Sodium Butyrate Reinforces Intestinal Homeostasis and Ameliorates Post-Resuscitation Neuroinflammation in Rat Model of Cardiac Arrest

Sodium Butyrate Reinforces Intestinal Homeostasis and Ameliorates Post-Resuscitation Neuroinflammation in Rat Model of Cardiac Arrest | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sodium Butyrate Reinforces Intestinal Homeostasis and Ameliorates Post-Resuscitation Neuroinflammation in Rat Model of Cardiac Arrest Haojun Zhang, Tianpeng Xu, He Li, Yufeng Zhu, Rongyi Shi, Xi Chen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6702701/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cardiac arrest (CA) poses a critical global public health challenge, with post-cardiac arrest syndrome (PCAS) remaining a leading cause of mortality despite advancements in resuscitation protocols. Systemic ischemia-reperfusion injury post-CA often triggers acute gastrointestinal injury (AGI), characterized by intestinal barrier disruption, dysbiosis, and endotoxin-driven inflammation, which correlates with poor clinical outcomes. This study investigated sodium butyrate, a gut microbiota-derived short-chain fatty acid, in a CA/CPR rat model, revealing its multifaceted therapeutic potential: it improved neurobehavioral recovery, preserved ileal epithelial tight junction integrity, and remodeled gut microbiota by enriching SCFA-producing taxa while suppressing pathogens. Furthermore, sodium butyrate significantly reduced neuroinflammatory markers (IL-1α, NLRP3 inflammasome) in brain tissues, suggesting modulation of the microbiota-gut-brain axis (MGBA). These findings underscore sodium butyrate’s role in mitigating PCAS through dual mechanisms—restoring intestinal homeostasis and dampening systemic inflammation—thereby offering a novel therapeutic strategy to improve post-resuscitation outcomes. Post-Cardiac Arrest Syndrome Intestinal Homeostasis Gut Microbiota Sodium Butyrate Microbiota-Gut-Brain Axis Neuroinflammation. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Cardiac Arrest (CA) represents one of the most critical clinical challenges in emergency and critical care medicine. Despite advancements in early implementation of the "chain of survival"—including prompt activation of emergency response systems, high-quality cardiopulmonary resuscitation (CPR), early defibrillation, and effective advanced life support—which have improved the probability of restoring spontaneous circulation (ROSC) in patients [ 1 , 2 ], post-ROSC multi-organ dysfunction remains a major hurdle in clinical management. Statistics indicate that approximately 60–70% of patients progress to post-cardiac arrest syndrome (PCAS) following ROSC [ 3 ], characterized by persistent brain injury, myocardial suppression, systemic ischemia-reperfusion (I/R) injury, and worsening of the underlying disease. Strategies to prevent and intervene in PCAS to improve patient outcomes have become a focal point of research in emergency medicine. Current therapeutic approaches for PCAS primarily focus on multi-organ support, such as respiratory and circulatory interventions, and neuroprotective hypothermia. Notably, the intestinal mucosal epithelium is one of the most sensitive tissues to systemic I/R injury. Clinically, over half of CA/CPR patients with ROSC exhibit upper gastrointestinal ischemic injury, and those with intestinal dysfunction demonstrate higher organ failure scores and poorer prognoses during ICU stays[ 4 , 5 ]. Manifestations include loss of bowel sounds and elevated intra-abdominal pressure. Research [ 6 – 8 ] suggests that post-CA intestinal I/R injury directly damages epithelial cells, disrupts tight junction proteins, and increases intestinal barrier permeability. Furthermore, gut dysbiosis post-CA activates Th17 cells, elevating IL-17 secretion and amplifying systemic inflammatory cascades, thereby accelerating multi-organ failure. Additionally, endotoxins from gut pathogens readily translocate through the compromised intestinal barrier via the portal system, activating hepatic Kupffer cells and exacerbating systemic inflammation. Thus, protecting gastrointestinal function and correcting intestinal dysregulation in CA/CPR patients are critical clinical strategies to improve outcomes and reduce multi-organ failure. The gastrointestinal tract harbors the body’s most extensive microbial reservoir, serving as a "driving force" for multi-organ dysfunction in critically ill patients [ 8 ]. The human gut microbiota is predominantly composed of Firmicutes and Bacteroidetes, with smaller proportions of Actinobacteria, Proteobacteria, and other microorganisms [ 9 ]. Current research on post-CPR gut microbiota is limited, Gut microbiota dysbiosis is closely linked to the prognosis of cerebral ischemic injury[ 10 ]. Existing studies reveal that ischemic injuries, such as stroke, lead to dysbiosis characterized by reduced abundance of Proteobacteria and specific Firmicutes (e.g., Clostridia and Lachnospiraceae )[ 11 ]. Animal studies in porcine CPR models demonstrate shifts in microbial composition and diversity, including increased abundance of Akkermansia muciniphila and Mucispirillum schaedleri , alongside decreased levels of anti-inflammatory, short-chain fatty acid (SCFA)-producing Bifidobacterium and Roseburia [ 12 ]. These findings underscore the urgent need to elucidate post-CPR gut microbial dynamics. Short-chain fatty acids (SCFAs), key metabolites of dietary fiber fermentation by gut microbes, play a vital role in intestinal protection. Major SCFAs include acetate, propionate, and butyrate [ 13 ]. Butyrate, the most critical SCFA for gut homeostasis, serves as the primary energy source for colonic epithelial cells. It mitigates intestinal I/R injury by reducing endotoxin leakage and suppressing oxidative stress [ 14 , 15 ]. SCFAs also modulate immune cell differentiation via G protein-coupled receptors (GPCRs) or histone deacetylase (HDAC) activity in intestinal epithelial cells. Beyond the gut, SCFAs regulate immunity in extra-intestinal organs (e.g., liver, lungs, reproductive tract, and brain) and are implicated in diverse pathologies, including inflammation, autoimmunity, allergies, and cancer [ 16 , 17 ]。 Sodium butyrate, the sodium salt of butyric acid, exhibits the strongest anti-inflammatory and immunomodulatory effects among SCFAs. Unlike butyrate, which undergoes significant hepatic first-pass metabolism, sodium butyrate—a stable derivative—prolongs biological half-life, enabling sustained systemic effects[ 13 , 18 , 19 ]. While most SCFA research focuses on probiotic therapies, challenges such as colonization efficiency, host heterogeneity, and disease-specific responses complicate direct validation of SCFAs as therapeutic mediators. Current studies on butyrate’s protective role in intestinal function emphasize inflammation regulation, primarily in single-organ I/R models (e.g., middle cerebral artery occlusion) and sepsis-induced gut injury. Preclinical evidence [ 20 – 22 ] shows that butyrate supplementation reduces chronic inflammation post-cerebral I/R injury, attenuates neuronal apoptosis via the GPR41-Gβγ-PI3K/Akt pathway, and corrects microglial polarization (M1/M2 imbalance) through GPR109A, downregulating the TLR4/NF-κB inflammatory axis and alleviating LPS-driven intestinal damage. Fecal microbiota transplantation (FMT) with butyrate-producing strains reduces ischemic lesion volume, cerebral edema, and neurobehavioral deficits in stroke models[ 23 ]. SCFA supplementation in microbiota-disrupted mice enhances gut T cell trafficking to the brain, increases regulatory T (Treg) cell populations, restores microglial homeostasis [ 24 ], and shifts the balance toward neuroprotective FoxP3 + Treg cells (via IL-10 secretion) and away from pro-inflammatory IL-17-producing γδT cells [ 25 ]。 This study aims to investigate sodium butyrate’s regulatory effects on intestinal homeostasis in a CA/CPR rat model. We will evaluate its impact on gut barrier integrity, microbiota diversity/structure, and inflammatory responses, while assessing CPR outcomes and 24-hour neurological function. Integrating 16S rRNA sequencing and immunological analyses, we seek to elucidate the role of the microbiota-gut-brain axis (MGBA) in PCAS pathogenesis, providing mechanistic insights and therapeutic targets for post-resuscitation multi-organ dysfunction. Methods Animals All experiments were performed following the National Institutes of Health Guide for the Care and Use of Laboratory Animals and ARRIVE guidelines. Experimental protocols were reviewed and approved by the Animal Ethics Committee of Naval Medical Center(2025Q034) and outlined in Fig. 1 .Male SPF-grade Sprague-Dawley (SD) rats (6–8 weeks old, body weight 300 ± 20 g) used in this experiment were purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd. The animals were housed under standardized conditions in the laboratory animal facility of Naval Medical University, with environmental parameters set as follows: 12-hour light/dark cycle, constant temperature maintained at 23 ± 2°C, relative humidity of 60%, and free access to standard rodent feed and autoclaved drinking water. After a 1-week acclimation period, all animals were fasted for 12 hours (with free access to water) prior to the establishment of the cardiac arrest/cardiopulmonary resuscitation (CA/CPR) model. Groups All Sprague-Dawley (SD) rats were randomly divided into 3 groups (6 rats per group). Sham group: Underwent anesthesia, tracheal intubation, and arteriovenous catheterization without further intervention; Control group: Underwent anesthesia, tracheal intubation, and arteriovenous catheterization, followed by 6-minute CA induced by asphyxia combined with potassium chloride injection, and subsequent CPR; Nab group: Underwent the same surgical procedures and CA/CPR protocol as the Control group, followed by sodium butyrate administration via oral gavage at 30 minutes, 6 hours, and 12 hours after return of spontaneous circulation(ROSC). Sodium butyrate administration Sodium butyrate powder was weighed at a dosage of 50 mg per 100 g of rat body weight, then dissolved in 3 mL of sterile injectable water to prepare a sterile sodium butyrate solution. Cardiac arrest model Following weight measurement, anesthesia was induced via intraperitoneal injection