disrupts the intestinal epithelial barrier by increasing myosin light chain kinase (MLCK1) activity, leading to endocytosis of the tight junction protein occludin and increased paracellular permeability \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In our study, TBZ alone had no effect on transcellular permeability. However, while IFN/TNF treatment reduced transepithelial electrical resistance (TEER), TBZ effectively restored this barrier function. Notably, TBZ exhibited divergent effects on paracellular permeability: it increased FITC-dextran influx in untreated Caco-2/TC7 cells and organoids but restored epithelial barrier integrity in cells stimulated with IFN/TNF. These findings align with previous reports highlighting the pleiotropic effects of H₂S, which can influence cellular homeostasis, inflammation, and mitochondrial function in a concentration- and context-dependent manner \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Thus, while our data reinforce prior evidence supporting the protective role of H₂S against inflammation-induced intestinal barrier disruption \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, further research is needed to determine the precise contexts in which H₂S could be clinically beneficial.\u003c/p\u003e\u003cp\u003eTo gain deeper insights into the molecular mechanisms involved in epithelial barrier protection by H\u003csub\u003e2\u003c/sub\u003eS in an inflammatory context, we performed an exploratory RNA-seq analysis followed by cross-validation by RT-qPCR. As mentioned, treatment with TBZ displayed the strongest transcriptional effect, surpassing the effect of IFN/TNF. It is unclear though if TBZ has a more dominant effect on transcription over IFN/TNF, since stimulation with these cytokines took place 24 h before cells were exposed to TBZ and further cultured for additional 72 h. Nevertheless, our findings do indicate that H\u003csub\u003e2\u003c/sub\u003eS has a regulatory action on gene expression that has not been reported before. Additionally, geneset enrichment analysis highlighted regulation of oxidative phosphorylation in both control and IFN/TNF-treated cells exposed to TBZ, indicating that H\u003csub\u003e2\u003c/sub\u003eS may serve as a master regulator of mitochondrial homeostasis through transcriptional regulation. This enrichment was driven by upregulation of multiple genes, including \u003cem\u003eCOX5A\u003c/em\u003e, \u003cem\u003eCOX6C\u003c/em\u003e, \u003cem\u003eNDUFA4\u003c/em\u003e, and \u003cem\u003eSLC25A3-5\u003c/em\u003e. The subunits \u003cem\u003eCOX5A\u003c/em\u003e and \u003cem\u003eCOX6C\u003c/em\u003e are components of cytochrome c oxidase (Complex IV), which plays a crucial role in the electron transport chain and ATP synthesis. \u003cem\u003eCOX5A\u003c/em\u003e modulates Complex IV activity, while \u003cem\u003eCOX6C\u003c/em\u003e maintains its structural integrity \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eNDUFA4\u003c/em\u003e, another component of Complex IV, contributes to electron transport and proton gradient formation, and its dysfunction is linked to mitochondrial disorders \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Furthermore, \u003cem\u003eSLC25A3-5\u003c/em\u003e are mitochondrial transporters that facilitate the exchange of essential metabolites like phosphate and ADP/ATP, thus ensuring efficient ATP production and cellular energy balance \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlthough no direct evidence in the existing literature links these specific genes to tight junction regulation or intestinal permeability, mitochondrial function, oxidative stress, and cellular energy balance are key regulators of epithelial barrier integrity \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Indeed, maintenance of this barrier is an energy-intensive process that relies on the coordination of tight junction proteins, cytoskeletal components, and other junctional elements \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Therefore, disruptions in mitochondrial activity can compromise tight junctions and increase permeability. A recent study demonstrated that STARD7, which mediates phosphatidylcholine transfer from the endoplasmic reticulum to mitochondria for membrane stabilization and respiration, is downregulated in the epithelium of patients with IBD. Moreover, its deficiency impairs tight junctions, the intestinal barrier, and worsens experimental colitis \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSeveral studies have assessed the regulation of H\u003csub\u003e2\u003c/sub\u003eS-synthesizing enzymes in IBD and in pre-clinical models of colitis, and although there are conflicting findings, the expression of H\u003csub\u003e2\u003c/sub\u003eS-synthesizing enzymes has been reported to be altered in these conditions, particularly