Dexmedetomidine modulates macrophage polarization by inhibiting ferroptosis to exert protective effects on intestinal barrier function in sepsis

Dexmedetomidine modulates macrophage polarization by inhibiting ferroptosis to exert protective effects on intestinal barrier function in sepsis | 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 Dexmedetomidine modulates macrophage polarization by inhibiting ferroptosis to exert protective effects on intestinal barrier function in sepsis Shijie Zhou, Bindan Zhang, Liyong Zou, Chunqiong Tang, Xiaowei Zhou, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6742785/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 The intestine is believed to play a pivotal role in the onset and progression of sepsis, serving as the driving force behind multiple organ dysfunction syndrome. The polarization state of macrophages is pivotal in the intestinal barrier dysfunction associated with sepsis. M1 macrophages initiate the degradation of barrier-sealing molecules, increasing intestinal barrier permeability. Studies have demonstrated that dexmedetomidine offers protection for organ function in sepsis; however, the mechanism behind its protective effect on the intestinal barrier remains unclear. We utilized cecal ligation and perforation surgery to establish sepsis models, along with lipopolysaccharide (LPS)-treated intestinal epithelial and RAW264.7 cell models, to explore the protective effects and mechanisms of dexmedetomidine on intestinal barrier function in rats with sepsis. Our study demonstrated that dexmedetomidine protects intestinal barrier function in septic rats by suppressing inflammatory responses, enhancing the expression of tight junction proteins between intestinal epithelial cells, and significantly reducing intestinal permeability. Additionally, dexmedetomidine markedly decreases the number of inflammatory M1 macrophages in the intestines of septic rats, facilitates the polarization of macrophages toward the anti-inflammatory M2 phenotype, and suppresses the secretion of inflammatory cytokines. Research has indicated that dexmedetomidine is closely linked to ferroptosis, influencing the transport protein xCT to increase the GSH content and GPX4 expression within macrophages. This, in turn, reduces intracellular ROS and lipid ROS levels, mitigates macrophage ferroptosis, and curtails the polarization of macrophages toward the proinflammatory M1 phenotype. sepsis macrophage dexmedetomidine intestinal barrier ferroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Sepsis is a life-threatening syndrome of organ dysfunction resulting from an uncoordinated host response to infection and is a common cause of death among patients admitted to intensive care units [ 1 – 3 ] . In the occurrence and development of sepsis, the gut has long been considered to play a crucial role and is the "motor" that causes multiple organ dysfunction syndrome. The gut acts as a defensive barrier between the body and the external environment, preventing toxic agents and pathogens from circulating in the gut. Damage to the intestinal barrier during sepsis predisposes bacteria and endotoxins and adversely affects distal organ structure and function, leading to multiple organ dysfunction syndrome [ 4 – 8 ] . Macrophages constitute a crucial element of the intestinal immune barrier, initiating and coordinating an effective immune response to bacteria that breach the intestinal epithelial barrier [ 9 – 10 ] . The majority of resident macrophages in the intestine are replenished by circulating blood mononuclear cells. Under physiological conditions, monocytes originating from the blood gradually develop into tissue-resident anti-inflammatory macrophages in the intestine, specifically known as M2 macrophages, which restrict inflammation through the secretion of anti-inflammatory factors such as IL-10. [ 11 ] . During sepsis, when the body is in an inflammatory state, blood-derived monocytes acquire more proinflammatory phenotypes in the intestine, namely, M1 macrophages, which secrete TNF-α, IL-1β and other proinflammatory cytokines, affecting the expression and localization of intestinal tight junction proteins [ 12 – 13 ] . M2-type macrophages promote intestinal epithelial barrier tightness, whereas M1-type macrophages lead to increased permeability of the intestinal barrier [ 14 ] . Therefore, the polarization phenotype of macrophages is closely related to intestinal barrier function in sepsis. Dexmedetomidine is a highly selective α2 adrenergic receptor agonist that results in good sedation, easy awakening without causing obvious respiratory depression, and can significantly reduce postoperative delirium in patients. Dexmedetomidine is widely used in severe and perioperative patients. In recent years, the protective effect of dexmedetomidine in preserving organ function during sepsis has become a hot topic of research. Some studies have shown that dexmedetomidine has a protective effect on the intestinal barrier damage caused by sepsis [ 15 – 18 ] . However, the mechanism by which it protects intestinal barrier function in sepsis remains unclear. Thus, we propose that dexmedetomidine can exert a protective effect on intestinal barrier function in sepsis by regulating the polarization phenotype of macrophages. In this study, we utilized cecal ligation and puncture to establish a sepsis model and treated intestinal epithelial cells and RAW264.7 cells with lipopolysaccharide (LPS) to explore the protective effects and mechanisms of dexmedetomidine on intestinal barrier function in septic rats both in vivo and ex vivo. 2. Materials and Methods 2.1 Ethical Statement All experimental protocols were approved by the Laboratory Animal Welfare and Ethics Committee of Army Medical University (No. AMUMEC-20224867) and were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experimental animals were supplied by the Experimental Animal Center of the Army Medical Center; their ages ranged from 12--14 weeks, and their body weights were 200 ± 20 g. The rats were fasted for 12 hours prior to surgery but had access to water ad libitum. The room temperature was maintained at approximately 25°C. 2.2 Reagents Dexmedetomidine was obtained from Yangtze River Pharmaceutical (Jiangsu, China). Evans blue (EB), lipopolysaccharide (LPS) and antibodies against iNOS were purchased from Sigma (St. Louis, MO, United States). ELISA kits for diamine oxidase (DAO), TNF-ɑ, IL-1β and IL-6 were obtained from Elabsicence. An ELISA kit for D-lactic acid was obtained from Nanjing Jiancheng Biological Engineering Research Institute (Nanjing, China). Antibodies against ZO-1, Occludin and β-actin were purchased from BOSTER (Wuhan, China). Antibodies for flow cytometry, including PE-conjugated anti-mouse CD86 and Qd605-conjugated anti-mouse Ly6c, were purchased from BD Biosciences. MDA was purchased from Bioss (Beijing, China). Glutathione and ROS detection kits were purchased from Beyotime Biotechnology (Shanghai, China). A lipid peroxidation kit and an antibody against CD206 were purchased from Thermo Fisher Scientific (Waltham MA, United States). 2.3 Establishment of the sepsis model Adult Sprague–Dawley (SD) rats (200–220 g) were anesthetized with 3% sodium pentobarbital (30 mg/kg intraperitoneally). The sepsis model was induced via cecal ligation and puncture (CLP) according to previous methods [19-20] . Briefly, the abdominal wall was incised, and then the cecum was exposed and ligated 0.7 cm from the distal end of the cecum with a 0 sike. A triangular needle with a diameter of approximately 1.5 mm was used to pierce the cecum, and feces were admitted to flow into the abdominal cavity. The rats were returned to their cages after the abdomen was closed and allowed water and food freely. After the experiment, the rats were euthanized by intraperitoneal injection of a lethal dose of pentobarbital sodium (100 mg/kg). 2.4 Cell Culture The IEC-6 intestinal epithelial cell line was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). IEC-6 cells and RAW 264.7 macrophages were maintained in DMEM supplemented with 10% FBS at 37°C in a 5% CO2 environment. 2.5 Intestinal Permeability Measurement The rats were anesthetized, and the abdomen was opened. A piece of small intestine approximately 7 cm in length was taken from each rat, the intestinal cavity was rinsed with PBS, and a capsule was prepared. A total of 0.2 mL of 1.5% EB solution was injected into the intestinal capsule, which was then incubated with a water bath at 37°C for 30 minutes. The intestinal capsule was rinsed with PBS until the washing solution was clarified, after which it was dried at 50°C for 24 hours. Next, the dry intestinal mass was recorded, formamide solution was added, and the mixture was incubated at 50°C for 24 hours. Finally, the supernatant was detected with an enzyme microplate meter at a wavelength of 650 nm, and the EB content in the intestinal tissue was calculated according to the obtained OD value. 2.6 Enzyme-linked immunosorbent assay Blood samples were collected from the femoral vein and centrifuged at 3500 r/min for 10 minutes. The supernatant was collected for ELISA analysis. The levels of DAO, D-lactate, TNF-ɑ and IL-1β in the serum and the concentrations of TNF-α and IL-6 in the cell supernatant were measured by corresponding ELISA kits according to the manufacturers’ instructions. 2.7 Tissue histology Twelve hours after CLP, the rats were anesthetized. The intestinal tissues were fixed in 4% paraformaldehyde for 24 h and cut into 4-μm thick sections, which were stained with hematoxylin and eosin (H&E). The pathological sections were observed via optical microscopy. 2.8 Immunofluorescence Staining The intestinal tissues were cut into 20-μm frozen sections, and RAW264.7 cells were seeded in the confocal chamber. The sections and cells were blocked with goat serum for 1 h at room temperature and then incubated with antibodies against iNOS (1:200) and CD206 (1:200) overnight at 4°C. After washing, the sections and cells were incubated with the corresponding fluorescent secondary antibodies for 1 h at room temperature. The slices and cells were subsequently washed again and dyed with DAPI. The samples were observed under a Leica microscope. 