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World J Gastroenterol. Jun 21, 2026; 32(23): 117320
Published online Jun 21, 2026. doi: 10.3748/wjg.v32.i23.117320
Esmolol alleviates lipopolysaccharide-induced intestinal injuries by enhancing autophagy through the AMPK/mTOR/ULK1 pathway
Yan-Bing Zhang, Fu-Hai Ji, Department of Anesthesiology and Pain Management, The First Affiliated Hospital, Soochow University, Suzhou 215006, Jiangsu Province, China
Zhe-Jun Yu, Jun Jin, Mao-Xia Liu, Xin-Jing Yang, Department of Intensive Care Unit, The First Affiliated Hospital, Soochow University, Suzhou 215006, Jiangsu Province, China
ORCID number: Yan-Bing Zhang (0000-0001-8172-5561); Fu-Hai Ji (0000-0001-6649-665X); Xin-Jing Yang (0009-0008-5791-0802).
Co-corresponding authors: Fu-Hai Ji and Xin-Jing Yang.
Author contributions: Zhang YB and Yang XJ designed and performed most of the investigation; Yu ZJ analyzed the data and Zhang YB wrote the original draft manuscript; Jin J and Liu MX contributed to interpretation of the data and analyses; Ji FH and Yang XJ contributed to revising the article and modifying the figures. All authors have read and approved the manuscript. We propose the designation of two co-corresponding authors to formally acknowledge their shared, equal responsibility for the scientific integrity, methodological rigor, and long-term reproducibility of this fully experimental study. Ji FH conceived the study, led the overall experimental design, secured funding, coordinated cross-functional resources and team execution, oversaw regulatory compliance and administrative approvals, and provided substantive guidance throughout manuscript drafting and revision. Yang XJ led the development and implementation of the experimental methodology-including protocol design, assay optimization, data workflow architecture, analytical robustness validation, data curation, and establishment of an end-to-end reproducibility framework (encompassing version-controlled datasets, comprehensive documentation, and auditable provenance trails). Correspondence regarding conceptual rationale, study interpretation, and editorial or reader inquiries will be jointly managed. Ji FH serves as the primary contact for questions related to study design, strategic oversight, and high-level scientific direction; Yang XJ serves as the primary contact for technical implementation, methodological details, data access, and reproducibility infrastructure. This co-corresponding authorship arrangement ensures authoritative, timely, and coordinated responses; maintains continuity across the research lifecycle; strengthens transparency; and accurately reflects their equivalent accountability for the validity, reliability, and enduring utility of the work.
Supported by National Natural Science Foundation of China, No. 82471281; Key Medical Research Projects in Jiangsu Province, No. ZD2022021; and Key R&D Program Projects in Jiangsu Province, No. BE2023709.
Institutional review board statement: This study does not involve any human experiments.
Institutional animal care and use committee statement: All animal procedures were approved by the Medical Ethics Committee of The First Affiliated Hospital of Soochow University with the Public Health Service Policy on Humane Care and Use of Laboratory Animals. All methods were conducted in accordance with relevant guidelines and regulations. All methods were reported in accordance with ARRIVE guidelines.
Conflict-of-interest statement: No conflict of interests.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Data sharing statement: Data are ethically restricted, and they cannot be shared publicly. Data are available from the corresponding author by request subject to ethical considerations.
Corresponding author: Xin-Jing Yang, MD, Department of Intensive Care Unit, The First Affiliated Hospital, Soochow University, No. 188 Shi-Zi Road, Suzhou 215006, Jiangsu Province, China. yangxinjingsuda@163.com
Received: December 5, 2025
Revised: February 5, 2026
Accepted: March 12, 2026
Published online: June 21, 2026
Processing time: 185 Days and 16.9 Hours

Abstract
BACKGROUND

Beta-1 receptor blockade is well characterized for its protective effects against septic symptoms, and esmolol (ES) is a selective β1-adrenoceptor antagonist.

AIM

To assess the effects of ES on lipopolysaccharide (LPS)-induced septic intestinal damage and explore the associated mechanism by focusing on the AMPK/mTOR/ULK1 pathway.

METHODS

Sepsis was induced via an intraperitoneal injection of LPS in male SD rats or LPS treatment in rat intestine epithelial cells. To assess their anti-sepsis effects, rats and cells were pretreated with ES, 3-methyladenine, rapamycin (RAPA), and/or compound C, 30 minutes before LPS exposure. Then, intestinal damage, intestinal fatty acid-binding protein (I-FABP) and diamine oxidase (DAO) levels in intestinal tissue, interleukin (IL)-6, IL-1, tumor necrosis factor-α (TNF-α), IL-17, and IL-10 levels, cell viability, autophagic processes, and AMPK/mTOR/ULK1-related signaling transduction were detected via a series of in vivo and in vitro assays.