of 5% pentobarbital sodium (45 mg/kg). Rats were positioned supine on a temperature-regulated surgical table (maintained by an infrared heating lamp) and underwent hair removal in the left groin area for surgical preparation. A 14G tracheal tube was inserted under direct visualization with an intelligent visual endoscope, secured to the oral skin with sutures, and connected to a small animal ventilator (ALC-V8D) set to controlled intermittent positive pressure ventilation with parameters: tidal volume 0.65 mL/100g, respiratory rate 80 breaths/min, and FiO₂ 21%. Simultaneously, a multi-channel physiological monitoring system was established: limb electrodes were attached for continuous lead II ECG monitoring via the ALCBioMPA2000 system; a PE-50 catheter was inserted into the left femoral artery for real-time mean arterial pressure (MAP) monitoring, while a venous catheter in the ipsilateral femoral vein provided drug administration access. All catheters were secured with double surgical sutures. Core body temperature (36.5–37.5°C) was maintained throughout the procedure and for 4 hours postoperatively. After 30 minutes of surgical stabilization, CA was induced by clamping the tracheal tube to induce asphyxia, followed by withdrawal of ventilator support and confirmation of apnea. CA was further verified by intravenous injection of 10% potassium chloride (0.12 mL/100g) until systolic pressure dropped to 25 mmHg. After 6 minutes of asystole, standardized CPR was initiated: manual chest compressions(160 compressions/min, depth 1/3 of anteroposterior chest diameter) were performed using the xiphoid landmark method, combined with mechanical ventilation(tidal volume 0.65 mL/100g, rate 80 breaths/min, FiO₂ 21%) at a compression-to-ventilation ratio of 2:1. Immediately upon CPR initiation, 0.5 mL of epinephrine(10 µg/kg) and 1.5 mL of 5% sodium bicarbonate were administered intravenously. ROSC was defined as spontaneous rhythm restoration with systolic pressure ≥ 60 mmHg sustained for ≥ 10 minutes; CPR duration to ROSC was recorded. Failed resuscitation was defined as persistent asystole after 12 minutes of CPR. At 30 minutes post-ROSC, post-resuscitation mean arterial pressure(aMAP) and heart rate(aHR) were monitored. Ventilator support and catheters were removed after spontaneous breathing resumed. Incisions were disinfected and sutured, and rats were housed individually with free access to water. All procedures strictly adhered to Utstein-style guidelines for experimental design and data recording[ 26 ]. Neurobehavioral assessment Neurobehavioral assessment among the groups was performed using the LONGA scoring method at 24 hours post-ROSC by placing rats in an open field to evaluate locomotor and behavioral parameters. Scores were recorded adhering to the LONGA criteria[ 27 ]: 0 point: no nerve function defect; 1 point: the paw on the paralytic side cannot be fully extended; 2 points: when walking, the rat circles to the paralytic side; 3 points: when walking, the rat body topples to the paralytic side; 4 points: unable to walk spontaneously, loss of consciousness. Euthanasia and tissue sampling A total of 19 rats (51.4%) could not be resuscitated,12 resuscitated rats and 6 Sham rats successfully survived 24 hours and were included in the study. Rats were anesthetized via intraperitoneal injection of 5% sodium pentobarbital (45 mg/kg). After achieving stable anesthesia, the right groin and abdominal regions were shaved and disinfected, followed by fixation on a sterile surgical table. A PE-50 catheter was inserted into the right femoral vein to collect 4 mL of whole blood, which was centrifuged at room temperature (3000 × g, 20 min) to isolate serum. The supernatant was aliquoted and stored at − 80°C for subsequent multiplex cytokine analysis. Following blood sampling, rats were euthanized by anesthetic overdose. The abdominal cavity was aseptically opened to dissect intestinal tissues, and a 2 cm ileal segment (2 cm proximal to the ileocecal junction) was excised. Fecal samples (~ 200 mg) were scraped from the mucosal surface, rapidly transferred to pre-chilled sterile EP tubes, flash-frozen in liquid nitrogen, and archived at − 80°C for 16S rDNA sequencing. Fresh ileal tissues were rinsed with saline, divided into two portions: one fixed for paraffin-embedded histomorphological analysis, and the other snap-frozen in liquid nitrogen for quantification of intestinal cytokines and injury biomarkers. All samples were maintained at − 80°C until further assays. Enzyme‑linked immunosorbent assay (ELISA) At 24 h post-ROSC, serum levels of inflammatory cytokines (IL-1α, IL-1β, IL-6, IL-10, IL-11, IL-17, TNF-α) were quantified using species-specific ELISA kits (Shanghai Xitang Biotechnology Co., Ltd., Shanghai, China) in accordance with the manufacturer’s protocol. For ileal tissue analysis, PBS-rinsed samples were homogenized with a 4°C pre-chilled tissue homogenizer, followed by centrifugation at 12,000 × g for 20 min (4°C) to obtain supernatants. Concentrations of inflammatory cytokines (IL-1α, IL-1β, IL-6, IL-10, IL-11, IL-17, TNF-α) and biomarkers (NLRP3, TLR4, MPO, Reg3α, I-FABP) in the supernatants were subsequently determined via the same ELISA methodology. Hematoxylin–eosin (HE) Staining and transmission electron microscopy (TEM) Fresh ileal specimens were fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin as 0.5 cm-thick blocks, and sectioned into 5-µm slices for HE staining per standard protocols, with subsequent digitization using a NanoZoomer S210 scanner (Hamamatsu, Japan). Parallel samples (1 mm³) underwent sequential processing including primary fixation in 2.5% glutaraldehyde, post-fixation with 1% OsO4 in PBS (pH 7.4, 2 hr RT), ethanol dehydration, propylene oxide-epoxy resin (1:1) infiltration at 70°C overnight, and ultramicrotomy-generated 60–80 nm sections for TEM imaging (Talos L120C, ThermoFisher) followed by identical NanoZoomer digitization. Blinded histological assessments were conducted by independent researchers to ensure objective evaluation. Histomorphological analysis - Chiu grading Paraffin-embedded jejunal tissue was sectioned(4µm) and stained with hematoxylin and eosin(H&E) according to standard protocols. The morphological integrity of the intestinal wall was classified by a blinded investigator using a modified protocol according to Chiu grading[ 28 ]: Grade 0: normal mucosa; Grade 1: development of a sub-epithelial space at the tips of the villi; Grade 2: more extended sub-epithelial space at the tips of the villi, development of Gruenhagen’s space at the tips of the villi; Grade 3: massive epithelial lifting down the sides of the villi, villus necrosis; Grade 4: villi are denuded of epithelial layer; Grade 5: loss of villi, mucosal ulceration and necrosis with invasion of the muscularis propria. DNA extraction and 16S rRNA high-throughput sequencing Sequencing was performed with the help of LC-Bio Technologies (Hangzhou) Co., Ltd. The total fecal microbial DNA was obtained through the Fecal Genome DNA Extraction Kit (AU46111-96, BioTeke, China) according to the manufacturer’ s instruction manual. The DNA was quantified by Qubit (Invitrogen, USA). Total DNA was amplified by PCR using the universal primer 341F/805R (341F: 5′-CCT ACGGGNGGCWGCAG-3′; 805R: 5′-GACTACHVGGG TATCTAATCC-3′). The PCR amplification conditions were pre-denaturation at 98℃ for 30s,denaturation at 98℃ for 10s, annealing at 54℃ for 30s, extension at 72℃ for 45s and 32 cycles. The final extension was at 72℃ for 10 min. The PCR product was purified using AMPure XP Beads (Beckman Coulter Genomics, Danvers, MA, USA) and quantified using Qubit (Invitrogen, USA). Qualified PCR products were evaluated using an Agilent 2100 Bioanalyzer (Agilent, USA) and Illumina library quantitative kits (Kapa Biosciences, Woburn, MA, USA), which were further pooled together and sequenced on an Illumina NovaSeq 6000(PE250), provided by LC-Bio Technology Co., Ltd, Hangzhou, China. The original raw data were spliced by overlapping quality control and chimera filtering to obtain 250-bp paired-end reads. A divisive amplicon denoising algorithm was used to dereplicate and establish ASVs, obtained through amplicon sequencing to conduct bacterial diversity analysis and annotation of species classification and differential analysis. Statistical analysis This study employed SPSS 26.0 for statistical analysis. Categorical data were described as frequency(percentage), while ordinal data were presented as medians. Median comparisons were performed using the Mann-Whitney U test, with a significance threshold set at p < 0.05. Normally distributed continuous data were expressed as mean ± standard deviation (x̄ ± s). Independent samples t -test was used for comparisons between two groups, and one-way analysis of variance (ANOVA) was applied for multi-group comparisons: the LSD method was selected when homogeneity of variance was satisfied, whereas the Games-Howell adjustment was employed for heterogeneous variance conditions. Sequencing primer were removed from de-multiplexed raw sequences using cutadapt (v1.9). Then, Pairedend reads were merged using FLASH (v1.2.8). The low-quality reads (quality scores < 20), short reads(< 100bp), and reads containing more than 5% “N” records were trimmed by using the sliding-window algorithm method in fqtrim (v 0.94). Quality filtering was performed to obtain high-quality clean tags according to fqtrim. Chimeric sequences were filtered using Vsearch software (v2.3.4). DADA2 was applied for denoising and generating amplicon sequence variants (ASVs). The sequence alignment of species annotation was performed by QIIME2 plugin feature-classifier, and the alignment database was SILVA and NT-16S. Alpha and beta diversities were calculated using QIIME2, Relative abundance was used in bacteria taxonomy. The Wilcox test was used to identify the differentially abundant genus, and significances were declared at p < 0.05. LDA effect size (LEfSe, LDA ≥ 3.0, p < 0.05) was performed using nsegata-lefse. Other diagrams were implemented using the R package (v3.4.4). Results Physiologic, hemodynamic, and resuscitation characteristics As detailed in Table 1 , a total of 37 rats underwent surgical procedures, including 6 in the Sham group, 15 in the Control group, and 16 in the Nab group