2.9 Tissue dissociation and flow cytometry Small intestine samples were minced with scissors before incubation with 1 mg/ml collagenase in DMEM for 30 min at 37°C. Single-cell suspensions were obtained by screening with a nylon filter strainer at 100 μm and 70 μm. To detect CD86+ and Ly6c+ macrophages, the cells were subsequently stained at room temperature for 20 min with appropriate dilutions of anti-CD86 (PE) and anti-Ly6c (Qd605) fluorochrome-conjugated antibodies. Flow cytometry was performed using a BD flow cytometer. 2.10 Metabolomic profiling Cells were flash frozen in liquid nitrogen and stored at -80°C. The extraction mixture (acetonitrile:methanol:water = 2:2:1, containing an isotope-labeled internal standard mixture) was added to the cells. The sample was subsequently incubated at -40°C for 1 hour and centrifuged at 12000 rpm for 15 minutes at 4°C. The sample was then analyzed via a UHPLC system (Vanquish, Thermo Fisher Scientific). The raw data were converted to mzXML format and processed via in-house programs for peak detection, extraction, and integration. 2.11 Bioinformatics analysis Principal component analysis (PCA) was employed to assess the intrinsic clustering of the metabolomics data. A 95% confidence interval (CI) served as a threshold to identify potential outliers across all samples. GraphPad Prism V.8.0.0 software was used to create volcano and violin plots, which clearly display upregulated and downregulated metabolites. 2.12 Statistical analysis Statistical analysis was conducted via SPSS 23.0 (SPSS Inc., Chicago, IL, USA), with the data represented as the means ± standard deviations (SDs). One-way analysis of variance (ANOVA) followed by the post hoc Tukey test was used for comparisons between experimental groups. Survival time was analyzed by the median and interquartile range, and Kaplan–Meier survival analysis and the log-rank test were used. P <0.05 was considered statistically significant. 3. Results 3.1 Dexmedetomidine improves intestinal permeability in septic rats After sepsis, intestinal tissue leakage in the rats was obvious, as indicated by a significant increase in EB permeability and serum DAO and D-lac contents. Compared with those in the sham group, EB permeability was increased by 244.12%, and the serum DAO and D-lac levels were increased by 159.39% and 148.19%, respectively ( P 0.05). After Dex treatment, the intestinal permeability of septic rats was significantly improved, EB leakage was reduced by 32.56%, and the serum DAO and D-lac levels were reduced by 26.42% and 13.76%, respectively ( P < 0.05) (Figure 1A). Additionally, the serum and intestinal inflammatory cytokines TNF-α, IL-1β and IL-6 were significantly greater in the sepsis group than in the sham group ( P 0.05). After treatment with dexmedetomidine, the serum and intestinal inflammatory cytokines TNF-α, IL-1β and IL-6 decreased significantly. Compared with those in the sepsis group, the serum inflammatory cytokine levels were 33.93%, 37.86% and 30.90% lower, respectively, and the intestinal inflammatory cytokine levels were 31.88%, 28.7% and 29.37% lower, respectively. Compared with those in the CT group, the reduction rates of the serum inflammatory cytokines were 26.13%, 29.86%, and 25.12%, and the reduction rates of the intestinal inflammatory cytokines were 24.27%, 26.96%, and 22.83%, respectively ( P <0.01) (Figure 1B, C), indicating that Dex could alleviate the intestinal inflammatory response. Finally, we observed the pathological staining structure of the intestinal tissue. The intestinal villi of the rats in the sham group were well structured and distinct, with columnar epithelial cells aligned neatly, and no infiltration of red blood cells or inflammatory cells was observed. In the sepsis group, the intestinal villi exhibited atrophy and disruption, accompanied by significant infiltration of red blood cells and inflammatory cells. No significant improvement was observed in the CT group. After treatment with dexmedetomidine, intestinal villus atrophy significantly improved, intestinal villi were neatly arranged, infiltration of red blood cells and inflammatory cells was reduced, and the intestinal structure was significantly restored, suggesting that dexmedetomidine can protect the intestinal structure in sepsis (Figure 1D). To further investigate the effect of dexmedetomidine on the permeability of intestinal epithelial cells in vitro, we observed the expression of tight junction proteins in these cells. The immunofluorescence results revealed that ZO-1 in the normal group was continuously and regularly distributed along intestinal epithelial cells, whereas ZO-1 expression in the LPS group was weak and obviously disrupted, indicating a loose distribution. Compared with that in the LPS group, the expression of ZO-1 in the dexmedetomidine group was markedly increased, and the morphology was relatively continuous and clear (Figure 1E). Compared with those in the normal group, tight junction protein expression in the LPS group was significantly lower, and ZO-1 and Occludin expression was 69% and 59.33% lower, respectively, than that in the normal group ( P <0.01). Compared with those in the LPS group, the expression of ZO-1 and occludin in the dexmedetomidine group increased by 127.96% and 72.95%, respectively ( P <0.05) (Figure 1F). These results further indicate that dexmedetomidine exerts a protective effect on intestinal barrier function in rats with sepsis. 3.2 Dexmedetomidine inhibits the M1-type polarization of macrophages to regulate intestinal permeability To assess the role of dexmedetomidine in regulating the polarization of intestinal macrophages following sepsis, we employed flow cytometry and immunofluorescence staining to detect biomarkers associated with M1 and M2 macrophages. M1 macrophages were identified as CD86+ Ly6c- macrophages, and M2 macrophages were identified as CD86-Ly6C+ macrophages. The flow cytometry results indicated that the proportion of M1 macrophages increased from 37.86% to 75.05% following sepsis. Dexmedetomidine administration notably reduced the number of M1 macrophages while increasing the number of M2 macrophages. Compared with the sepsis group, the dexmedetomidine group presented a reduction in the percentage of M1 macrophages from 75.05% to 51.70% and an increase in the percentage of M2 macrophages from 0.62% to 2.66% (Figure 2A). Immunofluorescence staining revealed increased iNOS expression in the intestinal macrophages of the rats in the sepsis group, whereas it was significantly decreased in the dexmedetomidine-treated group (Figure 2B). To further study the effects of dexmedetomidine on macrophage polarization, RAW264.7 cells were categorized into normal control, LPS, and Dex groups on the basis of distinct treatments. Various experimental approaches have been employed to assess the expression of M1 and M2 macrophage markers. The immunofluorescence results indicated that, relative to the control, LPS significantly enhanced M1 macrophage polarization by increasing iNOS expression (P<0.01). Following treatment with dexmedetomidine, the polarization of macrophages toward the M1 phenotype was markedly diminished, as evidenced by a reduction in iNOS expression (P<0.01) (Figure 2C). The ELISA results revealed that the levels of the proinflammatory cytokines TNF-α, IL-6, and IL-1β in the LPS group were significantly elevated compared with those in the normal control group (P<0.01), suggesting an increase in macrophage polarization toward the proinflammatory M1 type. After treatment with dexmedetomidine, the levels of TNF-α, IL-6, and IL-1β decreased, leading to a significant reduction in inflammation levels compared with those in the LPS group (Figure 2D). The above results suggest that dexmedetomidine can modulate macrophage polarization, curbing the rise of proinflammatory M1 macrophages postsepsis and fostering the shift toward anti-inflammatory M2 macrophages. 3.3 Dexmedetomidine inhibits macrophage ferroptosis by promoting glutathione production after sepsis The metabolomic profiles of macrophages following treatment with LPS and dexmedetomidine were examined to investigate the impact of dexmedetomidine on macrophage metabolism. Principal component analysis (PCA) revealed that the samples were largely distinct, yet all remained within the confidence interval (Figure 3A). The OPLS-DA results confirmed the model's stability and reliability (Figure 3B). The volcano plot revealed 247 distinct metabolites between the LPS and dexmedetomidine groups (Figure 3C) (differentially abundant metabolites were identified with P values 1). Significant differences were observed in the levels of glutathione and cysteine among the upregulated metabolites (Figure 3D-G). KEGG pathway enrichment analysis revealed that cysteine and glutathione are closely linked to the ferroptosis pathway (Figure 3H). Dex may modulate macrophage polarization through the promotion of glutathione production and its impact on macrophage ferroptosis. 