RESULTS

LPS induced intestinal damage in a time-dependent manner and suppressed autophagy at 12 and 24 hours. Pretreatment with ES or RAPA reduced I-FABP and DAO release; improved the damage score; and increased the expression of Beclin-1, LC3-II, p-AMPK, p-ULK1 and numbers of autophagosomes, and decreased the expression of p-mTOR at 12 and 24 hours, indicating amelioration of intestinal injury and augmentation of autophagy in rats. The results of in vivo assays were consistent with those in the IEC-6 intestinal epithelial cell line. Pre-treatment with ES reduced IL-1, TNF-α, IL-17, and IL-6 release and increased IL-10 release in cells.

CONCLUSION

The current findings demonstrate that ES ameliorates LPS-induced septic intestinal damage by activating autophagy through modulation of the AMPK/mTOR/ULK1 pathway.

Key Words: Esmolol; AMPK/mTOR/ULK1; Autophagy; Sepsis; Intestinal injury; Lipopolysaccharide

Core Tip: This study investigated the protective effects of esmolol (ES), a selective β1-blocker, against lipopolysaccharide-induced septic intestinal damage. Using both animal and cell models, researchers found that ES pretreatment reduced intestinal injury markers (intestinal fatty acid-binding protein and diamine oxidase), improved tissue damage scores, and suppressed inflammation by suppressing interleukin (IL)-1 tumor necrosis factor-α, IL-17, and IL-6 while enhancing IL-10. Mechanistically, ES enhanced autophagy through activation of the AMPK/mTOR/ULK1 signaling pathway, as shown by increasing the expression of Beclin-1, LC3-II, p-AMPK, p-ULK1 and numbers of autophagosomes, and decreasing the expression of p-mTOR. These effects were consistent in both in vivo and in vitro models. The findings suggest that ES alleviates septic intestinal injury by promoting autophagy via the AMPK/mTOR/ULK1 pathway.



INTRODUCTION

Sepsis, caused by the host’s imbalanced responses to infection, is a severe illness that results in organ malfunction and is conceived as a primary cause of high death rates in critical care units[1]. The disorder can cause many disturbances in the intestinal epithelium, including increased cytokine production, impaired barrier function, and increased epithelial apoptosis[2]. The gastrointestinal tract has been identified as crucial to the pathophysiology of sepsis and, more importantly, as the “motor” of systemic inflammatory responses[3]. Although advances have been made in treatment strategies targeting sepsis-induced intestinal damage, clinical outcomes remain limited, and the underlying mechanisms are still poorly understood. Thus, effects have being made to more comprehensively understand the pathogenesis of complications associated with sepsis and to promote the management of these complications. β-blockers, which are known for their ability to treat cardiovascular problems, have attracted attention in the field of sepsis research because of their potential to regulate the immune system, reduce inflammation, affect metabolism, and lower myocardial oxygen consumption[4-6]. In recent years, clinical trials investigating β-blockers have demonstrated promising outcomes in the treatment of sepsis. For instance, patients experiencing septic shock benefitted from reduced death rates and improved hemodynamics following treatment with esmolol (ES), a specific β1-adrenoceptor antagonist[7,8]. Additionally, other studies found that ES can improve sepsis outcomes by reducing local inflammatory responses and altering the integrity of the gut mucosa[9]. Following open abdominal surgery, the β1-blocker metoprolol reduced both local and systemic inflammatory responses while maintaining intestinal barrier function[10]. However, the exact molecular mechanisms by which β1-blockers such as ES ameliorate acute intestinal damage caused by lipopolysaccharide (LPS) remain unknown, and a comprehensive exploration of the mechanisms will definitely improve the clinical application of β1-blockers in the treatment of sepsis-related symptoms.

To maintain homeostasis and ensure life, cells degrade and recycle their own constituent parts through a process termed autophagy. Autophagy exhibits dual and time-dependent characteristics. Numerous investigations have demonstrated the role autophagy plays in the pathophysiology of intestinal damage induced by sepsis. For example, hesperetin can prevent neutrophil extracellular traps formation by regulating the reactive oxygen species (ROS)/autophagy pathway, thereby maintaining the integrity of the intestinal barrier during sepsis[11]. Many signaling pathways, such as the AMPK, autophagy-related gene, p53, Toll-like receptor, and mTOR signaling pathways[12], are implicated in the process of autophagy[13]. In addition, the ULK1 protein interacts with Atg13, Atg101, and FIP200 to promote autophagy, representing a crucial step in the process[14]. Of these components, AMPK, the primary energy sensor that maintains energy homeostasis by balancing cellular metabolism, triggers autophagy. Meanwhile, mTOR, a key regulator of cell development that absorbs signals from nutrients and growth factors, inhibits autophagy[15]. A recent study indicated that the AMPK/mTOR pathway might play a role in the activation of intestinal autophagy in mice following burns[16], and it is also reported that berberine reduces the expression and production of lysozyme and lessens ulcerative colitis caused by dextran sodium sulfate by promoting autophagy via the AMPK/mTOR/ULK1 pathway[17]. Given the key role of autophagy activation in the amelioration of sepsis and verified treatment effects of ES, it is reasonable to explore the potential involvement of autophagy in the anti-sepsis effects of ES. Our previous research also found that ES alleviates LPS-induced myocardial damage by activating AMPK/mTOR/ULK1 pathway-regulated autophagy[18]. To further explore the mechanism by which ES treats sepsis, this study investigated the effect of ES on sepsis-induced intestinal injury. Thus, in the current study, sepsis was induced using LPS both in vivo and in vitro, followed by treatment with ES, 3-methyladenine (3-MA), rapamycin (RAPA), and/or compound C (CC). Then, intestinal damage, intestinal permeability, cell viability, inflammatory factors, autophagic processes, and AMPK/mTOR/ULK1-related signaling transduction were detected via a series of in vivo and in vitro assays.