following supplemental experiments. No significant differences were observed in pre-operative baseline parameters among groups, including body weight, time to surgical preparation (T o ), heart rate (HR), and mean arterial pressure (MAP) measured at 30 min post-surgical preparation ( p > 0.05 for all comparisons). No statistically significant differences ( p > 0.05) were observed between Control and Nab rats in the time from the onset of asphyxia to cardiac arrest (T ca ), the duration of cardiopulmonary resuscitation (T cpr ), post-resuscitation average mean arterial pressure (aMAP), or heart rate (aHR) in Table 2 . Table 1 Basic characteristics Group Weight (g) HR (bpm) MAP (mmHg) T o (min) Sham 310.00 ± 7.75 429.17 ± 24.52 148.56 ± 11.94 41.45 ± 8.50 Control 306.67 ± 13.32 451.27 ± 34.22 156.20 ± 12.32 42.00 ± 5.99 Nab 305.00 ± 7.96 463.94 ± 37.37 156.63 ± 14.91 41.33 ± 7.86 P value 0.611 0.120 0.433 0.963 Values are mean ± SD. HR, heart rate; MAP, mean arterial pressure; T o , the time of surgical operation; g, gram; s, second; min, minute; bpm, beats per minute. Table 2 Resuscitation-associated characteristics Group T ca (s) T cpr (min) aMAP (mmHg) aHR (bpm) Control 66.67 ± 4.63 6.86 ± 2.23 108.67 ± 17.20 317.33 ± 43.92 Nab 67.50 ± 8.19 7.96 ± 2.22 103.16 ± 14.17 354.50 ± 65.06 P value 0.833 0.411 0.559 0.273 Values are mean ± SD, bpm, Score are midline; aMAP, mean arterial pressure after ROSC; aHR, heart rate after ROSC; bpm, beats per minute. SB altered the ileal microbiota composition after CA To investigate the alterations in intestinal flora during cardiac arrest (CA) and the regulatory effects of sodium butyrate (SB) on post-resuscitation gut microbiota in rats, we conducted 16S rDNA gene amplicon sequencing analysis on ileal fecal samples from rats (6 samples per group). Compared to the Sham group, the number of operational taxonomic units (OTUs) in the gut microbiota was significantly reduced in the control group after CA/CPR, whereas it was significantly increased in the Nab group post-CA/CPR. This result aligns with the design objectives of our study (Fig. 2 A). Alpha-diversity analysis showed no significant difference in ileal community richness (Chao1 index, Fig. 2 B) between Control and Sham groups ( p = 0.132), but a significant increase in community diversity (Shannon index, Fig. 2 C) in the Control group ( p = 0.026). The Nab group exhibited significantly enhanced ileal richness (Chao1 index) and community diversity (Shannon index) compared to both Sham and Control groups ( p < 0.05). Beta-diversity analysis using Weighted_Unifrac-based PCoA and Anosim demonstrated distinct clustering of gut microbiota communities among the three groups (Fig. 2 D&E, p = 0.001, R = 0.5045). At the phylum level (Fig. 2 F), Control rats showed significantly reduced relative abundance of Firmicutes and increased Proteobacteria and Fusobacteriota compared to Sham group. Genus-level analysis (Fig. 2 G&H) revealed elevated relative abundance of Escherichia-Shigella in Control versus Sham group. Compared to Control, Nab group exhibited increased abundance of short-chain fatty acid (SCFA)-producing genera: Ruminococcus, Rothia, Lachnospiraceae_NK4A136_group, Oscillibacter, Romboutsia, and decreased abundance of Escherichia-Shigella. LEfSe analysis (Fig. 2 I&J) identified opportunistic pathogens Fusobacterium and Escherichia-Shigella as biomarkers in Control group, while short-chain fatty acid (SCFA)-producing Romboutsia, Eubacterium, and Ruminococcus were characteristic of Nab group. These findings suggest that Nab administration may remodel post-CA ileal microbiota by upregulating SCFA-producing genera with potential intestinal barrier-enhancing properties while suppressing pathogenic bacteria, indicating therapeutic potential for gut microbiota modulation after resuscitation. SB maintains ileal epithelial homeostasis in rats after CA After preparing hematoxylin-eosin (HE)-stained and electron microscopy (EM) sections of ileal tissues from rats 24 hours post-resuscitation, the ileal tissue slices from the sham, Control, and Nab groups were scanned into images using randomized blinding methods and blinded evaluation, as shown in Fig. 3 A&B. The ileal mucosa of control group rats exhibited villus tip denudation, with severe cases showing Gruenhagen's space, along with mild perivascular edema, epithelial cell swelling, and scattered pyknotic cells. In contrast, ileal tissues from the Nab group at 24 hours post-ROSC showed no pathological injuries exceeding Chiu grade 1. Furthermore, as shown in Fig. 3 C&D, the median Chiu’s score of ileal tissues in the Nab group was significantly reduced compared to the Control group (0.5 vs. 2, p = 0.011). Under electron microscopy, ileal epithelial cells in the Control group at 24 hours post-cardiopulmonary resuscitation (CPR) demonstrated widened gaps in tight junctions between cells (as indicated by arrows in Fig. 3 E. In contrast, such phenomena were not observed in the Nab group. SB improve neurobehavioral deficits after CA 24 hours post-resuscitation, rats were placed in an open field to observe their motor activity and behavior. Scores were recorded based on the observations, with the neurobehavioral assessment conducted using the LONGA scoring criteria. In the Nab group, the median neurofunctional deficit score (LONGA score) at 24 hours post-cardiopulmonary resuscitation (CPR) was 0, which was significantly lower than the median score of 2 in the Control group (0 vs. 2, p = 0.009, Fig. 3 F). SB alleviated neuroinflammatory response at 24 hours post-CPR As shown in Fig. 3 G &H , the concentrations of cytokines and injury-related biomarkers in brain tissues were analyzed. The IL-1α level in the Nab group was significantly lower than that in the Control group (32.10 ± 13.32 pg/ml vs. 64.84 ± 17.28 pg/ml, p = 0.011). Additionally, the NLRP3 inflammasome level in the Nab group was markedly reduced compared to the Control group (0.79 ± 0.05 ng/ml vs. 1.51 ± 0.43 ng/ml, p = 0.021). Discussion This study demonstrated that sodium butyrate (SB) can remodel the ileal microbiota structure post-cardiac arrest/cardiorespiratory resuscitation (CA/CPR) in rats by reducing the relative abundance of pathogenic Escherichia-Shigella and increasing short-chain fatty acid (SCFA)-producing genera, thereby alleviating intestinal barrier damage, reducing neuroinflammation, and improving post-resuscitation neurobehavioral deficits. Furthermore, SB may exert its neuroprotective effects through the microbiota-gut-brain axis (MGBA) by downregulating the IL-1α/NLRP3 signaling pathway in the nervous system. Intestinal dysfunction following CA/CPR constitutes a critical component of post-cardiac arrest syndrome (PCAS). Intestinal mucosal epithelial cells are among the most sensitive organs to systemic ischemia/reperfusion (I/R) injury. Intestinal I/R injury triggers epithelial barrier disruption, inflammatory cytokine release, systemic inflammatory responses, and multi-organ failure. Additionally, trillions of gut microbes undergo structural and diversity alterations during intestinal damage, exacerbating mucosal permeability and promoting bacterial translocation—a key catalyst for post-resuscitation multi-organ dysfunction[5, 7, 29]. Human retrospective studies and animal CA models[11, 12] have confirmed that cerebral I/R injury induces dysbiosis, characterized by reduced abundance of dominant phyla such as Proteobacteria, Firmicutes (Clostridia and Lachnospiraceae), and Bacteroidetes (Bifidobacterium and anti-inflammatory SCFA-producing Roseburia ). This dysbiosis drives elevated serum IL-17 levels, promotes Th17 cell infiltration into brain tissues[6] , and facilitates pathogenic microbiota-derived lipopolysaccharide (LPS)-mediated inflammatory cascades, ultimately worsening clinical outcomes[30]. The human gut microbiota is predominantly composed of Firmicutes and Bacteroidetes, with minor contributions from Actinobacteria, Proteobacteria, and other microorganisms[9]. Post-cerebral ischemic injury, patients exhibit decreased abundance of Proteobacteria and specific Firmicutes (e.g., Clostridia and Lachnospiraceae)[11] . In animal models, swine post-CPR models show reduced microbial diversity, particularly diminished abundance of Bifidobacterium and SCFA-producing Roseburia [12] . While SCFA-producing microbiota have demonstrated anti-inflammatory and antioxidative effects in post-resuscitation brain injury[31, 32] and myocardial protection against I/R injury[33] , evidence remains limited. Our findings reveal that oral SB administration reduces post-CPR ileal Escherichia-Shigella abundance while enhancing SCFA-producing microbiota in rats. Short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are key metabolites of dietary fiber fermentation by gut microbiota[13] . Emerging evidence highlights butyrate's critical role in maintaining intestinal immune homeostasis and epithelial barrier integrity[34-37] . Preclinical and clinical studies indicate that butyrate supplementation alleviates colitis via immunomodulation and barrier enhancement[36, 38, 39] , showing therapeutic potential in inflammatory bowel disease (IBD)[35] . However, research on butyrate's regulation of post-CA/CPR intestinal homeostasis remains scarce, with prior studies primarily focusing on its unvalidated anti-inflammatory effects in focal cerebral I/R models[20-22] . We hypothesize that endogenous SB may mitigate CA/CPR-induced global I/R-related neuroinflammation through MGBA-mediated intestinal homeostasis modulation. Our data show SB significantly improves 24-hour neurobehavioral scores and reduces cerebral IL-1α/NLRP3 levels in resuscitated rats. Notably, IL-1α correlates with all-cause mortality in out-of-hospital cardiac arrest (OHCA) patients[40] , while NLRP3 inflammasome inhibition attenuates microglial activation[41] and improves resuscitation outcomes by mitigating post-resuscitation myocardial dysfunction[42]。