3.4 Dexmedetomidine regulates macrophage polarization by inhibiting ferroptosis via xCT To validate the metabolomics findings, we employed a GSH assay kit to measure GSH levels in macrophages postsepsis. The results indicated that following LPS stimulation, both the GSH level and the GSH/GSSG ratio significantly decreased (P<0.01). In contrast, the Dex group presented a significant increase in the level of intracellular GSH (P<0.01) and the GSH/GSSG ratio (P<0.05) compared with those of the LPS group (Figure 4A-B). Given that intracellular cysteine levels impact GSH production and that xCT is the primary transporter protein for cysteine entry into cells, Dex treatment led to a significant upregulation of xCT expression compared with LPS treatment (Figure 4C), suggesting a correlation between the increase in GSH and xCT. Moreover, GSH activates the core regulatory enzyme GPX4 of the glutathione antioxidant system, contributing to the generation of ROS. We next examined the changes in GPX4, revealing an increase in expression within the DEX group (Figure 4D) accompanied by a reduction in ROS and lipid-ROS production (Figures 4E-F). Compared with those in the normal control group, the expression of xCT and GPX4 in the LPS group was decreased by 68.33% and 60%, respectively (P<0.01), the ROS level was increased by 452.91% (P<0.01), and the lipid ROS level was significantly increased. Compared with those in the LPS group, the expression of xCT and GPX4 in the dexmedetomidine group was increased by 69.47% and 52.5%, respectively (P<0.01), the ROS level was reduced by 40.29% (P<0.01), and the lipid ROS level was significantly decreased (Figure 4C-D), indicating that Dex can inhibit ferroptosis in macrophages. In addition to the xCT inhibitor SAS, the population of M1 macrophages increased in comparison with those in the xCT intervention group, suggesting an intensification of macrophage polarization toward M1 postxCT inhibition (Figure 4G). These results suggest that dexmedetomidine prevents macrophage ferroptosis via xCT and diminishes macrophage polarization toward the M1 phenotype. 3.5 Effects of dexmedetomidine on organ function and survival time in septic rats To assess whether dexmedetomidine, by mitigating intestinal leakage in sepsis, can safeguard the functions of additional vital organs and prolong survival, we initially assessed markers of liver, kidney, and myocardial function, along with arterial blood gases and mean arterial pressure. These findings revealed that dexmedetomidine substantially enhanced organ function in rats with sepsis. Compared with those in the standard treatment group, the ALT, AST, creatinine (Crea), and CK-MB levels decreased by 30.36% (P<0.01), 34.31% (P<0.01), 21.31% (P<0.05), and 23.55% (P<0.05), respectively. The pH and oxygen partial pressure significantly increased, whereas the carbon dioxide partial pressure markedly decreased (P<0.05) (Figure 5A-G). The mean arterial blood pressure results indicated a significant decrease in rats after sepsis surgery, decreasing to approximately 60 mm Hg. Following fluid resuscitation, the blood pressure in the sepsis group decreased to approximately 46 mm Hg. Moreover, in the conventional treatment group, the arterial blood pressure gradually increased to approximately 72 mm Hg, and in the dexmedetomidine group, it increased to approximately 100 mm Hg (Figure 5H). Subsequent observations of the 24-hour survival duration and rate revealed that only one rat in the sepsis group survived beyond 24 hours, representing a survival rate of 6.25%, with an average survival time of 5.70 ±6.36 hours. Following conventional fluid resuscitation, two rats survived, with a survival rate of 12.50% and an average survival time of 10.10 ±7.80 hours, indicating an increase in survival time compared with that of the sepsis group. After Dex treatment, the number of rats surviving 24 hours increased to six, with a survival rate of 37.50% and a survival time of 18.30 ± 5.80 hours, indicating a significant improvement in the survival rate and a substantial extension in survival time compared with those of the conventional treatment group (P<0.05, Figure 5I‒J). 4. Discussion Sepsis is a frequently encountered critical illness in clinical practice, where a dysregulated immune response can lead to fatal organ dysfunction. If not promptly treated, it can progress to multiple organ dysfunction syndrome or even septic shock, resulting in an extremely high mortality rate [21] . The gut plays an important role in sepsis. Under physiological conditions, the gut acts as a defensive barrier between the body and the external environment, preventing the entry of toxic agents and pathogens into the circulation. During sepsis, the cascading release of numerous inflammatory factors impairs the intestinal barrier, leading to necrosis of epithelial cells, disruption of intercellular junctions, and severe intestinal leakage. This allows toxic agents, including bacteria and endotoxins, to transgress the intestinal epithelial barrier abnormally, triggering systemic inflammation. This, in turn, intensifies the progression of sepsis, ultimately leading to multiorgan dysfunction and patient fatality [22-26] . However, targeted prevention and treatment methods for intestinal leakage postsepsis are lacking. Consequently, investigating effective strategies to protect the intestinal barrier is crucial for sepsis treatment. Macrophages, highly heterogeneous immune cells, differentiate into various types in response to diverse pathological stimuli, thereby playing a pivotal role in the body's inflammatory response. There are two types of macrophage polarization, namely, classically activated macrophages (M1) and alternately activated macrophages (M2). M1 macrophages, which are primarily induced by lipopolysaccharides (LPS), secrete proinflammatory cytokines such as TNF-α, IL-1β, and IL-6, which boost the body's immune response during sepsis's excessive inflammatory phase, leading to immune dysfunction. M2 macrophages, which are primarily activated by IL-4, release anti-inflammatory factors such as IL-10 to limit inflammation, foster tissue repair, and facilitate the healing of injured areas. [27-29] . The polarization state of macrophages is considered pivotal in maintaining intestinal homeostasis. During sepsis, there is an increase in the number of M1 macrophages, which secrete high levels of proinflammatory cytokines such as TNF-α, in the intestine. Proinflammatory cytokines are considered key regulators of the expression and localization of tight junction proteins. TNF-α has been demonstrated to enhance intestinal permeability by influencing the expression of ZO-1 and inducible nitric oxide synthase (iNOS) [24-25] . Consequently, M1 macrophages exacerbate the intestinal damage associated with sepsis, whereas shifting polarized macrophages from the M1 phenotype to the M2 phenotype can effectively mitigate inflammatory dysregulation and intestinal damage [13-14] . Dexmedetomidine, a novel anesthetic and sedative, has emerged as a focal point of research in recent years, largely because of its anti-inflammatory properties. CHEN [30] reported that Dex could inhibit the activation of the NLRP3 inflammasome, reduce lung inflammation, and play a protective role in the lung. In ZI’s study [31] , Dex significantly inhibited the release of inflammatory factors through a cholinergic anti-inflammatory mechanism and alleviated acute liver injury in sepsis. Another study [32] reported that dexmedetomidine can inhibit the myocardial inflammatory response and alleviate myocardial dysfunction caused by sepsis by inducing cardiomyocyte autophagy. Our findings demonstrate that sepsis is followed by a significant increase in intestinal M1 macrophages. Dexmedetomidine administration significantly reduces the number of intestinal M1 macrophages and promotes M2 polarization. Similarly, cellular experiments confirmed pronounced M1 polarization of macrophages after LPS stimulation, with significant increases in the inflammatory cytokines TNF-α, IL-6, and IL-β (P<0.01). Following dexmedetomidine treatment, the M1 marker iNOS was significantly reduced, inflammatory cytokines were markedly decreased, and the M2 markers CD206 and Arg1 were significantly increased (P<0.05). These results suggest that dexmedetomidine exerts a protective effect on intestinal barrier function in sepsis by modulating macrophage polarization and suppressing the intestinal inflammatory response. To further elucidate how dexmedetomidine regulates macrophage polarization, we analyzed the metabolomic profiles of macrophages treated with LPS and dexmedetomidine. We found that dexmedetomidine significantly increased cysteine and GSH levels, and KEGG pathway enrichment analysis indicated that glutathione and cysteine are involved in ferroptosis. Ferroptosis, a newly identified form of programmed cell death, is characterized by lipid peroxidation and increased ROS levels due to the accumulation of ferrous ions within cells [33-35] . The primary mechanism for inducing ferroptosis is the inactivation of GPX4 due to GSH depletion. By inhibiting the membrane transporter xCT and reducing the levels of cysteine, a precursor for GSH synthesis, the intracellular GSH levels decrease, resulting in the inactivation of GPX4 [36] . GPX4 is the core regulatory enzyme of the antioxidant system (glutathione system) and converts peroxide bonds involved in lipid peroxidation into hydroxyl groups, thus inhibiting ferroptosis [37] . Studies indicate that ferroptosis is closely linked to M1 polarization in two ways: (1) During ferroptosis in macrophages, excess intracellular ferrous iron elevates the levels of M1 macrophage markers, including IL-6, TNF-α, and IL-1β, while reducing the levels of M2 markers, such as TGM2 [38] . Moreover, ROS accumulation and p53 acetylation due to iron overload facilitate M1 polarization [39] . (2) Cells undergoing ferroptosis facilitate the recruitment of M1 inflammatory macrophages by releasing HMGB1 and inducing the expression of inflammatory genes such as CCL2 and CCL7 [40-43] . To validate the metabolomics findings, we employed a GSH assay kit to measure GSH levels in macrophages postsepsis. Dexmedetomidine notably elevated GSH levels in macrophages from patients with sepsis (P<0.01). We hypothesize that dexmedetomidine may regulate macrophage polarization by adjusting GSH levels via xCT. Consequently, we assessed the levels of GSH, xCT, GPX4, and ROS. The results indicated that, relative to the LPS group, dexmedetomidine significantly increased the GSH level and increased the protein expression of xCT and GPX4 (P<0.01) while also reducing the levels of intracellular and lipid-bound ROS (P<0.01), thereby inhibiting ferroptosis in macrophages. Following the addition of xCT inhibitors, dexmedetomidine is unable to inhibit M1 macrophage polarization. Thus, after establishing that dexmedetomidine influences GSH synthesis via xCT and that GSH, as a substrate for GPX4, inhibits lipid peroxidation and reduces ROS levels in macrophages, we hypothesize that Dex may reduce macrophage polarization toward the proinflammatory M1 phenotype by suppressing ferroptosis. This study has several limitations. First, this study demonstrated that dexmedetomidine can inhibit intestinal leakage in rats with sepsis both in vivo and in vitro, but it remains unclear whether dexmedetomidine consistently protects intestinal barrier function in patients with sepsis. Second, in addition to protecting intestinal barrier function by inhibiting macrophage polarization toward the M1 phenotype through the ferroptosis pathway, further research is needed to determine whether dexmedetomidine influences macrophage polarization and function through other metabolic pathways and metabolites. 