MATERIALS AND METHODS
Experimental animals

SD rats (male, 2 months old, weighing 250-300 g) were provided by Zhejiang Vital River Laboratory Animal Technology Co., Ltd. (Zhejiang, China). Additionally, LPS (Sigma-Aldrich Co., Ltd., St. Louis, MO, United States), 3-MA (MedChemExpress, Monmouth Junction, NJ, Pharmaceutical Co., Ltd., Shanghai, China), RAPA (MedChemExpress), CC (Sigma-Aldrich), and normal saline (NS; Kelun Pharmaceutical Co., Ltd. Chengdu, China) were generously donated by the pharmacy at the First Affiliated Hospital of Soochow University.

A schematic of the experimental overview is presented in Figure 1. After treatment with sodium pentobarbital (10 mg/kg) for anesthesia, the rats underwent internal jugular vein catheterization, and the operative time was controlled within 15 minutes. Following surgery, rats were randomly divided into five groups: Sham, LPS, LPS + ES, LPS + 3-MA, and LPS + RAPA. The Sham and LPS groups were further divided into four subgroups, respectively, whereas the LPS + ES, LPS + 3-MA, and LPS + RAPA groups were each divided into two subgroups. Each subgroup included eight rats. Regarding the specifics of treatment, NS was continuously infused at a rate of 0.5 mL/h over varying spans of time depending on the group. Rats in the Sham group received NS over intervals of 3, 6, 12, and 24 hours (Figure 1A). Rats in the LPS group received a NS infusion at 0.5 mL/h for 3, 6, 12, and 24 hours (Figure 1B). Rats in the LPS + ES group received ES (15 mg/kg/h) continuously at a rate of 0.5 mL/h for 12 and 24 hours (Figure 1C). Rats in the LPS + 3-MA and LPS + RAPA groups received 3-MA (15 mg/kg) and RAPA (4 mg/kg), respectively, via intraperitoneal (ip) administration, along with NS at 0.5 mL/hour for 12 and 24 hours (Figure 1D). To establish the sepsis model, LPS (10 mg/kg)[19] was administered by ip injection for 30 minutes. The rats were treated for their specified times and then sacrificed. Serum samples and jejunum specimens were immediately collected for further analysis. In our methodology, we used 3-MA (an autophagy inhibitor) and RAPA (an autophagy promoter) to ascertain whether ES had any impact on autophagy modulation in LPS-induced intestinal injury.

Figure 1
Figure 1 Schematic of the study design. A: Rats in the Sham group continuously received normal saline (NS) for 3, 6, 12, or 24 hours after surgery; B: Rats in the lipopolysaccharide (LPS) group continuously received NS for 3, 6, 12, or 24 hours, and LPS was administered 30 minutes after the start of the NS infusion; C: Rats in the LPS + ES group continuously received ES for 12 or 24 hours, and LPS was administered 30 minutes after the start of ES and LPS treatment; D: Rats in the LPS + 3-methyladenine (3-MA) and LPS + rapamycin (RAPA) groups continuously received with NS for 12 or 24 hours as well as an intraperitoneal injection of 3-MA or RAPA, followed by an intraperitoneal injection of LPS 30 minutes after the start of the NS infusion. 3-MA: 3-methyladenine; ES: Esmolol; NS: Normal saline; RAPA: Rapamycin; LPS: Lipopolysaccharide.

This study adhered to ethical guidelines and received approval from the Experimental Animal Ethics Committee of Soochow University, No. SUDA20251118A01.