While this study provides phenotypic evidence of SB's intestinal-protective effects post-CPR, the specific regulatory pathways and SB's role within the MGBA framework require further elucidation. As the "second genome" and "ninth organ system," the gut microbiota plays a pivotal role in systemic health. SCFAs, particularly butyrate as a key MGBA messenger, hold significant therapeutic potential for post-resuscitation intestinal homeostasis and neuroprotection. In summary, this study confirms that oral SB alleviates CA/CPR-induced neurobehavioral deficits in rats by restoring gut microbiota balance, mitigating intestinal injury, and reducing neuroinflammation, potentially mediated through MGBA. These findings highlight SB's capacity to modulate remote organ systems (e.g., the nervous system), warranting further investigation into its precise mechanisms of action. Declarations Funding Declaration The study was financially supported by the National Key Clinical Specialty Discipline Construction Program of China (2022YFC3103-001004) Author Contribution Haojun Zhang and Tianpeng Xu participated in the design of this study, performed most of the experiments and statistical analysis and drafted the main manuscript. He Li prepared figures 1-4. Yufeng Zhu, Rongyi Shi and Xi Chen were the assistants during all of the experiments. Yi Shan supervised the work. All authors have read and approved the final version of the manuscript. 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Neurocrit Care, 2024. Martin-Gallausiaux, C., et al., SCFA: mechanisms and functional importance in the gut. Proc Nutr Soc, 2021. 80 (1): p. 37-49. Qiao, Y., et al., Protective effects of butyrate on intestinal ischemia-reperfusion injury in rats. J Surg Res, 2015. 197 (2): p. 324-30. Liu, B., et al., Butyrate protects rat liver against total hepatic ischemia reperfusion injury with bowel congestion. PLoS One, 2014. 9 (8): p. e106184. Mann, E.R., Y.K. Lam, and H.H. Uhlig, Short-chain fatty acids: linking diet, the microbiome and immunity. Nat Rev Immunol, 2024. 24 (8): p. 577-595. Juul, F.E., et al., Fecal Microbiota Transplantation for Primary Clostridium difficile Infection. N Engl J Med, 2018. 378 (26): p. 2535-2536. Corrêa-Oliveira, R., et al., Regulation of immune cell function by short-chain fatty acids. Clin Transl Immunology, 2016. 5 (4): p. e73. Silva, Y.P., A. Bernardi, and R.L. Frozza, The Role of Short-Chain Fatty Acids From Gut Microbiota in Gut-Brain Communication. Front Endocrinol (Lausanne), 2020. 11 : p. 25. Zhou, Z., et al., Sodium butyrate attenuated neuronal apoptosis via GPR41/Gβγ/PI3K/Akt pathway after MCAO in rats. J Cereb Blood Flow Metab, 2021. 41 (2): p. 267-281. Wei, H., et al., Butyrate ameliorates chronic alcoholic central nervous damage by suppressing microglia-mediated neuroinflammation and modulating the microbiome-gut-brain axis. Biomed Pharmacother, 2023. 160 : p. 114308. Ma, B.D.Y., T.Y.H. Chan, and B.W.Y. Lo, Unveiling the hidden culprit: How the brain-gut axis fuels neuroinflammation in ischemic stroke. Surg Neurol Int, 2024. 15 : p. 394. Pasokh, A., et al., The effect of fecal microbiota transplantation on stroke outcomes: A systematic review. J Stroke Cerebrovasc Dis, 2022. 31 (11): p. 106727. Celorrio, M., et al., Short-chain fatty acids are a key mediator of gut microbial regulation of T cell trafficking and differentiation after traumatic brain injury. Res Sq, 2024. Benakis, C. and A. Liesz, The gut-brain axis in ischemic stroke: its relevance in pathology and as a therapeutic target. Neurol Res Pract, 2022. 4 (1): p. 57. Antman, E.M., et al., ACC/AHA guidelines for the management of patients with ST-elevation myocardial infarction--executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Revise the 1999 Guidelines for the Management of Patients With Acute Myocardial Infarction). Circulation, 2004. 110 (5): p. 588-636. Longa, E.Z., et al., Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989. 20 (1): p. 84-91. Chiu, C.J., et al., Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal. Arch Surg, 1970. 101 (4): p. 478-83. Piton, G., et al., Enterocyte Damage: A Piece in the Puzzle of Post-Cardiac Arrest Syndrome. Shock, 2015. 44 (5): p. 438-44. Li, X., et al., Hydrogen sulfide inhibits lipopolysaccharide-based neuroinflammation-induced astrocyte polarization after cerebral ischemia/reperfusion injury. Eur J Pharmacol, 2023. 949 : p. 175743. Cao, F., et al., Artificial-enzymes-armed Bifidobacterium longum probiotics for alleviating intestinal inflammation and microbiota dysbiosis. Nature Nanotechnology, 2023. 18 (6): p. 617-627. Wang, Y.-H., et al., Gut microbiota-derived succinate aggravates acute lung injury after intestinal ischemia/reperfusion in mice. European Respiratory Journal, 2022: p. 2200840. Chen, H.C., et al., Gut butyrate-producers confer post-infarction cardiac protection. Nat Commun, 2023. 14 (1): p. 7249. Peng, L., et al., Butyrate enhances the intestinal barrier by facilitating tight junction assembly via activation of AMP-activated protein kinase in Caco-2 cell monolayers. J Nutr, 2009. 139 (9): p. 1619-25. Recharla, N., R. Geesala, and X.Z. Shi, Gut Microbial Metabolite Butyrate and Its Therapeutic Role in Inflammatory Bowel Disease: A Literature Review. Nutrients, 2023. 15 (10). Liang, L., et al., Gut microbiota-derived butyrate regulates gut mucus barrier repair by activating the macrophage/WNT/ERK signaling pathway. Clin Sci (Lond), 2022. 136 (4): p. 291-307. Arpaia, N., et al., Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature, 2013. 504 (7480): p. 451-5. Velasquez-Manoff, M., Gut microbiome: the peacekeepers. Nature, 2015. 518 (7540): p. S3-11. Rivière, A., et al., Bifidobacteria and Butyrate-Producing Colon Bacteria: Importance and Strategies for Their Stimulation in the Human Gut. Front Microbiol, 2016. 7 : p. 979. Zelniker, T.A., et al., Relationship between markers of inflammation and hemodynamic stress and death in patients with out-of-hospital cardiac arrest. Scientific Reports, 2021. 11 (1): p. 9954. Wang, M., et al., Microglia-Mediated Neuroinflammation: A Potential Target for the Treatment of Cardiovascular Diseases. J Inflamm Res, 2022. 15 : p. 3083-3094. Zheng, G., et al., The Selective NLRP3-inflammasome inhibitor MCC950 Mitigates Post-resuscitation Myocardial Dysfunction and Improves Survival in a Rat Model of Cardiac Arrest and Resuscitation. Cardiovasc Drugs Ther, 2023. 37 (3): p. 423-433. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6702701","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":463443416,"identity":"81e92eb6-cc6f-413a-b45b-4053c20cd3d6","order_by":0,"name":"Haojun Zhang","email":"","orcid":"","institution":"PLA Naval Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Haojun","middleName":"","lastName":"Zhang","suffix":""},{"id":463443417,"identity":"b192b816-fe42-408c-91e8-4ca3f01e89cc","order_by":1,"name":"Tianpeng Xu","email":"","orcid":"","institution":"Shanghai Changzheng Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tianpeng","middleName":"","lastName":"Xu","suffix":""},{"id":463443418,"identity":"9f2803b3-44d7-4c85-b0f7-c39d16d95d18","order_by":2,"name":"He Li","email":"","orcid":"","institution":"PLA Naval Medical Center","correspondingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Li","suffix":""},{"id":463443419,"identity":"bc2f2cc3-2897-40c5-ab0e-766152d8d6d1","order_by":3,"name":"Yufeng Zhu","email":"","orcid":"","institution":"Shanghai Changzheng Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yufeng","middleName":"","lastName":"Zhu","suffix":""},{"id":463443420,"identity":"540d1b92-4365-4ec6-b8e5-67be11bef6fe","order_by":4,"name":"Rongyi Shi","email":"","orcid":"","institution":"Shanghai Changzheng Hospital","correspondingAuthor":false,"prefix":"","firstName":"Rongyi","middleName":"","lastName":"Shi","suffix":""},{"id":463443421,"identity":"4bad2d67-025f-48b6-b766-6f4f9b594c90","order_by":5,"name":"Xi Chen","email":"","orcid":"","institution":"Shanghai Changzheng Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xi","middleName":"","lastName":"Chen","suffix":""},{"id":463443422,"identity":"3ffb0d4d-e310-48b1-a69c-6804808c9081","order_by":6,"name":"Yi Shan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYDACCRBhYGPHxt7Y+PADCVrSkvl4DjcbSxCvheEw4zyJ9DYBHmJ0yM9ufvaYpyCNmU3yYRtQv52cbgMBLQZ3jpkb8xjY8LFJJ7Y9KGBINjY7QEiLRIKZNI8B0BbpxHYDCYYDidsIaZGfkf4NqOUwY5vkwTYJHmK0MNzIMYNokWAkUovBjZwyyTnAQGbjSQQGsgERfgE6bJvEmz82dvLtxx8+/FBhJ0dQCwgwIaLDgAjlIMD4g0iFo2AUjIJRMEIBAIa2PAAVa93zAAAAAElFTkSuQmCC","orcid":"","institution":"Shanghai Changzheng Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yi","middleName":"","lastName":"Shan","suffix":""}],"badges":[],"createdAt":"2025-05-20 01:53:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6702701/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6702701/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83734158,"identity":"f31f17d1-8f56-4faa-8c0e-8256134a4668","added_by":"auto","created_at":"2025-06-01 15:25:51","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":240940,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental protocol. Abstract od flow and timing of experiments including grouping, cardiac arrest, cardiopulmonary resuscitation and drug administration.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6702701/v1/b0356f70dff9db3b4c71f628.jpg"},{"id":83734159,"identity":"98b63634-104d-43d7-9514-19a4dbf11bdc","added_by":"auto","created_at":"2025-06-01 15:25:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2891471,"visible":true,"origin":"","legend":"\u003cp\u003eComposition analysis of ileal microbiota based on 16S rDNA sequencing. (A) Venn diagram based on the abundance of OTUs. The numbers and precent represent the values of OTUs that can be detected in all rats in a group. (B and C) Alpha-diversity analysis of ileal bacteria (Chao1, Shannon index). (D and E) PCoA plot based on Weighted Unifrac distance matrix and Anosim analysis. (F and G) Stacking plot analysis of bacteria at the phylum and Genu level for clustering aims to show the proportion of the top 20 species in the ranking and the changing trend. (H) Heatmap plot of bacteria at the genus level in different groups. (I-J) Cladogram and distribution histograms showing the results of LEfSe analysis (LDA ≥ 3.0, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6702701/v1/daf858c94b31bb51f4a45109.jpg"},{"id":83734161,"identity":"f827d3a9-5928-453f-8bca-0ca09e27f779","added_by":"auto","created_at":"2025-06-01 15:25:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3855133,"visible":true,"origin":"","legend":"\u003cp\u003ePathological Evaluation, Inflammatory Cytokine Profiles, Biomarker Levels of Ileal Epithelium and Neurobehavioral Outcomes Following Cardiopulmonary Resuscitation. (A-B) Histopathological evaluation demonstrated marked preservation of ileal architecture in the Nab group compared to Control group, with light microscopic analysis revealing apical villous denudation, Gruenhagen's spaces formation, perivascular edema, and pyknotic nuclei in control specimens (median Chiu score: 2 [IQR 1.5-2.5]), whereas Nab group exhibited intact mucosal integrity without lesions exceeding Chiu Grade I (subepithelial space at villus tip only; median score: 0.5 [IQR 0-1], \u003cem\u003ep\u003c/em\u003e=0.013 by Mann-Whitney U test), indicating significant mitigation of post-resuscitation intestinal ischemia-reperfusion injury through the intervention，Electron microscopy revealed widened intercellular spaces at ileal epithelial tight junctions in Control rats, while no such abnormalities were observed in the Nab group. (C) Neurobehavioral Assessment at 24 Hours Post-Resuscitation. (D-Q) Multiplex detection of inflammatory cytokines and injury biomarkers at 24 Hours Post-Resuscitation. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6702701/v1/302e5465ac3d560dcd4d67aa.jpg"},{"id":83734160,"identity":"e50a6332-269d-45ba-aac3-d2b328429e9e","added_by":"auto","created_at":"2025-06-01 15:25:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1060273,"visible":true,"origin":"","legend":"\u003cp\u003eSB protects against CA/CPR via the microbiome-gut-brain axis.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6702701/v1/f1676ea524891e76f88fc5b0.jpg"},{"id":85857362,"identity":"0f9f5af5-a7d1-4c18-9878-c61d4a10544e","added_by":"auto","created_at":"2025-07-02 11:47:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8912430,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6702701/v1/96158ed7-24fe-4a09-ad7e-efb8af1b489a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sodium Butyrate Reinforces Intestinal Homeostasis and Ameliorates Post-Resuscitation Neuroinflammation in Rat Model of Cardiac Arrest","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiac Arrest (CA) represents one of the most critical clinical challenges in emergency and critical care medicine. Despite advancements in early implementation of the \"chain of survival\"\u0026mdash;including prompt activation of emergency response systems, high-quality cardiopulmonary resuscitation (CPR), early defibrillation, and effective advanced life support\u0026mdash;which have improved the probability of restoring spontaneous circulation (ROSC) in patients [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], post-ROSC multi-organ dysfunction remains a major hurdle in clinical management. Statistics indicate that approximately 60\u0026ndash;70% of patients progress to post-cardiac arrest syndrome (PCAS) following ROSC [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], characterized by persistent brain injury, myocardial suppression, systemic ischemia-reperfusion (I/R) injury, and worsening of the underlying disease. Strategies to prevent and intervene in PCAS to improve patient outcomes have become a focal point of research in emergency medicine.\u003c/p\u003e \u003cp\u003eCurrent therapeutic approaches for PCAS primarily focus on multi-organ support, such as respiratory and circulatory interventions, and neuroprotective hypothermia. Notably, the intestinal mucosal epithelium is one of the most sensitive tissues to systemic I/R injury. Clinically, over half of CA/CPR patients with ROSC exhibit upper gastrointestinal ischemic injury, and those with intestinal dysfunction demonstrate higher organ failure scores and poorer prognoses during ICU stays[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Manifestations include loss of bowel sounds and elevated intra-abdominal pressure. Research [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] suggests that post-CA intestinal I/R injury directly damages epithelial cells, disrupts tight junction proteins, and increases intestinal barrier permeability. Furthermore, gut dysbiosis post-CA activates Th17 cells, elevating IL-17 secretion and amplifying systemic inflammatory cascades, thereby accelerating multi-organ failure. Additionally, endotoxins from gut pathogens readily translocate through the compromised intestinal barrier via the portal system, activating hepatic Kupffer cells and exacerbating systemic inflammation. Thus, protecting gastrointestinal function and correcting intestinal dysregulation in CA/CPR patients are critical clinical strategies to improve outcomes and reduce multi-organ failure.\u003c/p\u003e \u003cp\u003eThe gastrointestinal tract harbors the body\u0026rsquo;s most extensive microbial reservoir, serving as a \"driving force\" for multi-organ dysfunction in critically ill patients [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The human gut microbiota is predominantly composed of Firmicutes and Bacteroidetes, with smaller proportions of Actinobacteria, Proteobacteria, and other microorganisms [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Current research on post-CPR gut microbiota is limited, Gut microbiota dysbiosis is closely linked to the prognosis of cerebral ischemic injury[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Existing studies reveal that ischemic injuries, such as stroke, lead to dysbiosis characterized by reduced abundance of Proteobacteria and specific Firmicutes (e.g., \u003cem\u003eClostridia\u003c/em\u003e and \u003cem\u003eLachnospiraceae\u003c/em\u003e)[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Animal studies in porcine CPR models demonstrate shifts in microbial composition and diversity, including increased abundance of \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e and \u003cem\u003eMucispirillum schaedleri\u003c/em\u003e, alongside decreased levels of anti-inflammatory, short-chain fatty acid (SCFA)-producing \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eRoseburia\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These findings underscore the urgent need to elucidate post-CPR gut microbial dynamics.\u003c/p\u003e \u003cp\u003eShort-chain fatty acids (SCFAs), key metabolites of dietary fiber fermentation by gut microbes, play a vital role in intestinal protection. Major SCFAs include acetate, propionate, and butyrate [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Butyrate, the most critical SCFA for gut homeostasis, serves as the primary energy source for colonic epithelial cells. It mitigates intestinal I/R injury by reducing endotoxin leakage and suppressing oxidative stress [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. SCFAs also modulate immune cell differentiation via G protein-coupled receptors (GPCRs) or histone deacetylase (HDAC) activity in intestinal epithelial cells. Beyond the gut, SCFAs regulate immunity in extra-intestinal organs (e.g., liver, lungs, reproductive tract, and brain) and are implicated in diverse pathologies, including inflammation, autoimmunity, allergies, and cancer [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]。\u003c/p\u003e \u003cp\u003eSodium butyrate, the sodium salt of butyric acid, exhibits the strongest anti-inflammatory and immunomodulatory effects among SCFAs. Unlike butyrate, which undergoes significant hepatic first-pass metabolism, sodium butyrate\u0026mdash;a stable derivative\u0026mdash;prolongs biological half-life, enabling sustained systemic effects[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. While most SCFA research focuses on probiotic therapies, challenges such as colonization efficiency, host heterogeneity, and disease-specific responses complicate direct validation of SCFAs as therapeutic mediators. Current studies on butyrate\u0026rsquo;s protective role in intestinal function emphasize inflammation regulation, primarily in single-organ I/R models (e.g., middle cerebral artery occlusion) and sepsis-induced gut injury. Preclinical evidence [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] shows that butyrate supplementation reduces chronic inflammation post-cerebral I/R injury, attenuates neuronal apoptosis via the GPR41-Gβγ-PI3K/Akt pathway, and corrects microglial polarization (M1/M2 imbalance) through GPR109A, downregulating the TLR4/NF-κB inflammatory axis and alleviating LPS-driven intestinal damage. Fecal microbiota transplantation (FMT) with butyrate-producing strains reduces ischemic lesion volume, cerebral edema, and neurobehavioral deficits in stroke models[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. SCFA supplementation in microbiota-disrupted mice enhances gut T cell trafficking to the brain, increases regulatory T (Treg) cell populations, restores microglial homeostasis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and shifts the balance toward neuroprotective FoxP3\u0026thinsp;+\u0026thinsp;Treg cells (via IL-10 secretion) and away from pro-inflammatory IL-17-producing γδT cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]。\u003c/p\u003e \u003cp\u003eThis study aims to investigate sodium butyrate\u0026rsquo;s regulatory effects on intestinal homeostasis in a CA/CPR rat model. We will evaluate its impact on gut barrier integrity, microbiota diversity/structure, and inflammatory responses, while assessing CPR outcomes and 24-hour neurological function. Integrating 16S rRNA sequencing and immunological analyses, we seek to elucidate the role of the microbiota-gut-brain axis (MGBA) in PCAS pathogenesis, providing mechanistic insights and therapeutic targets for post-resuscitation multi-organ dysfunction.