5. Conclusion This study demonstrated that dexmedetomidine exerts a protective effect on intestinal barrier function in rats with sepsis. The mechanism involves Dex regulating the production of GSH/GPX4, which in turn inhibits macrophage ferroptosis and suppresses macrophage polarization toward the proinflammatory M1 phenotype. This study offers novel therapeutic insights and targets for preserving intestinal barrier function in sepsis. Declarations Acknowledgements None. Author contributions TL, LW, HY and SJZ designed the study. SJZ, BDZ, LYZ, CQT, and XWZ analyzed the data. TL acquired the financial support. SJZ drafted the manuscript. LW revised the manuscript. All the authors performed the experimental procedures. Funding This study was supported by the Chongqing Talent Program Lump-sum Project (cstc2022ycjh-bgzxm0011). Data availability The original data described in the research are included in the article and supplementary materials. For further inquiries, please directly contact the corresponding authors. Ethics approval and consent to participate The animal experimental processes were approved by the Ethnic Committee of Laboratory Animal Welfare and Ethics Committee Of the Third Military Medical University hospital and conducted in strict accordance to the standard of the Guide for the Care and Use of Laboratory Animals published by the Ministry of Science and Technology of the People's Republic of China in 2006. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Singer M, Deutschman CS, Seymour CW, et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3)[J]. JAMA, 2016,315(8):801. Kellum JA, Formeck CL, Kernan KF, et al. Subtypes and Mimics of Sepsis. Crit Care Clin, 2022 Apr;38(2):195-211. Oczkowski S, Alshamsi F, Belley-Cote E . Surviving Sepsis Campaign Guidelines 2021: highlights for the practicing clinician. 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Cell Metab. 2023 Jan 3;35(1):84-100.e8. Jiang XJ, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol. 2021 Apr;22(4):266-282. Koppula P, Zhuang L, Gan B.Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy[J]. Protein Cell,2021 ,12(8):599-620. Seibt TM, Proneth B, Conrad M.Role of GPX4 in ferroptosis and its pharmacological implication[J]. Free Radical Biology and Medicine,2019,133:144-152. Handa P, Thomas S, Morgan-Stevenson V, et al. Iron alters macrophage polarization status and leads to steatohepatitis and fibrogenesis[J]. Journal of Leukocyte Biology,2019，105:1015–26. Zhou Y, Que KT, Zhang Z, et al. Iron overloaded polarizes macrophage to proinflammation phenotype through ROS/acetyl-p53 pathway[J].Cancer Medicine, 2018,7:4012–22. Wen Q, Liu J, Kang R,et al. The release and activity of HMGB1 in ferroptosis[J]. Biochemical and Biophysical Research Communications,2019,510:278–83. Luo X, Gong HB, Gao HY, et al. Oxygenated phosphati-dylethanolamine navigates phagocytosis of ferroptotic cells by interacting with TLR2[J]. Cell Death Differentiaton,2021,28:1971–89. Djudjaj S, Martin IV, Buhl EM, et al. Macrophage migration inhibitory factor limits renal inflammation and fibrosis by counteracting tubular cell cycle arrest[J]. Journal of the American Society Nephrology,2017,28:3590–604. Lv LL, Feng Y, Wen Y, et al. Exosomal CCL2 from tubular epithelial cells is critical for albumin-induced tubulointerstitial inflammation. Journal of the American Society Nephrology, 2018,29:919–35. Additional Declarations No competing interests reported. Supplementary Files additionaldocument.docx 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. 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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-6742785","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":475368140,"identity":"13d019cd-1b34-44b0-8d14-e081a38c8f04","order_by":0,"name":"Shijie Zhou","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shijie","middleName":"","lastName":"Zhou","suffix":""},{"id":475368141,"identity":"698778ab-cb27-400e-ab44-54c10255ef80","order_by":1,"name":"Bindan Zhang","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bindan","middleName":"","lastName":"Zhang","suffix":""},{"id":475368142,"identity":"990b0396-45f2-4cfb-ad68-9177cf306786","order_by":2,"name":"Liyong Zou","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Liyong","middleName":"","lastName":"Zou","suffix":""},{"id":475368146,"identity":"558c89b6-84f1-42c1-bfd7-60b54c6da419","order_by":3,"name":"Chunqiong Tang","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chunqiong","middleName":"","lastName":"Tang","suffix":""},{"id":475368147,"identity":"94a48bea-4f86-4909-bfc0-48bbef115dcb","order_by":4,"name":"Xiaowei Zhou","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaowei","middleName":"","lastName":"Zhou","suffix":""},{"id":475368148,"identity":"cf650d21-ec69-4d66-9b17-d051a666dd27","order_by":5,"name":"Hong Yan","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Yan","suffix":""},{"id":475368150,"identity":"991a5c37-cf57-42ab-bc9d-39ebfab81285","order_by":6,"name":"Tao Li","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Li","suffix":""},{"id":475368152,"identity":"b643cb90-2aed-4e4c-be2e-750f739107d3","order_by":7,"name":"Li Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYLCCBwYScvLsDVDeAWK0JBhYGBv2HCZJC0NFYsONZCK1GBw/e/hFQoEEY+PM90c33WxjkOO7kcD4uQCfljN5aRYJBhLM7NLJbLdz2xiMJW8kMEvPwKPF7ECOmQFQCxvjbIiWxA03EtiYefBpOf8GrIWH4eZhsJZ6wlpu5Bg/AGqRYLjBDNaSYEBIi/2NN2ZAZRIGhj3JZrdzzkkYzjzzsFkanxbJ/hzjDx/+1NXPZz/47HZOmY083/Hkg5/xaQECNgkkDojN2IBfAwMD8wdCKkbBKBgFo2CEAwCKaU00RvSAbQAAAABJRU5ErkJggg==","orcid":"","institution":"Army Medical University","correspondingAuthor":true,"prefix":"","firstName":"Li","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-05-25 09:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6742785/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6742785/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85616174,"identity":"a1adb36c-96c6-4b9f-a18c-1edbfc7e609b","added_by":"auto","created_at":"2025-06-29 14:35:14","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":708009,"visible":true,"origin":"","legend":"\u003cp\u003eThe influence of Dex on intestinal permeability in rats with sepsis. Leakage of Evans blue from the intestine and serum DAO and D-Lac levels of rats in different groups, n=8; B: Serum proinflammatory cytokine levels of rats in different groups, n=8; C: Intestine proinflammatory cytokine levels of rats in different groups, n=8; D: Pathological structure of intestinal tissue of rats in different groups (HE×200), n=3; E: ZO-1 expression was determined by immunofluorescence, n=3; F: ZO-1 and Occludin expression was determined by WB, n=3. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, compared with the sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, compared with the CT group;\u003csup\u003e ##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, compared with the CT group; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, compared with the control group;\u003csup\u003e ##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, compared with the LPS group.\u003c/p\u003e","description":"","filename":"1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6742785/v1/ed641f7546ad98dcd0ca14ef.jpeg"},{"id":85616153,"identity":"a1b1ee82-1dd0-48c6-b364-3d4d989cad0d","added_by":"auto","created_at":"2025-06-29 14:35:12","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1312444,"visible":true,"origin":"","legend":"\u003cp\u003eThe influence of dexmedetomidine on macrophage polarization after sepsis. A: Flow cytometric analysis of intestinal single cells, n=3; B: Measurement of the expression of iNOS after sepsis in the small intestine by immunofluorescence, n=3. C: Measurement of the expression of iNOS after sepsis in RAW264.7 cells by immunofluorescence, n=3; D: Measurement of TNF-α and IL-6 levels after sepsis in RAW264.7 cells, n=3. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, compared with the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, compared with the LPS group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, compared with the LPS group.\u003c/p\u003e","description":"","filename":"2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6742785/v1/918aae0ce77f16084d701c4e.jpeg"},{"id":85616173,"identity":"98e85066-1602-4fc3-b2b4-b42156dc07e7","added_by":"auto","created_at":"2025-06-29 14:35:14","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":557949,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolomic analysis.\u003cstrong\u003e \u003c/strong\u003eA:\u003cstrong\u003e \u003c/strong\u003eCell metabolites were significantly different between the LPS and Dex groups according to principal component analysis. B: OPLS‒DA permutation test to assess the favorable stability of the sample data\u003cstrong\u003e. \u003c/strong\u003eC:\u003cstrong\u003e \u003c/strong\u003eVolcano plot of the metabolomics data of RAW264.7 cells\u003cstrong\u003e. \u003c/strong\u003eD:\u003cstrong\u003e \u003c/strong\u003eHeatmap of differentially expressed metabolites affected by Dex and LPS.\u003cstrong\u003e \u003c/strong\u003eE: Radar chart analysis of differentially expressed metabolites in the Dex vs LPS groups.\u003cstrong\u003e \u003c/strong\u003eF:\u003cstrong\u003e \u003c/strong\u003eFerroptosis pathway map enriched by KEGG. \u003csup\u003e**\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, compared with the LPS group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, compared with the LPS group.