in those sites exhibiting active inflammation \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. For instance, a recent study reported that 3-MST expression was markedly decreased in colonic samples collected from both patients with IBD and DSS-treated mice, while 3-MST deficiency led to increased colitis severity \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn order to get better insights into the cellular and subcellular distribution of H\u003csub\u003e2\u003c/sub\u003eS-synthesizing enzymes in the human intestinal epithelium, we performed immunofluorescence on human colorectal tissue sections from healthy volunteers. One key finding in these experiments was the observation that 3-MST and CSE immunoreactivities were overall enriched in the apical pole of colonic epithelial cells, therefore, coincidently with the localization of junctional complexes. This subcellular enrichment in colonic epithelial cells might have interesting functional purposes in regulating mitochondrial homeostasis and junctional complex integrity mediated through H\u003csub\u003e2\u003c/sub\u003eS synthesis. In other polarized cells, such as neurons, there is growing evidence pointing to mitochondrial morphology and functional diversity within subcellular domains \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Nevertheless, this aspect remains uncharted in intestinal epithelial cells.\u003c/p\u003e\u003cp\u003eOur study underpins cellular and molecular mechanisms underlying the protective effect of TBZ towards intestinal epithelial barrier integrity in inflammation. Of note, we provide evidence indicating that H\u003csub\u003e2\u003c/sub\u003eS may represent a master regulator of genes involved in oxidative phosphorylation and mitochondrial homeostasis. These findings highlight promising therapeutic prospects for H\u003csub\u003e2\u003c/sub\u003eS-releasing compounds to treat conditions rooted in mitochondrial and intestinal epithelial barrier dysfunction. However, a deeper understanding of H₂S\u0026rsquo;s dual effects is essential to harness its benefits while mitigating potential risks.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eIEC culture\u003c/h2\u003e\u003cp\u003eThe human colon adenocarcinoma cell line Caco-2/TC7 (Sigma Aldrich, Germany; cat. #SCC209), and the normal human small intestinal epithelial cell line HIEC-6 (ATCC, United States; cat. #CRL-3266) were genotyped by the commercial suppliers through STR analysis to verify the unique identity of the cell lines. The cells were mycoplasma free and were grown under hypoxia (5% O\u003csub\u003e2\u003c/sub\u003e, 95% CO\u003csub\u003e2\u003c/sub\u003e, 37\u0026ordm;C and 90% humidity, CBF260; Binder, GmbH). Caco-2/TC7 cells were grown in DMEM/F12 medium, supplemented with 1% Glutamax, 10% FBS, and 1% penicillin/streptomycin. HIEC-6 cells were grown in Opti-MEM\u0026reg;I supplemented with 1% Glutamax, 10% Fetal Bovine Serum (FBS), and 1% penicillin/streptomycin.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExperimental design\u003c/h3\u003e\n\u003cp\u003eCaco-2/TC7 and HIEC-6 cells were grown to 80\u0026ndash;90% confluency before starting experiments. Cells were treated with TBZ (50 \u0026micro;M) or vehicle (DMSO 0.1%) for 72 hours. For the inflammatory conditions, cells were initially exposed to IFNγ (2.5 ng/mL) for 3 hours to induce expression of the receptor for TNFα, followed by treatment with TNFα (10 ng/mL) or vehicle (culture medium) for a total period of 24 hours. After which cells were treated with TBZ or vehicle for an additional 72 hours.\u003c/p\u003e\n\u003ch3\u003eCellular viability assessment\u003c/h3\u003e\n\u003cp\u003eTh1 cytokines such as IFNγ and TNFα are key mediators in gastrointestinal lesions and have been reported to cause IEC apoptosis in a synergistic way \u003csup\u003e\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Therefore, we assessed the effect of the H\u003csub\u003e2\u003c/sub\u003eS donor on IEC viability in normal and inflammatory conditions. Firstly, we employed the RealTime-Glo\u0026trade; MT Cell Viability Assay (Promega; Cat. G9711) with Caco-2/TC7 cells, which measures the reductive capacity of cells, and is a proxy for cell viability. Secondly, we employed the CellTiter-Glo\u0026reg; Luminescent Cell Viability Assay (Promega; Cat. G7570) with both Caco-2/TC7 and HIEC-6 cells, which measures ATP levels, and is also a measure of cell viability. In brief, Caco-2/TC7 and HIEC-6 cells were plated in 96-well plates, containing 5x10\u003csup\u003e4\u003c/sup\u003e cells per well. After 1 week of growth, the experimental design aforementioned was followed. Afterwards, the cells were treated for 30 minutes with either RealTime-Glo\u0026trade; MT Cell Viability reagent to measure the reductive capacity of cells, or with CellTiter-Glo\u0026reg; reagent to measure the amount of ATP, after which luminescence was measured using a luminometer (FLUOstar Omega, BMG Labtech, Hepatology Research Unit, KU Leuven). RealTime-Glo\u0026trade; MT Cell viability assay readings were plotted against baseline. Raw CellTiter-Glo\u0026reg; assay readings were interpolated against an ATP standard curve (0.001\u0026ndash;10 \u0026micro;M). Experiments were repeated independently 3 times.