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All experiments were performed following the National Institutes of Health Guide for the Care and Use of Laboratory Animals and ARRIVE guidelines. Experimental protocols were reviewed and approved by the Animal Ethics Committee of Naval Medical Center(2025Q034) and outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.Male SPF-grade Sprague-Dawley (SD) rats (6–8 weeks old, body weight 300 ± 20 g) used in this experiment were purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd. The animals were housed under standardized conditions in the laboratory animal facility of Naval Medical University, with environmental parameters set as follows: 12-hour light/dark cycle, constant temperature maintained at 23 ± 2°C, relative humidity of 60%, and free access to standard rodent feed and autoclaved drinking water. After a 1-week acclimation period, all animals were fasted for 12 hours (with free access to water) prior to the establishment of the cardiac arrest/cardiopulmonary resuscitation (CA/CPR) model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGroups\u003c/h3\u003e\n\u003cp\u003eAll Sprague-Dawley (SD) rats were randomly divided into 3 groups (6 rats per group). Sham group: Underwent anesthesia, tracheal intubation, and arteriovenous catheterization without further intervention; Control group: Underwent anesthesia, tracheal intubation, and arteriovenous catheterization, followed by 6-minute CA induced by asphyxia combined with potassium chloride injection, and subsequent CPR; Nab group: Underwent the same surgical procedures and CA/CPR protocol as the Control group, followed by sodium butyrate administration via oral gavage at 30 minutes, 6 hours, and 12 hours after return of spontaneous circulation(ROSC).\u003c/p\u003e\n\u003ch3\u003eSodium butyrate administration\u003c/h3\u003e\n\u003cp\u003eSodium butyrate powder was weighed at a dosage of 50 mg per 100 g of rat body weight, then dissolved in 3 mL of sterile injectable water to prepare a sterile sodium butyrate solution.\u003c/p\u003e\n\u003ch3\u003eCardiac arrest model\u003c/h3\u003e\n\u003cp\u003eFollowing weight measurement, anesthesia was induced via intraperitoneal injection of 5% pentobarbital sodium (45 mg/kg). Rats were positioned supine on a temperature-regulated surgical table (maintained by an infrared heating lamp) and underwent hair removal in the left groin area for surgical preparation. A 14G tracheal tube was inserted under direct visualization with an intelligent visual endoscope, secured to the oral skin with sutures, and connected to a small animal ventilator (ALC-V8D) set to controlled intermittent positive pressure ventilation with parameters: tidal volume 0.65 mL/100g, respiratory rate 80 breaths/min, and FiO₂ 21%. Simultaneously, a multi-channel physiological monitoring system was established: limb electrodes were attached for continuous lead II ECG monitoring via the ALCBioMPA2000 system; a PE-50 catheter was inserted into the left femoral artery for real-time mean arterial pressure (MAP) monitoring, while a venous catheter in the ipsilateral femoral vein provided drug administration access. All catheters were secured with double surgical sutures. Core body temperature (36.5–37.5°C) was maintained throughout the procedure and for 4 hours postoperatively.\u003c/p\u003e \u003cp\u003eAfter 30 minutes of surgical stabilization, CA was induced by clamping the tracheal tube to induce asphyxia, followed by withdrawal of ventilator support and confirmation of apnea. CA was further verified by intravenous injection of 10% potassium chloride (0.12 mL/100g) until systolic pressure dropped to 25 mmHg. After 6 minutes of asystole, standardized CPR was initiated: manual chest compressions(160 compressions/min, depth 1/3 of anteroposterior chest diameter) were performed using the xiphoid landmark method, combined with mechanical ventilation(tidal volume 0.65 mL/100g, rate 80 breaths/min, FiO₂ 21%) at a compression-to-ventilation ratio of 2:1. Immediately upon CPR initiation, 0.5 mL of epinephrine(10 µg/kg) and 1.5 mL of 5% sodium bicarbonate were administered intravenously. ROSC was defined as spontaneous rhythm restoration with systolic pressure ≥ 60 mmHg sustained for ≥ 10 minutes; CPR duration to ROSC was recorded. Failed resuscitation was defined as persistent asystole after 12 minutes of CPR.\u003c/p\u003e \u003cp\u003eAt 30 minutes post-ROSC, post-resuscitation mean arterial pressure(aMAP) and heart rate(aHR) were monitored. Ventilator support and catheters were removed after spontaneous breathing resumed. Incisions were disinfected and sutured, and rats were housed individually with free access to water. All procedures strictly adhered to Utstein-style guidelines for experimental design and data recording[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eNeurobehavioral assessment\u003c/h3\u003e\n\u003cp\u003eNeurobehavioral assessment among the groups was performed using the LONGA scoring method at 24 hours post-ROSC by placing rats in an open field to evaluate locomotor and behavioral parameters. Scores were recorded adhering to the LONGA criteria[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]: 0 point: no nerve function defect; 1 point: the paw on the paralytic side cannot be fully extended; 2 points: when walking, the rat circles to the paralytic side; 3 points: when walking, the rat body topples to the paralytic side; 4 points: unable to walk spontaneously, loss of consciousness.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEuthanasia and tissue sampling\u003c/h2\u003e \u003cp\u003eA total of 19 rats (51.4%) could not be resuscitated,12 resuscitated rats and 6 Sham rats successfully survived 24 hours and were included in the study. Rats were anesthetized via intraperitoneal injection of 5% sodium pentobarbital (45 mg/kg). After achieving stable anesthesia, the right groin and abdominal regions were shaved and disinfected, followed by fixation on a sterile surgical table. A PE-50 catheter was inserted into the right femoral vein to collect 4 mL of whole blood, which was centrifuged at room temperature (3000 × g, 20 min) to isolate serum. The supernatant was aliquoted and stored at − 80°C for subsequent multiplex cytokine analysis. Following blood sampling, rats were euthanized by anesthetic overdose. The abdominal cavity was aseptically opened to dissect intestinal tissues, and a 2 cm ileal segment (2 cm proximal to the ileocecal junction) was excised. Fecal samples (~ 200 mg) were scraped from the mucosal surface, rapidly transferred to pre-chilled sterile EP tubes, flash-frozen in liquid nitrogen, and archived at − 80°C for 16S rDNA sequencing. Fresh ileal tissues were rinsed with saline, divided into two portions: one fixed for paraffin-embedded histomorphological analysis, and the other snap-frozen in liquid nitrogen for quantification of intestinal cytokines and injury biomarkers. All samples were maintained at − 80°C until further assays.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEnzyme‑linked immunosorbent assay (ELISA)\u003c/h3\u003e\n\u003cp\u003e At 24 h post-ROSC, serum levels of inflammatory cytokines (IL-1α, IL-1β, IL-6, IL-10, IL-11, IL-17, TNF-α) were quantified using species-specific ELISA kits (Shanghai Xitang Biotechnology Co., Ltd., Shanghai, China) in accordance with the manufacturer’s protocol. For ileal tissue analysis, PBS-rinsed samples were homogenized with a 4°C pre-chilled tissue homogenizer, followed by centrifugation at 12,000 × \u003cem\u003eg\u003c/em\u003e for 20 min (4°C) to obtain supernatants. Concentrations of inflammatory cytokines (IL-1α, IL-1β, IL-6, IL-10, IL-11, IL-17, TNF-α) and biomarkers (NLRP3, TLR4, MPO, Reg3α, I-FABP) in the supernatants were subsequently determined via the same ELISA methodology.\u003c/p\u003e\n\u003ch3\u003eHematoxylin–eosin (HE) Staining and transmission electron microscopy (TEM)\u003c/h3\u003e\n\u003cp\u003eFresh ileal specimens were fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin as 0.5 cm-thick blocks, and sectioned into 5-µm slices for HE staining per standard protocols, with subsequent digitization using a NanoZoomer S210 scanner (Hamamatsu, Japan). Parallel samples (1 mm³) underwent sequential processing including primary fixation in 2.5% glutaraldehyde, post-fixation with 1% OsO4 in PBS (pH 7.4, 2 hr RT), ethanol dehydration, propylene oxide-epoxy resin (1:1) infiltration at 70°C overnight, and ultramicrotomy-generated 60–80 nm sections for TEM imaging (Talos L120C, ThermoFisher) followed by identical NanoZoomer digitization. Blinded histological assessments were conducted by independent researchers to ensure objective evaluation.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHistomorphological analysis - Chiu grading\u003c/h2\u003e \u003cp\u003eParaffin-embedded jejunal tissue was sectioned(4µm) and stained with hematoxylin and eosin(H\u0026amp;E) according to standard protocols. The morphological integrity of the intestinal wall was classified by a blinded investigator using a modified protocol according to Chiu grading[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]: Grade 0: normal mucosa; Grade 1: development of a sub-epithelial space at the tips of the villi; Grade 2: more extended sub-epithelial space at the tips of the villi, development of Gruenhagen’s space at the tips of the villi; Grade 3: massive epithelial lifting down the sides of the villi, villus necrosis; Grade 4: villi are denuded of epithelial layer; Grade 5: loss of villi, mucosal ulceration and necrosis with invasion of the muscularis propria.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction and 16S rRNA high-throughput sequencing\u003c/h2\u003e \u003cp\u003eSequencing was performed with the help of LC-Bio Technologies (Hangzhou) Co., Ltd. The total fecal microbial DNA was obtained through the Fecal Genome DNA Extraction Kit (AU46111-96, BioTeke, China) according to the manufacturer’ s instruction manual. The DNA was quantified by Qubit (Invitrogen, USA). Total DNA was amplified by PCR using the universal primer 341F/805R (341F: 5′-CCT ACGGGNGGCWGCAG-3′; 805R: 5′-GACTACHVGGG TATCTAATCC-3′). The PCR amplification conditions were pre-denaturation at 98℃ for 30s,denaturation at 98℃ for 10s, annealing at 54℃ for 30s, extension at 72℃ for 45s and 32 cycles. The final extension was at 72℃ for 10 min. The PCR product was purified using AMPure XP Beads (Beckman Coulter Genomics, Danvers, MA, USA) and quantified using Qubit (Invitrogen, USA). Qualified PCR products were evaluated using an Agilent 2100 Bioanalyzer (Agilent, USA) and Illumina library quantitative kits (Kapa Biosciences, Woburn, MA, USA), which were further pooled together and sequenced on an Illumina NovaSeq 6000(PE250), provided by LC-Bio Technology Co., Ltd, Hangzhou, China. The original raw data were spliced by overlapping quality control and chimera filtering to obtain 250-bp paired-end reads. A divisive amplicon denoising algorithm was used to dereplicate and establish ASVs, obtained through amplicon sequencing to conduct bacterial diversity analysis and annotation of species classification and differential analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThis study employed SPSS 26.0 for statistical analysis. Categorical data were described as frequency(percentage), while ordinal data were presented as medians. Median comparisons were performed using the Mann-Whitney U test, with a significance threshold set at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. Normally distributed continuous data were expressed as mean ± standard deviation (x̄ ± s). Independent samples \u003cem\u003et\u003c/em\u003e-test was used for comparisons between two groups, and one-way analysis of variance (ANOVA) was applied for multi-group comparisons: the LSD method was selected when homogeneity of variance was satisfied, whereas the Games-Howell adjustment was employed for heterogeneous variance conditions.