\u003c/p\u003e","description":"","filename":"3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6742785/v1/a68cb33e28a186f02ea22080.jpeg"},{"id":85616147,"identity":"0480bf6a-ce5c-4162-9c80-41926be655f9","added_by":"auto","created_at":"2025-06-29 14:35:12","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":922286,"visible":true,"origin":"","legend":"\u003cp\u003eDextetomidine regulates macrophage polarization by affecting metabolism. A: The expression of xCT was determined via WB, n=3. B: The expression of GPX4 was determined via WB, n=3. C: The content of ROS was determined via immunofluorescence, n=3. D: The content of lipid ROS was determined via immunofluorescence, n=3. E: Measurement of the expression of iNOS after sepsis in RAW264.7 cells via immunofluorescence, n=3.\u003csup\u003e **\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, compared with the control group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, compared with the LPS group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, compared with the LPS group; \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, compared with the LPS+Dex group;\u003csup\u003e \u0026amp;\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, compared with the LPS+Dex group.\u003c/p\u003e","description":"","filename":"4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6742785/v1/76b8222e22c4e9192dfe1e0d.jpeg"},{"id":85618696,"identity":"04c80adc-c93f-4e4a-8c85-5c2d2380648f","added_by":"auto","created_at":"2025-06-29 14:51:12","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":231937,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of dexmedetomidine on organ function and survival time in septic rats. A-B: The influence of dexmedetomidine on the liver function of rats in different groups, n=8; C: The influence of dexmedetomidine on the kidney function of rats in different groups, n=8; D: The influence of dexmedetomidine on the myocardial function of rats in different groups, n=8; E-G: The influence of dexmedetomidine on the pH, PaO2 and PaCO2 of rats in different groups, n=8; H: The influence of dexmedetomidine on the blood pressure of rats in different groups, n=8. I: survival rate, n=16; J: survival time, n=16. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, compared with the sham group;\u003csup\u003e #\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, compared with the CT group;\u003csup\u003e ##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, compared with the CT group.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6742785/v1/d6d6d109605db25915a75150.jpg"},{"id":102961418,"identity":"ae0b3c72-e71e-4f12-9b9e-950917002de7","added_by":"auto","created_at":"2026-02-19 03:39:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4546522,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6742785/v1/3f7af520-ff99-44f8-94fd-251db370d912.pdf"},{"id":85617042,"identity":"eb87a7b7-0568-444f-a1fa-34c77fbd8d60","added_by":"auto","created_at":"2025-06-29 14:43:12","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":755169,"visible":true,"origin":"","legend":"","description":"","filename":"additionaldocument.docx","url":"https://assets-eu.researchsquare.com/files/rs-6742785/v1/42339d9e8325191751aa75f8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dexmedetomidine modulates macrophage polarization by inhibiting ferroptosis to exert protective effects on intestinal barrier function in sepsis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSepsis is a life-threatening syndrome of organ dysfunction resulting from an uncoordinated host response to infection and is a common cause of death among patients admitted to intensive care units \u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. In the occurrence and development of sepsis, the gut has long been considered to play a crucial role and is the \"motor\" that causes multiple organ dysfunction syndrome. The gut acts as a defensive barrier between the body and the external environment, preventing toxic agents and pathogens from circulating in the gut. Damage to the intestinal barrier during sepsis predisposes bacteria and endotoxins and adversely affects distal organ structure and function, leading to multiple organ dysfunction syndrome\u003csup\u003e[\u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMacrophages constitute a crucial element of the intestinal immune barrier, initiating and coordinating an effective immune response to bacteria that breach the intestinal epithelial barrier\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. The majority of resident macrophages in the intestine are replenished by circulating blood mononuclear cells. Under physiological conditions, monocytes originating from the blood gradually develop into tissue-resident anti-inflammatory macrophages in the intestine, specifically known as M2 macrophages, which restrict inflammation through the secretion of anti-inflammatory factors such as IL-10. \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. During sepsis, when the body is in an inflammatory state, blood-derived monocytes acquire more proinflammatory phenotypes in the intestine, namely, M1 macrophages, which secrete TNF-α, IL-1β and other proinflammatory cytokines, affecting the expression and localization of intestinal tight junction proteins\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. M2-type macrophages promote intestinal epithelial barrier tightness, whereas M1-type macrophages lead to increased permeability of the intestinal barrier \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Therefore, the polarization phenotype of macrophages is closely related to intestinal barrier function in sepsis.\u003c/p\u003e \u003cp\u003eDexmedetomidine is a highly selective α2 adrenergic receptor agonist that results in good sedation, easy awakening without causing obvious respiratory depression, and can significantly reduce postoperative delirium in patients. Dexmedetomidine is widely used in severe and perioperative patients. In recent years, the protective effect of dexmedetomidine in preserving organ function during sepsis has become a hot topic of research. Some studies have shown that dexmedetomidine has a protective effect on the intestinal barrier damage caused by sepsis\u003csup\u003e[\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. However, the mechanism by which it protects intestinal barrier function in sepsis remains unclear. Thus, we propose that dexmedetomidine can exert a protective effect on intestinal barrier function in sepsis by regulating the polarization phenotype of macrophages.\u003c/p\u003e \u003cp\u003eIn this study, we utilized cecal ligation and puncture to establish a sepsis model and treated intestinal epithelial cells and RAW264.7 cells with lipopolysaccharide (LPS) to explore the protective effects and mechanisms of dexmedetomidine on intestinal barrier function in septic rats both in vivo and ex vivo.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Ethical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental protocols were approved by the Laboratory Animal Welfare and Ethics Committee of Army Medical University (No. AMUMEC-20224867) and were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experimental animals were supplied by the Experimental Animal Center of the Army Medical Center; their ages ranged from 12--14 weeks, and their body weights were 200 \u0026plusmn; 20 g. The rats were fasted for 12 hours prior to surgery but had access to water ad libitum. The room temperature was maintained at approximately 25\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDexmedetomidine was obtained from Yangtze River Pharmaceutical (Jiangsu, China). Evans blue (EB), lipopolysaccharide (LPS) and antibodies against iNOS were purchased from Sigma (St. Louis, MO, United States). ELISA kits for diamine oxidase (DAO), TNF-ɑ, IL-1\u0026beta; and IL-6 were obtained from Elabsicence. An ELISA kit for D-lactic acid was obtained from Nanjing Jiancheng Biological Engineering Research Institute (Nanjing, China). Antibodies against ZO-1, Occludin and \u0026beta;-actin were purchased from BOSTER (Wuhan, China). Antibodies for flow cytometry, including PE-conjugated anti-mouse CD86 and Qd605-conjugated anti-mouse Ly6c, were purchased from BD Biosciences. MDA was purchased from Bioss (Beijing, China). Glutathione and ROS detection kits were purchased from Beyotime Biotechnology (Shanghai, China). A lipid peroxidation kit and an antibody against CD206 were purchased from Thermo Fisher Scientific (Waltham MA, United States).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Establishment of the sepsis model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdult Sprague\u0026ndash;Dawley (SD) rats (200\u0026ndash;220 g) were anesthetized with 3% sodium pentobarbital (30 mg/kg intraperitoneally). The sepsis model was induced via cecal ligation and puncture (CLP) according to previous methods\u003csup\u003e[19-20]\u003c/sup\u003e. Briefly, the abdominal wall was incised, and then the cecum was exposed and ligated 0.7 cm from the distal end of the cecum with a 0 sike. A triangular needle with a diameter of approximately 1.5 mm was used to pierce the cecum, and feces were admitted to flow into the abdominal cavity. The rats were returned to their cages after the abdomen was closed and allowed water and food freely. After the experiment, the rats were euthanized by intraperitoneal injection of a lethal dose of pentobarbital sodium (100 mg/kg).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Cell Culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe IEC-6 intestinal epithelial cell line was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). IEC-6 cells and RAW 264.7 macrophages were maintained in DMEM supplemented with 10% FBS at 37\u0026deg;C in a 5% CO2 environment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Intestinal Permeability Measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rats were anesthetized, and the abdomen was opened. A piece of small intestine approximately 7 cm in length was taken from each rat, the intestinal cavity was rinsed with PBS, and a capsule was prepared. A total of 0.2 mL of 1.5% EB solution was injected into the intestinal capsule, which was then incubated with a water bath at 37\u0026deg;C for 30 minutes. The intestinal capsule was rinsed with PBS until the washing solution was clarified, after which it was dried at 50\u0026deg;C for 24 hours. Next, the dry intestinal mass was recorded, formamide solution was added, and the mixture was incubated at 50\u0026deg;C for 24 hours. Finally, the supernatant was detected with an enzyme microplate meter at a wavelength of 650 nm, and the EB content in the intestinal tissue was calculated according to the obtained OD value.