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of epithelial barrier integrity\u003c/h2\u003e\u003cp\u003eThe non-tumoral cell line HIEC-6 expresses certain tight junction proteins, but lacks occludin, an important component of junctional complexes, and a major target of TNFα-induced barrier dysfunction \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Therefore, the assessment of the effects of the H\u003csub\u003e2\u003c/sub\u003eS donor on transcellular permeability was done with Caco-TC7 cells, while paracellular permeability was assessed in this cell line as well as in human intestinal organoids.\u003c/p\u003e\u003cp\u003eIn brief, 5x10\u003csup\u003e5\u003c/sup\u003e Caco-2/TC7 cells were seeded on a Costar 6.5 mm\u003csup\u003e2\u003c/sup\u003e Transwell system with polyester membrane inserts with 0.4 \u0026micro;m pores (StemCell Technologies) and differentiated for 21 days. 750 \u0026micro;L and 250 \u0026micro;L media were added to the basal and apical compartment of each well, respectively, and changed every 2 days. A well only containing culture medium was used as a blank. After 21 days, stimuli and treatment of intestinal epithelial were conducted, as described in the experimental design. Transepithelial electrical resistance (TEER) was measured with the EVOM device (EVM-MT-03-01, WPI, GmbH). The values were measured in Ω*cm\u003csup\u003e2\u003c/sup\u003e and blank-corrected. Moreover, paracellular permeability was measured by the influx of FITC-dextran (MW: 3 kDa) from the apical to the basal compartment. Briefly, after the last TEER measurement, wells were washed twice with DMEM/F12 without phenol red. FITC-dextran (250 \u0026micro;L; 100 \u0026micro;M in medium without phenol red) was added to the apical compartment and then 750 \u0026micro;L of culture medium was added to the basal compartment. Aliquots (100 \u0026micro;L) of the basal compartment were collected, and fluorescence intensity was measured using a microplate reader (FLUOstar Omega, BMG Labtec) with excitation at 496 nm, emission at 524 nm, and a cutoff wavelength at 515 nm.\u003c/p\u003e\u003cp\u003eAdditionally, intestinal organoids were used to assess paracellular epithelial barrier integrity. In short, organoids from diverse intestinal regions were cultured based on previously described methods \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. For experimental setups, organoids were digested into single cells, 500 of which were plated in BME drops. Organoids were treated according to the experimental design after four days of growth. 72 hours after initiation of TBZ treatment, organoids were overlaid with advanced DMEM/F12 containing 100 \u0026micro;M FITC-dextran (MW: 3kDa). One hour afterwards, organoids were washed 3 times with fresh advanced DMEM/F12 and treated with DAPI to assess cell death. Imaging was performed using a Perkin Elmer Operetta CLS microscope (VIB BioImaging Core Leuven), after which the mean FITC-dextran and DAPI signal per organoid was analyzed using ImageJ.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eBulk RNA-sequencing (RNA-seq)\u003c/h2\u003e\u003cp\u003eCaco-2/TC7 cells were treated according to the aforementioned experimental design. Three biological replicates per condition were pooled and subjected to total RNA extraction using the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer\u0026rsquo;s instructions. Since biological replicates were pooled during RNA extraction, the RNA-seq analysis is exploratory (FDR\u0026thinsp;=\u0026thinsp;1). RNA-seq was performed by the Genomics Core KU Leuven. Briefly, cDNA libraries were synthesized with the QuantSeq 3\u0026rsquo; mRNA Seq Kit (Lexogen, Inc.) and sequenced on an Illumina HiSeq4000 system. The raw sequencing reads were initially checked for quality using FastQC, and adapter sequences and low-quality bases were trimmed using Trimmomatic. Reads were then aligned to the reference genome (GRCh38) using Hisat2. Following normalization, counts and sample tables were uploaded into Omics Playground v3 (BigOmics Analytics, Switzerland) for further assessment of quality control, differential gene expression, and geneset enrichment. A heatmap was generated using the ComplexHeatmap R/Bioconductor package \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e on scaled log-expression values (z-score) using Euclidean distance and Ward linkage using the fastcluster R package. The