\u003c/p\u003e \u003cp\u003eSequencing primer were removed from de-multiplexed raw sequences using cutadapt (v1.9). Then, Pairedend reads were merged using FLASH (v1.2.8). The low-quality reads (quality scores \u0026lt; 20), short reads(\u0026lt; 100bp), and reads containing more than 5% “N” records were trimmed by using the sliding-window algorithm method in fqtrim (v 0.94). Quality filtering was performed to obtain high-quality clean tags according to fqtrim. Chimeric sequences were filtered using Vsearch software (v2.3.4). DADA2 was applied for denoising and generating amplicon sequence variants (ASVs). The sequence alignment of species annotation was performed by QIIME2 plugin feature-classifier, and the alignment database was SILVA and NT-16S. Alpha and beta diversities were calculated using QIIME2, Relative abundance was used in bacteria taxonomy. The Wilcox test was used to identify the differentially abundant genus, and significances were declared at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. LDA effect size (LEfSe, LDA ≥ 3.0, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) was performed using nsegata-lefse. Other diagrams were implemented using the R package (v3.4.4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":"\u003ch2\u003ePhysiologic, hemodynamic, and resuscitation characteristics\u003c/h2\u003e\u003cp\u003eAs detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, a total of 37 rats underwent surgical procedures, including 6 in the Sham group, 15 in the Control group, and 16 in the Nab group following supplemental experiments. No significant differences were observed in pre-operative baseline parameters among groups, including body weight, time to surgical preparation (T\u003csub\u003eo\u003c/sub\u003e), heart rate (HR), and mean arterial pressure (MAP) measured at 30 min post-surgical preparation (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05 for all comparisons).\u003c/p\u003e\u003cp\u003eNo statistically significant differences (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05) were observed between Control and Nab rats in the time from the onset of asphyxia to cardiac arrest (T\u003csub\u003eca\u003c/sub\u003e), the duration of cardiopulmonary resuscitation (T\u003csub\u003ecpr\u003c/sub\u003e), post-resuscitation average mean arterial pressure (aMAP), or heart rate (aHR) in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBasic characteristics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWeight\u003c/p\u003e \u003cp\u003e(g)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHR\u003c/p\u003e \u003cp\u003e(bpm)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMAP\u003c/p\u003e \u003cp\u003e(mmHg)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT\u003csub\u003eo\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(min)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSham\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e310.00 ± 7.75\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e429.17 ± 24.52\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e148.56 ± 11.94\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41.45 ± 8.50\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e306.67 ± 13.32\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e451.27 ± 34.22\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e156.20 ± 12.32\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42.00 ± 5.99\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNab\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e305.00 ± 7.96\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e463.94 ± 37.37\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e156.63 ± 14.91\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41.33 ± 7.86\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.611\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.120\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.433\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.963\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003cem\u003eValues are mean ± SD. HR, heart rate; MAP, mean arterial pressure; T\u003c/em\u003e \u003csub\u003e \u003cem\u003eo\u003c/em\u003e \u003c/sub\u003e, \u003cem\u003ethe time of surgical operation; g, gram; s, second; min, minute; bpm, beats per minute.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResuscitation-associated characteristics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT\u003csub\u003eca\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(s)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT\u003csub\u003ecpr\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(min)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eaMAP\u003c/p\u003e \u003cp\u003e(mmHg)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eaHR\u003c/p\u003e \u003cp\u003e(bpm)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.67 ± 4.63\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.86 ± 2.23\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e108.67 ± 17.20\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e317.33 ± 43.92\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNab\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e67.50 ± 8.19\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.96 ± 2.22\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e103.16 ± 14.17\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e354.50 ± 65.06\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.833\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.411\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.559\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.273\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003cem\u003eValues are mean ± SD, bpm, Score are midline; aMAP, mean arterial pressure after ROSC; aHR, heart rate after ROSC; bpm, beats per minute.\u003c/em\u003e \u003c/p\u003e\u003ch2\u003eSB altered the ileal microbiota composition after CA\u003c/h2\u003e\u003cp\u003eTo investigate the alterations in intestinal flora during cardiac arrest (CA) and the regulatory effects of sodium butyrate (SB) on post-resuscitation gut microbiota in rats, we conducted 16S rDNA gene amplicon sequencing analysis on ileal fecal samples from rats (6 samples per group). Compared to the Sham group, the number of operational taxonomic units (OTUs) in the gut microbiota was significantly reduced in the control group after CA/CPR, whereas it was significantly increased in the Nab group post-CA/CPR. This result aligns with the design objectives of our study (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Alpha-diversity analysis showed no significant difference in ileal community richness (Chao1 index, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) between Control and Sham groups (\u003cem\u003ep\u003c/em\u003e = 0.132), but a significant increase in community diversity (Shannon index, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) in the Control group (\u003cem\u003ep\u003c/em\u003e = 0.026). The Nab group exhibited significantly enhanced ileal richness (Chao1 index) and community diversity (Shannon index) compared to both Sham and Control groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Beta-diversity analysis using Weighted_Unifrac-based PCoA and Anosim demonstrated distinct clustering of gut microbiota communities among the three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u0026amp;E, \u003cem\u003ep =\u003c/em\u003e 0.001, R = 0.5045). At the phylum level (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), Control rats showed significantly reduced relative abundance of Firmicutes and increased Proteobacteria and Fusobacteriota compared to Sham group. Genus-level analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG\u0026amp;H) revealed elevated relative abundance of Escherichia-Shigella in Control versus Sham group. Compared to Control, Nab group exhibited increased abundance of short-chain fatty acid (SCFA)-producing genera: Ruminococcus, Rothia, Lachnospiraceae_NK4A136_group, Oscillibacter, Romboutsia, and decreased abundance of Escherichia-Shigella. LEfSe analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI\u0026amp;J) identified opportunistic pathogens Fusobacterium and Escherichia-Shigella as biomarkers in Control group, while short-chain fatty acid (SCFA)-producing Romboutsia, Eubacterium, and Ruminococcus were characteristic of Nab group. These findings suggest that Nab administration may remodel post-CA ileal microbiota by upregulating SCFA-producing genera with potential intestinal barrier-enhancing properties while suppressing pathogenic bacteria, indicating therapeutic potential for gut microbiota modulation after resuscitation.