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Enzyme-linked immunosorbent assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples were collected from the femoral vein and centrifuged at 3500 r/min for 10 minutes. The supernatant was collected for ELISA analysis. The levels of DAO, D-lactate, TNF-ɑ and IL-1\u0026beta; in the serum and the concentrations of TNF-\u0026alpha; and IL-6 in the cell supernatant were measured by corresponding ELISA kits according to the manufacturers\u0026rsquo; instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Tissue histology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwelve hours after CLP, the rats were anesthetized. The intestinal tissues were fixed in 4% paraformaldehyde for 24 h and cut into 4-\u0026mu;m thick sections, which were stained with hematoxylin and eosin (H\u0026amp;E). The pathological sections were observed via optical microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Immunofluorescence Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe intestinal tissues were cut into 20-\u0026mu;m frozen sections, and RAW264.7 cells were seeded in the confocal chamber. The sections and cells were blocked with goat serum for 1 h at room temperature and then incubated with antibodies against iNOS (1:200) and CD206 (1:200) overnight at 4\u0026deg;C. After washing, the sections and cells were incubated with the corresponding fluorescent secondary antibodies for 1 h at room temperature. The slices and cells were subsequently washed again and dyed with DAPI. The samples were observed under a Leica microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Tissue dissociation and flow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSmall intestine samples were minced with scissors before incubation with 1 mg/ml collagenase in DMEM for 30 min at 37\u0026deg;C. Single-cell suspensions were obtained by screening with a nylon filter strainer at 100 \u0026mu;m and 70 \u0026mu;m. To detect CD86+ and Ly6c+ macrophages, the cells were subsequently stained at room temperature for 20 min with appropriate dilutions of anti-CD86 (PE) and anti-Ly6c (Qd605) fluorochrome-conjugated antibodies. Flow cytometry was performed using a BD flow cytometer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10\u0026nbsp;\u003c/strong\u003eMetabolomic profiling Cells were flash frozen in liquid nitrogen and stored at -80\u0026deg;C. The extraction mixture (acetonitrile:methanol:water = 2:2:1, containing an isotope-labeled internal standard mixture) was added to the cells. The sample was subsequently incubated at -40\u0026deg;C for 1 hour and centrifuged at 12000 rpm for 15 minutes at 4\u0026deg;C. The sample was then analyzed via a UHPLC system (Vanquish, Thermo Fisher Scientific). The raw data were converted to mzXML format and processed via in-house programs for peak detection, extraction, and integration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11 Bioinformatics analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrincipal component analysis (PCA) was employed to assess the intrinsic clustering of the metabolomics data. A 95% confidence interval (CI) served as a threshold to identify potential outliers across all samples. GraphPad Prism V.8.0.0 software was used to create volcano and violin plots, which clearly display upregulated and downregulated metabolites.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.12 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was conducted via SPSS 23.0 (SPSS Inc., Chicago, IL, USA), with the data represented as the means \u0026plusmn; standard deviations (SDs). One-way analysis of variance (ANOVA) followed by the post hoc Tukey test was used for comparisons between\u0026nbsp;experimental\u0026nbsp;groups.\u0026nbsp;Survival time was analyzed by the median and interquartile range, and Kaplan\u0026ndash;Meier survival analysis and the log-rank test were used.\u0026nbsp;\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1\u003c/strong\u003e \u003cstrong\u003eDexmedetomidine improves intestinal permeability in septic rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAfter sepsis, intestinal tissue leakage in the rats was obvious, as indicated by a significant increase in EB permeability and serum DAO and D-lac contents.\u0026nbsp;Compared with those in the sham group, EB permeability was increased by 244.12%, and the serum DAO and D-lac levels were increased by 159.39% and 148.19%, respectively (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). After routine fluid resuscitation, intestinal permeability improved only partially (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05). After Dex treatment, the intestinal permeability of septic rats was significantly improved, EB leakage was reduced by 32.56%, and the serum DAO and D-lac levels were reduced by 26.42% and 13.76%, respectively (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Figure 1A).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;Additionally, the serum and intestinal inflammatory cytokines TNF-\u0026alpha;, IL-1\u0026beta; and IL-6 were significantly greater in the sepsis group than in the sham group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). The serum and intestinal inflammatory cytokines TNF-\u0026alpha;, IL-1\u0026beta; and IL-6 were decreased to a certain extent in the CT group (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05). After treatment with dexmedetomidine, the serum and intestinal inflammatory cytokines TNF-\u0026alpha;, IL-1\u0026beta; and IL-6 decreased significantly. Compared with those in the sepsis group, the serum inflammatory cytokine levels were 33.93%, 37.86% and 30.90% lower, respectively, and the intestinal inflammatory cytokine levels were 31.88%, 28.7% and 29.37% lower, respectively. Compared with those in the CT group, the reduction rates of the serum inflammatory cytokines were 26.13%, 29.86%, and 25.12%, and the reduction rates of the intestinal inflammatory cytokines were 24.27%, 26.96%, and 22.83%, respectively (\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01) (Figure 1B, C), indicating that Dex could alleviate the intestinal inflammatory response.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;Finally, we observed the pathological staining structure of the intestinal tissue. The intestinal villi of the rats in the sham group were well structured and distinct, with columnar epithelial cells aligned neatly, and no infiltration of red blood cells or inflammatory cells was observed. In the sepsis group, the intestinal villi exhibited atrophy and disruption,\u0026nbsp;accompanied by significant infiltration of red blood cells and inflammatory cells. No significant improvement was observed in the CT group. After treatment with dexmedetomidine, intestinal villus atrophy significantly improved, intestinal villi were neatly arranged, infiltration of red blood cells and inflammatory cells was reduced, and the intestinal structure was significantly restored, suggesting that dexmedetomidine can protect the intestinal structure in sepsis (Figure 1D).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;To further investigate the effect of dexmedetomidine on the permeability of intestinal epithelial cells in vitro, we observed the expression of tight junction proteins in these cells. The immunofluorescence results revealed that ZO-1 in the normal group was continuously and regularly distributed along intestinal epithelial cells, whereas ZO-1 expression in the LPS group was weak and obviously disrupted, indicating a loose distribution. Compared with that in the LPS group, the expression of ZO-1 in the dexmedetomidine group was markedly increased, and the morphology was relatively continuous and clear (Figure 1E). Compared with those in the normal group, tight junction protein expression in the LPS group was significantly lower, and ZO-1 and Occludin expression was 69% and 59.33% lower, respectively, than that in the normal group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). Compared with those in the LPS group, the expression of ZO-1 and occludin in the dexmedetomidine group increased by 127.96% and 72.95%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) (Figure 1F). These results further indicate that dexmedetomidine exerts a protective effect on intestinal barrier function in rats with sepsis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Dexmedetomidine inhibits the M1-type polarization of macrophages to regulate intestinal permeability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the role of dexmedetomidine in regulating the polarization of intestinal macrophages following sepsis, we employed flow cytometry and immunofluorescence staining to detect biomarkers associated with M1 and M2 macrophages. M1 macrophages were identified as CD86+ Ly6c- macrophages, and M2 macrophages were identified as CD86-Ly6C+ macrophages. The flow cytometry results indicated that the proportion of M1 macrophages increased from 37.86% to 75.05% following sepsis. Dexmedetomidine administration notably reduced the number of M1 macrophages while increasing the number of M2 macrophages. Compared with the sepsis group, the dexmedetomidine group presented a reduction in the percentage of M1 macrophages from 75.05% to 51.70% and an increase in the percentage of M2 macrophages from 0.62% to 2.66% (Figure 2A). Immunofluorescence staining revealed increased iNOS expression in the intestinal macrophages of the rats in the sepsis group, whereas it was significantly decreased in the dexmedetomidine-treated group (Figure 2B).