available methods to select the top features are sd (standard deviation) - features with the highest standard deviation across all the samples, marker - features that are overexpressed in each phenotype class compared to the rest, or by PCA - principal component analysis (performed using the irlba R package). Geneset enrichment was performed using CAMERA \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, GSEA \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, ssGSEA \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, fGSEA, GSVA \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, and fry \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. The q-values yielded by the different methods were then combined into a meta-q value, where the meta value corresponds to the maximum. Hence, geneset enrichment analysis focused on the HALLMARK collection \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence\u003c/h2\u003e\u003cp\u003eHuman colorectal mucosa samples were collected from healthy volunteers. Tissue sections (10 \u0026micro;m thickness) were fixed with 4% paraformaldehyde (PFA) for 20 min at RT. The sections were then permeabilized with 0.03% Triton X-100 in PBS at room temperature for 5 min and then incubated with a blocking solution containing 3% BSA in PBS for 60 min at room temperature. The sections were subsequently incubated (overnight at 4\u0026deg;C) with primary antibodies: mouse anti-cystathionine γ-lyase (CSE; Invitrogen, cat. #MA5-25423,1:50), rabbit anti-3-mercaptopyruvate sulfurtransferase (3MST; Invitrogen, cat. #PA5-51548, 1:50) and goat anti-epithelial cell adhesion molecule (EPCAM; R\u0026amp;D Systems, cat. #AF960, 1:100). After primary incubation, the sections were incubated with secondary antibodies conjugated to the fluorophores AlexaFluor 488, 594, and 647 (Invitrogen) for 1.5 h at room temperature, and then stained for 5 minutes with DAPI. Cover slides were mounted using mounting medium (ProLong Gold, Thermo Fisher).\u003c/p\u003e\u003cp\u003ePhotomicrographs were captured using a confocal microscopy (Zeiss LSM 880) equipped with Zen software. Images were analyzed using Fiji Image J software (1.53 t, NIH, Baltimore, MD, USA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.nih.gov/ij/\u003c/span\u003e\u003cspan address=\"https://imagej.nih.gov/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (S.E.M). With the aid of GraphPad Prism v10 software, mean group differences were analyzed by either 1- or 2-way ANOVA, followed by post-tests with correction for multiple comparisons by controlling the false discovery rate. Values of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the KU Leuven startup grant STG/22/023 and C1 grant C14/23/135. JPO received a scholarship from the Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico - CNPq (CNPq, 312514/2019-0) and from the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal e N\u0026iacute;vel Superior - Brazil (CAPES; 88887.694612/2022-00). We would like to acknowledge the support from FAPESP (processes 2016/06146-3 and 2019/14051-2). MNM and SKPC are recipients of scientific productivity scholarships from the Brazilian National Council for Scientific and Technological Development (CNPq; processes 306294/2019-2 and 312514/2019-0, respectively).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImages were recorded on a Zeiss LSM 880 \u0026ndash; Airyscan (Cell and Tissue Imaging Cluster (CIC), supported by Hercules AKUL/15/37_GOH1816N and FWO G.0929.15 to Pieter Vanden Berghe, University of Leuven. Figures 1A and 2A and the graphical abstract were created with BioRender.com. We thank Prof. Dr. Guy Boeckxstaens for providing rectal tissue samples from healthy volunteers and Dr. Hannelie Korf for reviewing the manuscript. Marc Ferrante is a Senior Clinical Investigator of the Research Foundation - Flanders (FWO), Belgium.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJPO, MvS, SC and ADS: study conceptualization and design. JPO, MvS, MW, VFA and ADS: contributed to data acquisition. JPO, ADS, MvS, SC and CA: involved in data analysis and/or interpretation. JLW, AV, TV: resources. JPO and ADS: drafted the manuscript. ADS, JPO, MvS, CA, MNM, SvdM, MF, JLW and SC: Reviewing, Editing, Resources. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA-seq datasets generated and analyzed during the present study are publicly available in the European Genome-Phenome Archive (EGA) under accession number EGAS50000001237. Any additional original data reported in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the Ethics Committee of the University Hospital Leuven (Ethics protocols S62059 and S68855). The study was performed in accordance with the ethical standards established in the Declaration of Helsinki and written informed consent was obtained from all participants prior to the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest disclosure:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMarc Ferrante received research grants from AbbVie, EG Pharma, Janssen, Pfizer, Takeda and Viatris; consultancy fees from AbbVie, AgomAb Therapeutics, Boehringer Ingelheim, Celgene, Celltrion, Eli Lilly, Janssen-Cilag, Merck Sharp and Dohme, MRM Health, Pfizer, Takeda and ThermoFisher; and speakers\u0026rsquo; fees from AbbVie, Biogen, Boehringer Ingelheim, Dr Falk Pharma, Ferring, Janssen-Cilag, Merck Sharp and Dohme, Pfizer, Takeda, Truvion Healthcare and Viatris. John L. Wallace is a co-founder of Antibe Therapeutics Inc.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSoderholm, A.T., and Pedicord, V.A. (2019). 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Cell Syst \u003cem\u003e1\u003c/em\u003e, 417-425. 10.1016/j.cels.2015.12.004.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7324506/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7324506/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe intestinal epithelial barrier is essential for protecting against pathogens and toxins while permitting nutrient and water absorption. Barrier dysfunction is a hallmark of inflammatory diseases affecting the gastrointestinal (GI) tract and beyond. Hydrogen sulfide (H₂S) has emerged as a critical regulator of intestinal homeostasis. This study examines the effects of the H₂S-releasing compound 4-hydroxithiobenzamide (TBZ) on epithelial barrier integrity. While TBZ did not prevent interferon-γ and tumor necrosis factor-α (IFN/TNF)-induced epithelial cell death, it reversed cytokine-induced increases in transepithelial permeability. Interestingly, TBZ alone elevated paracellular permeability, yet normalized it under inflammatory conditions, indicating a context-dependent effect. H₂S-producing enzymes localized apically in intestinal epithelial cells, suggesting spatial regulation. Transcriptomic analysis implicated oxidative phosphorylation as a pathway mediating TBZ\u0026rsquo;s effects. These findings advance our understanding of H₂S in intestinal barrier regulation and support TBZ as a candidate therapeutic agent for conditions marked by barrier dysfunction in an inflammatory context.\u003c/p\u003e","manuscriptTitle":"Functional and Transcriptional Effects of a Hydrogen Sulfide Donor on the Intestinal Epithelial Barrier","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-27 17:46:22","doi":"10.21203/rs.3.rs-7324506/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-21T20:28:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-21T19:24:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-20T15:25:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"17619503348999330321177642503049821100","date":"2025-10-14T00:17:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21433496291204510109265628165593038578","date":"2025-10-07T16:27:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"239524906176369598556804045965305486942","date":"2025-10-07T10:09:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"43017438240830902557554042114401907589","date":"2025-10-07T07:14:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"227298344031505982519185408239837334512","date":"2025-10-05T14:04:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-03T14:29:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273215642076408535306051484303920147334","date":"2025-09-20T06:52:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-18T00:21:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-18T00:03:08+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-17T19:44:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-16T05:30:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-16T05:27:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f16ce9ca-77fe-4bb6-886c-ba15a5ad624a","owner":[],"postedDate":"September 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55265506,"name":"Biological sciences/Cell biology"},{"id":55265507,"name":"Health sciences/Diseases"},{"id":55265508,"name":"Health sciences/Gastroenterology"},{"id":55265509,"name":"Biological sciences/Immunology"},{"id":55265510,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-03-02T16:01:07+00:00","versionOfRecord":{"articleIdentity":"rs-7324506","link":"https://doi.org/10.1038/s41598-026-40348-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-02-23 15:57:56","publishedOnDateReadable":"February 23rd, 2026"},"versionCreatedAt":"2025-09-27 17:46:22","video":"","vorDoi":"10.1038/s41598-026-40348-2","vorDoiUrl":"https://doi.org/10.1038/s41598-026-40348-2","workflowStages":[]},"version":"v1","identity":"rs-7324506","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7324506","identity":"rs-7324506","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}