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003eSB maintains ileal epithelial homeostasis in rats after CA\u003c/h2\u003e\u003cp\u003eAfter preparing hematoxylin-eosin (HE)-stained and electron microscopy (EM) sections of ileal tissues from rats 24 hours post-resuscitation, the ileal tissue slices from the sham, Control, and Nab groups were scanned into images using randomized blinding methods and blinded evaluation, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026amp;B. The ileal mucosa of control group rats exhibited villus tip denudation, with severe cases showing Gruenhagen's space, along with mild perivascular edema, epithelial cell swelling, and scattered pyknotic cells. In contrast, ileal tissues from the Nab group at 24 hours post-ROSC showed no pathological injuries exceeding Chiu grade 1. Furthermore, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026amp;D, the median Chiu’s score of ileal tissues in the Nab group was significantly reduced compared to the Control group (0.5 vs. 2, \u003cem\u003ep\u003c/em\u003e = 0.011). Under electron microscopy, ileal epithelial cells in the Control group at 24 hours post-cardiopulmonary resuscitation (CPR) demonstrated widened gaps in tight junctions between cells (as indicated by arrows in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE. In contrast, such phenomena were not observed in the Nab group.\u003c/p\u003e\u003ch2\u003eSB improve neurobehavioral deficits after CA\u003c/h2\u003e\u003cp\u003e24 hours post-resuscitation, rats were placed in an open field to observe their motor activity and behavior. Scores were recorded based on the observations, with the neurobehavioral assessment conducted using the LONGA scoring criteria. In the Nab group, the median neurofunctional deficit score (LONGA score) at 24 hours post-cardiopulmonary resuscitation (CPR) was 0, which was significantly lower than the median score of 2 in the Control group (0 vs. 2, \u003cem\u003ep\u003c/em\u003e = 0.009, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e\u003ch2\u003eSB alleviated neuroinflammatory response at 24 hours post-CPR\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG\u003cb\u003e\u0026amp;H\u003c/b\u003e, the concentrations of cytokines and injury-related biomarkers in brain tissues were analyzed. The IL-1α level in the Nab group was significantly lower than that in the Control group (32.10 ± 13.32 pg/ml vs. 64.84 ± 17.28 pg/ml, \u003cem\u003ep\u003c/em\u003e = 0.011). Additionally, the NLRP3 inflammasome level in the Nab group was markedly reduced compared to the Control group (0.79 ± 0.05 ng/ml vs. 1.51 ± 0.43 ng/ml, \u003cem\u003ep\u003c/em\u003e = 0.021).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrated that sodium butyrate (SB) can remodel the ileal microbiota structure post-cardiac arrest/cardiorespiratory resuscitation (CA/CPR) in rats by reducing the relative abundance of pathogenic \u003cem\u003eEscherichia-Shigella\u003c/em\u003e and increasing short-chain fatty acid (SCFA)-producing genera, thereby alleviating intestinal barrier damage, reducing neuroinflammation, and improving post-resuscitation neurobehavioral deficits. Furthermore, SB may exert its neuroprotective effects through the microbiota-gut-brain axis (MGBA) by downregulating the IL-1\u0026alpha;/NLRP3 signaling pathway in the nervous system.\u003c/p\u003e\n\u003cp\u003eIntestinal dysfunction following CA/CPR constitutes a critical component of post-cardiac arrest syndrome (PCAS). Intestinal mucosal epithelial cells are among the most sensitive organs to systemic ischemia/reperfusion (I/R) injury. Intestinal I/R injury triggers epithelial barrier disruption, inflammatory cytokine release, systemic inflammatory responses, and multi-organ failure. Additionally, trillions of gut microbes undergo structural and diversity alterations during intestinal damage, exacerbating mucosal permeability and promoting bacterial translocation\u0026mdash;a key catalyst for post-resuscitation multi-organ dysfunction[5, 7, 29]. Human retrospective studies and animal CA models[11, 12] have confirmed that cerebral I/R injury induces dysbiosis, characterized by reduced abundance of dominant phyla such as Proteobacteria, Firmicutes (Clostridia and Lachnospiraceae), and Bacteroidetes (Bifidobacterium and anti-inflammatory SCFA-producing \u003cem\u003eRoseburia\u003c/em\u003e). This dysbiosis drives elevated serum IL-17 levels, promotes Th17 cell infiltration into brain tissues[6] , and facilitates pathogenic microbiota-derived lipopolysaccharide (LPS)-mediated inflammatory cascades, ultimately worsening clinical outcomes[30].\u003c/p\u003e\n\u003cp\u003eThe human gut microbiota is predominantly composed of Firmicutes and Bacteroidetes, with minor contributions from Actinobacteria, Proteobacteria, and other microorganisms[9]. Post-cerebral ischemic injury, patients exhibit decreased abundance of Proteobacteria and specific Firmicutes (e.g., Clostridia and Lachnospiraceae)[11] . In animal models, swine post-CPR models show reduced microbial diversity, particularly diminished abundance of Bifidobacterium and SCFA-producing \u003cem\u003eRoseburia\u003c/em\u003e[12] . While SCFA-producing microbiota have demonstrated anti-inflammatory and antioxidative effects in post-resuscitation brain injury[31, 32] and myocardial protection against I/R injury[33] , evidence remains limited. Our findings reveal that oral SB administration reduces post-CPR ileal \u003cem\u003eEscherichia-Shigella\u003c/em\u003e abundance while enhancing SCFA-producing microbiota in rats.\u003c/p\u003e\n\u003cp\u003eShort-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are key metabolites of dietary fiber fermentation by gut microbiota[13] . Emerging evidence highlights butyrate\u0026apos;s critical role in maintaining intestinal immune homeostasis and epithelial barrier integrity[34-37] . Preclinical and clinical studies indicate that butyrate supplementation alleviates colitis via immunomodulation and barrier enhancement[36, 38, 39] , showing therapeutic potential in inflammatory bowel disease (IBD)[35] . However, research on butyrate\u0026apos;s regulation of post-CA/CPR intestinal homeostasis remains scarce, with prior studies primarily focusing on its unvalidated anti-inflammatory effects in focal cerebral I/R models[20-22] . We hypothesize that endogenous SB may mitigate CA/CPR-induced global I/R-related neuroinflammation through MGBA-mediated intestinal homeostasis modulation. Our data show SB significantly improves 24-hour neurobehavioral scores and reduces cerebral IL-1\u0026alpha;/NLRP3 levels in resuscitated rats. Notably, IL-1\u0026alpha; correlates with all-cause mortality in out-of-hospital cardiac arrest (OHCA) patients[40] , while NLRP3 inflammasome inhibition attenuates microglial activation[41] and improves resuscitation outcomes by mitigating post-resuscitation myocardial dysfunction[42]。While this study provides phenotypic evidence of SB\u0026apos;s intestinal-protective effects post-CPR, the specific regulatory pathways and SB\u0026apos;s role within the MGBA framework require further elucidation. As the \u0026quot;second genome\u0026quot; and \u0026quot;ninth organ system,\u0026quot; the gut microbiota plays a pivotal role in systemic health. SCFAs, particularly butyrate as a key MGBA messenger, hold significant therapeutic potential for post-resuscitation intestinal homeostasis and neuroprotection.\u003c/p\u003e\n\u003cp\u003eIn summary, this study confirms that oral SB alleviates CA/CPR-induced neurobehavioral deficits in rats by restoring gut microbiota balance, mitigating intestinal injury, and reducing neuroinflammation, potentially mediated through MGBA. These findings highlight SB\u0026apos;s capacity to modulate remote organ systems (e.g., the nervous system), warranting further investigation into its precise mechanisms of action.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch1\u003eFunding Declaration\u003c/h1\u003e\n\u003cp\u003eThe study was financially supported by the National Key Clinical Specialty Discipline Construction Program of China (2022YFC3103-001004)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHaojun Zhang and Tianpeng Xu participated in the design of this study, performed most of the experiments and statistical analysis and drafted the main manuscript. He Li prepared figures 1-4. Yufeng Zhu, Rongyi Shi and Xi Chen were the assistants during all of the experiments. Yi Shan supervised the work. All authors have read and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGreif, R., et al., \u003cem\u003eEducation, Implementation, and Teams: 2020 International Consensus on Cardiopulmonary Resuscitation and Emergency Cardiovascular Care Science With Treatment Recommendations.\u003c/em\u003e Resuscitation, 2020. \u003cstrong\u003e156\u003c/strong\u003e: p. A188-a239.\u003c/li\u003e\n\u003cli\u003eSoar, J., et al., \u003cem\u003eAdult Advanced Life Support: 2020 International Consensus on Cardiopulmonary Resuscitation and Emergency Cardiovascular Care Science with Treatment Recommendations.\u003c/em\u003e Resuscitation, 2020. \u003cstrong\u003e156\u003c/strong\u003e: p. 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Systemic ischemia-reperfusion injury post-CA often triggers acute gastrointestinal injury (AGI), characterized by intestinal barrier disruption, dysbiosis, and endotoxin-driven inflammation, which correlates with poor clinical outcomes. This study investigated sodium butyrate, a gut microbiota-derived short-chain fatty acid, in a CA/CPR rat model, revealing its multifaceted therapeutic potential: it improved neurobehavioral recovery, preserved ileal epithelial tight junction integrity, and remodeled gut microbiota by enriching SCFA-producing taxa while suppressing pathogens. Furthermore, sodium butyrate significantly reduced neuroinflammatory markers (IL-1α, NLRP3 inflammasome) in brain tissues, suggesting modulation of the microbiota-gut-brain axis (MGBA). These findings underscore sodium butyrate\u0026rsquo;s role in mitigating PCAS through dual mechanisms\u0026mdash;restoring intestinal homeostasis and dampening systemic inflammation\u0026mdash;thereby offering a novel therapeutic strategy to improve post-resuscitation outcomes.\u003c/p\u003e","manuscriptTitle":"Sodium Butyrate Reinforces Intestinal Homeostasis and Ameliorates Post-Resuscitation Neuroinflammation in Rat Model of Cardiac Arrest","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-01 15:25:47","doi":"10.21203/rs.3.rs-6702701/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aceaf5d1-b254-416f-952c-ecebf3c5c66b","owner":[],"postedDate":"June 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-02T11:38:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-01 15:25:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6702701","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6702701","identity":"rs-6702701","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}