\u003c/p\u003e\n\u003cp\u003eTo further study the effects of dexmedetomidine on macrophage polarization, RAW264.7 cells were categorized into normal control, LPS, and Dex groups on the basis of distinct treatments. Various experimental approaches have been employed to assess the expression of M1 and M2 macrophage markers. The immunofluorescence results indicated that, relative to the control, LPS significantly enhanced M1 macrophage polarization by increasing iNOS expression (P\u0026lt;0.01). Following treatment with dexmedetomidine, the polarization of macrophages toward the M1 phenotype was markedly diminished, as evidenced by a reduction in iNOS expression (P\u0026lt;0.01) (Figure 2C). The ELISA results revealed that the levels of the proinflammatory cytokines TNF-\u0026alpha;, IL-6, and IL-1\u0026beta; in the LPS group were significantly elevated compared with those in the normal control group (P\u0026lt;0.01), suggesting an increase in macrophage polarization toward the proinflammatory M1 type. After treatment with dexmedetomidine, the levels of TNF-\u0026alpha;, IL-6, and IL-1\u0026beta; decreased, leading to a significant reduction in inflammation levels compared with those in the LPS group (Figure 2D).\u003c/p\u003e\n\u003cp\u003eThe above results suggest that dexmedetomidine can modulate macrophage polarization, curbing the rise of proinflammatory M1 macrophages postsepsis and fostering the shift toward anti-inflammatory M2 macrophages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Dexmedetomidine inhibits macrophage ferroptosis by promoting glutathione production after sepsis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe metabolomic profiles of macrophages following treatment with LPS and dexmedetomidine were examined to investigate the impact of dexmedetomidine on macrophage metabolism. Principal component analysis (PCA) revealed that the samples were largely distinct, yet all remained within the confidence interval (Figure 3A). The OPLS-DA results confirmed the model\u0026apos;s stability and reliability (Figure 3B). The volcano plot revealed 247 distinct metabolites between the LPS and dexmedetomidine groups (Figure 3C) (differentially abundant metabolites were identified with P values \u0026lt;0.05 and VIP values \u0026gt;1). Significant differences were observed in the levels of glutathione and cysteine among the upregulated metabolites (Figure 3D-G). KEGG pathway enrichment analysis revealed that cysteine and glutathione are closely linked to the ferroptosis pathway (Figure 3H). Dex may modulate macrophage polarization through the promotion of glutathione production and its impact on macrophage ferroptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Dexmedetomidine regulates macrophage polarization by inhibiting ferroptosis via xCT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the metabolomics findings, we employed a GSH assay kit to measure GSH levels in macrophages postsepsis. The results indicated that following LPS stimulation, both the GSH level and the GSH/GSSG ratio significantly decreased (P\u0026lt;0.01). In contrast, the Dex group presented a significant increase in the level of intracellular GSH (P\u0026lt;0.01) and the GSH/GSSG ratio (P\u0026lt;0.05) compared with those of the LPS group (Figure 4A-B). Given that intracellular cysteine levels impact GSH production and that xCT is the primary transporter protein for cysteine entry into cells, Dex treatment led to a significant upregulation of xCT expression compared with LPS treatment (Figure 4C), suggesting a correlation between the increase in GSH and xCT. Moreover, GSH activates the core regulatory enzyme GPX4 of the glutathione antioxidant system, contributing to the generation of ROS. We next examined the changes in GPX4, revealing an increase in expression within the DEX group (Figure 4D) accompanied by a reduction in ROS and lipid-ROS production (Figures 4E-F). Compared with those in the normal control group, the expression of xCT and GPX4 in the LPS group was decreased by 68.33% and 60%, respectively (P\u0026lt;0.01), the ROS level was increased by 452.91% (P\u0026lt;0.01), and the lipid ROS level was significantly increased. Compared with those in the LPS group, the expression of xCT and GPX4 in the dexmedetomidine group was increased by 69.47% and 52.5%, respectively (P\u0026lt;0.01), the ROS level was reduced by 40.29% (P\u0026lt;0.01), and the lipid ROS level was significantly decreased (Figure 4C-D), indicating that Dex can inhibit ferroptosis in macrophages. In addition to the xCT inhibitor SAS, the population of M1 macrophages increased in comparison with those in the xCT intervention group, suggesting an intensification of macrophage polarization toward M1 postxCT inhibition (Figure 4G). These results suggest that dexmedetomidine prevents macrophage ferroptosis via xCT and diminishes macrophage polarization toward the M1 phenotype.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Effects of dexmedetomidine on organ function and survival time in septic rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eTo assess whether dexmedetomidine, by mitigating intestinal leakage in sepsis, can safeguard the functions of additional vital organs and prolong survival, we initially assessed markers of liver, kidney, and myocardial function, along with arterial blood gases and mean arterial pressure. These findings revealed that dexmedetomidine substantially enhanced organ function in rats with sepsis. Compared with those in the standard treatment group, the ALT, AST, creatinine (Crea), and CK-MB levels decreased by 30.36% (P\u0026lt;0.01), 34.31% (P\u0026lt;0.01), 21.31% (P\u0026lt;0.05), and 23.55% (P\u0026lt;0.05), respectively. The pH and oxygen partial pressure significantly increased, whereas the carbon dioxide partial pressure markedly decreased (P\u0026lt;0.05) (Figure 5A-G). The mean arterial blood pressure results indicated a significant decrease in rats after sepsis surgery, decreasing to approximately 60 mm Hg. Following fluid resuscitation, the blood pressure in the sepsis group decreased to approximately 46 mm Hg. Moreover, in the conventional treatment group, the arterial blood pressure gradually increased to approximately 72 mm Hg, and in the dexmedetomidine group, it increased to approximately 100 mm Hg (Figure 5H). Subsequent observations of the 24-hour survival duration and rate revealed that only one rat in the sepsis group survived beyond 24 hours, representing a survival rate of 6.25%, with an average survival time of 5.70 \u0026plusmn;6.36 hours. Following conventional fluid resuscitation, two rats survived, with a survival rate of 12.50% and an average survival time of 10.10 \u0026plusmn;7.80 hours, indicating an increase in survival time compared with that of the sepsis group. After Dex treatment, the number of rats surviving 24 hours increased to six, with a survival rate of 37.50% and a survival time of 18.30 \u0026plusmn; 5.80 hours, indicating a significant improvement in the survival rate and a substantial extension in survival time compared with those of the conventional treatment group (P\u0026lt;0.05, Figure 5I‒J).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eSepsis is a frequently encountered critical illness in clinical practice, where a dysregulated immune response can lead to fatal organ dysfunction. If not promptly treated, it can progress to multiple organ dysfunction syndrome or even septic shock, resulting in an extremely high mortality rate \u003csup\u003e[21]\u003c/sup\u003e. The gut plays an important role in sepsis. Under physiological conditions, the gut acts as a defensive barrier between the body and the external environment, preventing the entry of toxic agents and pathogens into the circulation. During sepsis, the cascading release of numerous inflammatory factors impairs the intestinal barrier, leading to necrosis of epithelial cells, disruption of intercellular junctions, and severe intestinal leakage.\u0026nbsp;This allows toxic agents, including bacteria and endotoxins, to transgress the intestinal epithelial barrier abnormally, triggering systemic inflammation. This, in turn, intensifies the progression of sepsis, ultimately leading to\u0026nbsp;multiorgan\u0026nbsp;dysfunction and patient fatality \u003csup\u003e[22-26]\u003c/sup\u003e. However, targeted prevention and treatment\u0026nbsp;methods\u0026nbsp;for intestinal leakage\u0026nbsp;postsepsis are\u0026nbsp;lacking. Consequently, investigating effective strategies to protect the intestinal barrier is crucial for sepsis treatment.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;Macrophages, highly heterogeneous immune cells, differentiate into various types in response to diverse pathological stimuli, thereby playing a pivotal role in the body\u0026apos;s inflammatory response. There are two types of macrophage polarization, namely, classically activated macrophages (M1) and alternately activated macrophages (M2). M1 macrophages, which are primarily induced by lipopolysaccharides (LPS), secrete proinflammatory cytokines such as TNF-\u0026alpha;, IL-1\u0026beta;, and IL-6, which boost the body\u0026apos;s immune response during sepsis\u0026apos;s excessive inflammatory phase, leading to immune dysfunction. M2 macrophages, which are primarily activated by IL-4, release anti-inflammatory factors such as IL-10 to limit inflammation, foster tissue repair, and facilitate the healing of injured areas. \u003csup\u003e[27-29]\u003c/sup\u003e. The polarization state of macrophages is considered pivotal in maintaining intestinal homeostasis. During sepsis, there is an increase in the number of M1 macrophages, which secrete high levels of proinflammatory cytokines such as TNF-\u0026alpha;, in the intestine. Proinflammatory cytokines are considered key regulators of the expression and localization of tight junction proteins. TNF-\u0026alpha; has been demonstrated to enhance intestinal permeability by influencing the expression of ZO-1 and inducible nitric oxide synthase (iNOS)\u003csup\u003e[24-25]\u003c/sup\u003e. Consequently, M1 macrophages exacerbate the intestinal damage associated with sepsis, whereas shifting polarized macrophages from the M1 phenotype to the M2 phenotype can effectively mitigate inflammatory dysregulation and intestinal damage\u003csup\u003e[13-14]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;Dexmedetomidine, a novel anesthetic and sedative, has emerged as a focal point of research in recent years, largely because of its anti-inflammatory properties. CHEN \u003csup\u003e[30]\u003c/sup\u003e reported that Dex could inhibit the activation of the NLRP3 inflammasome, reduce lung inflammation, and play a protective role in the lung. In ZI\u0026rsquo;s study\u003csup\u003e[31]\u003c/sup\u003e, Dex significantly inhibited the release of inflammatory factors through a cholinergic anti-inflammatory mechanism and alleviated acute liver injury in sepsis. Another study \u003csup\u003e[32]\u0026nbsp;\u003c/sup\u003ereported that dexmedetomidine can inhibit the myocardial inflammatory response and alleviate myocardial dysfunction caused by sepsis by inducing cardiomyocyte autophagy. Our findings demonstrate that sepsis is followed by a significant increase in intestinal M1 macrophages. Dexmedetomidine administration significantly reduces the number of intestinal M1 macrophages and promotes M2 polarization. Similarly, cellular experiments confirmed pronounced M1 polarization of macrophages after LPS stimulation, with significant increases in the inflammatory cytokines TNF-\u0026alpha;, IL-6, and IL-\u0026beta; (P\u0026lt;0.01). Following dexmedetomidine treatment, the M1 marker iNOS was significantly reduced, inflammatory cytokines were markedly decreased, and the M2 markers CD206 and Arg1 were significantly increased (P\u0026lt;0.05). These results suggest that dexmedetomidine exerts a protective effect on intestinal barrier function in sepsis by modulating macrophage polarization and suppressing the intestinal inflammatory response.\u003c/p\u003e\n\u003cp\u003eTo further elucidate how dexmedetomidine regulates macrophage polarization, we analyzed the metabolomic profiles of macrophages treated with LPS and dexmedetomidine. We found that dexmedetomidine significantly increased cysteine and GSH levels, and KEGG pathway enrichment analysis indicated that glutathione and cysteine are involved in ferroptosis. Ferroptosis, a newly identified form of programmed cell death, is characterized by lipid peroxidation and increased ROS levels due to the accumulation of ferrous ions within cells \u003csup\u003e[33-35]\u003c/sup\u003e. The primary mechanism for inducing ferroptosis is the inactivation of GPX4 due to GSH depletion. By inhibiting the membrane transporter xCT and reducing the levels of cysteine, a precursor for GSH synthesis, the intracellular GSH levels decrease, resulting in the inactivation of GPX4 \u003csup\u003e[36]\u003c/sup\u003e. GPX4 is the core regulatory enzyme of the antioxidant system (glutathione system) and converts peroxide bonds involved in lipid peroxidation into hydroxyl groups, thus inhibiting ferroptosis\u003csup\u003e\u0026nbsp;[37]\u003c/sup\u003e. Studies indicate that ferroptosis is closely linked to M1 polarization in two ways: (1) During ferroptosis in macrophages, excess intracellular ferrous iron elevates the levels of M1 macrophage markers, including IL-6, TNF-\u0026alpha;, and IL-1\u0026beta;, while reducing the levels of M2 markers, such as TGM2 \u003csup\u003e[38]\u003c/sup\u003e. Moreover, ROS accumulation and p53 acetylation due to iron overload facilitate M1 polarization \u003csup\u003e[39]\u003c/sup\u003e. (2) Cells undergoing ferroptosis facilitate the recruitment of M1 inflammatory macrophages by releasing HMGB1 and inducing the expression of inflammatory genes such as CCL2 and CCL7 \u003csup\u003e[40-43]\u003c/sup\u003e. To validate the metabolomics findings, we employed a GSH assay kit to measure GSH levels in macrophages postsepsis. Dexmedetomidine notably elevated GSH levels in macrophages from patients with sepsis (P\u0026lt;0.01). We hypothesize that dexmedetomidine may regulate macrophage polarization by adjusting GSH levels via xCT. Consequently, we assessed the levels of GSH, xCT, GPX4, and ROS. The results indicated that, relative to the LPS group, dexmedetomidine significantly increased the GSH level and increased the protein expression of xCT and GPX4 (P\u0026lt;0.01) while also reducing the levels of intracellular and lipid-bound ROS (P\u0026lt;0.01), thereby inhibiting ferroptosis in macrophages. Following the addition of xCT inhibitors, dexmedetomidine is unable to inhibit M1 macrophage polarization. Thus, after establishing that dexmedetomidine influences GSH synthesis via xCT and that GSH, as a substrate for GPX4, inhibits lipid peroxidation and reduces ROS levels in macrophages, we hypothesize that Dex may reduce macrophage polarization toward the proinflammatory M1 phenotype by suppressing ferroptosis.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;This study has several limitations. First, this study demonstrated that dexmedetomidine can inhibit intestinal leakage in rats with sepsis both in vivo and in vitro, but it remains unclear whether dexmedetomidine consistently protects intestinal barrier function in patients with sepsis. Second, in addition to protecting intestinal barrier function by inhibiting macrophage polarization toward the M1 phenotype through the ferroptosis pathway, further research is needed to determine whether dexmedetomidine influences macrophage polarization and function through other metabolic pathways and metabolites.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study demonstrated that dexmedetomidine exerts a protective effect on intestinal barrier function in rats with sepsis. The mechanism involves Dex regulating the production of GSH/GPX4, which in turn inhibits macrophage ferroptosis and suppresses macrophage polarization toward the proinflammatory M1 phenotype. This study offers novel therapeutic insights and targets for preserving intestinal barrier function in sepsis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTL, LW, HY and SJZ designed the study. SJZ, BDZ, LYZ, CQT, and XWZ analyzed the data. TL acquired the financial support. SJZ drafted the manuscript. LW revised the manuscript. All the authors performed the experimental procedures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Chongqing Talent Program Lump-sum Project (cstc2022ycjh-bgzxm0011).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original data described in the research are included in the article and supplementary materials. For further inquiries, please directly contact the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experimental processes were approved by the Ethnic Committee of Laboratory Animal Welfare and Ethics Committee Of the Third Military Medical University hospital and conducted in strict accordance to the standard of the Guide for the Care and Use of Laboratory Animals published by the Ministry of Science and Technology of the People\u0026apos;s Republic of China in 2006.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSinger M, Deutschman CS, Seymour CW, et al. 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Journal of the American Society Nephrology, 2018,29:919\u0026ndash;35.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"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},"keywords":"sepsis, macrophage, dexmedetomidine, intestinal barrier, ferroptosis","lastPublishedDoi":"10.21203/rs.3.rs-6742785/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6742785/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe intestine is believed to play a pivotal role in the onset and progression of sepsis, serving as the driving force behind multiple organ dysfunction syndrome. The polarization state of macrophages is pivotal in the intestinal barrier dysfunction associated with sepsis. M1 macrophages initiate the degradation of barrier-sealing molecules, increasing intestinal barrier permeability. Studies have demonstrated that dexmedetomidine offers protection for organ function in sepsis; however, the mechanism behind its protective effect on the intestinal barrier remains unclear. We utilized cecal ligation and perforation surgery to establish sepsis models, along with lipopolysaccharide (LPS)-treated intestinal epithelial and RAW264.7 cell models, to explore the protective effects and mechanisms of dexmedetomidine on intestinal barrier function in rats with sepsis. Our study demonstrated that dexmedetomidine protects intestinal barrier function in septic rats by suppressing inflammatory responses, enhancing the expression of tight junction proteins between intestinal epithelial cells, and significantly reducing intestinal permeability. Additionally, dexmedetomidine markedly decreases the number of inflammatory M1 macrophages in the intestines of septic rats, facilitates the polarization of macrophages toward the anti-inflammatory M2 phenotype, and suppresses the secretion of inflammatory cytokines. Research has indicated that dexmedetomidine is closely linked to ferroptosis, influencing the transport protein xCT to increase the GSH content and GPX4 expression within macrophages. This, in turn, reduces intracellular ROS and lipid ROS levels, mitigates macrophage ferroptosis, and curtails the polarization of macrophages toward the proinflammatory M1 phenotype.\u003c/p\u003e","manuscriptTitle":"Dexmedetomidine modulates macrophage polarization by inhibiting ferroptosis to exert protective effects on intestinal barrier function in sepsis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-29 14:35:07","doi":"10.21203/rs.3.rs-6742785/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":"be7b6570-28ac-40c7-bdcf-14012c823f78","owner":[],"postedDate":"June 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-19T03:39:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-29 14:35:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6742785","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6742785","identity":"rs-6742785","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}