Cell modeling and grouping

The IEC-6 rat intestinal crypt cell line was acquired from the American Type Culture Collection and cultured in an incubator with RPMI 1640 (HyClone, GE, United States) containing 10% fetal bovine serum, 10 U/mL penicillin-streptomycin, and 0.06% insulin at 37 °C in a 5% CO2 atmosphere. For the in vitro model, the cells were treated with various concentrations of LPS (Sigma-Aldrich, 0, 1, 5, 10, 50, 100 μg/mL) for 6 hours. Then, the cells were further treated with LPS (100 μg/mL) for 0, 0.5, 1, 2, 6, 12, or 24 hours. In the LPS + ES group, IEC-6 cells were pre-treated with ES at concentrations of 1 × 10-3, 1 × 10-4, and 1 × 10-5 g/mL for 30 minutes before incubation with LPS (100 μg/mL) for 6 hours. In the LPS + 3-MA/RAPA/CC group, cells were treated with 3-MA (2 × 10-3 mol/L), RAPA (10 μmol/L), and CC (10 μmol/L) for 30 minutes before LPS (100 μg/mL) stimulation for 6 hours. In the LPS + ES + CC group, cells were treated with ES (1 × 10-4 g/mL) and CC (10 μmol/L) for 30 minutes before LPS (100 μg/mL) stimulation for 6 hours.

Hematoxylin-eosin staining

Fresh jejunal tissues were cleaned three times with NS, preserved in 10% neutral formaldehyde buffer, dried, paraffin-embedded, and cut with a microtome. After dewaxing and staining with hematoxylin-eosin, the samples were examined under a digital light microscope (Olympus, Tokyo, Japan), and the length of the intestinal villi was measured using ImageJ (NIH).

MTT cell viability assay

To determine the viability of cells, 200 μL of a cell suspension was cultivated in 96-well flat-bottom culture plates at a density of 1 × 105 cells/well. Then, cells were treated with or without ES (1 × 10-4 g/mL)/CC (10 μmol/L) for 30 minutes, followed by incubation with or without LPS (100 μg/mL) for 6 hours. Afterward, 100 μL of MTT solution (1 mg/mL) was added to each well, followed by incubation at 37 °C in a 5% CO2 atmosphere for 4 hours. The supernatant was extracted, and 100 μL of dimethyl sulfoxide was added into each well, followed by incubation for 15 minutes. Cell viability was determined by the optical density measured at 490 nm using a microplate reader (Multiskan FC, Thermo Fisher Scientific, Waltham, MA, United States).

Western blot analysis

Intestinal tissues were homogenized and lysed for 30 minutes on ice in radioimmunoprecipitation assay buffer. Following pretreatment, the tissues were washed three times with cold phosphate-buffered saline (PBS) and RIPA buffer supplemented with phosphatase and protease inhibitors to obtain whole-cell lysates. The protein concentration was ascertained using BCA protein assay kits (Com Win Biotech), and protein samples of the same quantity were separated using 10% SDS-PAGE and then transferred to PVDF membranes (Amersham Pharmacia Biotech, United Kingdom). After blocking the membranes for 2 hours at room temperature using 5% skim milk in PBS and 0.1% Tween 20, the membranes were incubated with the following primary antibodies: Anti-β-actin, anti-Beclin-1, anti-p-AMPK (Thr172) (40H9), anti-p-ULK1 (Ser757) (D7O6U), anti-p-mTOR (Ser2448) (D9C2), and anti-LC3-II (abc62721). Protein bands were developed using enhanced chemiluminescence (Amersham Pharmacia Biotech) and quantified using ImageJ. The relative expression of proteins was presented as the fold change vs the control group using β-actin as the internal reference.

Enzyme-linked immunosorbent assay

The levels of intestinal fatty acid-binding protein (I-FABP) and diamine oxidase (DAO) in rat serum; levels of IL-1, IL-6, and IL-10 in intestinal tissue; and levels of IL-1, IL-17, TNF-α, IL-6, and IL-10 in IEC-6 cell culture supernatant were detected using enzyme-linked immunosorbent assay (ELISA) kits (LIANKE Technology Co., Ltd.) following the manufacturer’s instructions. Briefly, 1 g of fresh tissue was ground into tissue homogenate and centrifuged at 3000 × g for 10 minutes. IEC-6 cells (1 × 106 cells) were collected via centrifugation at 2000 × g for 20 minutes. The supernatant of intestinal tissue and IEC-6 cells was then retained, and the levels of targets were measured by a microplate reader using corresponding kits (Thermo Fisher Scientific).

Transmission electron microscopy

Fresh intestinal tissue samples (2 mm × 2 mm × 2 mm) were quickly and carefully obtained after the rats were sacrificed and incubated in tubes containing a fixation solution for 4 hours. Then, the intestinal tissues underwent penetration, dehydration, and embedding overnight, followed by detection using a transmission electron microscope (Dmetrix, China). The ultrastructural damage to the mitochondria was evaluated by determining the number of autophagosomes in different groups.

RNA sequencing

Total RNA was extracted using a TRIzol reagent kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States) and confirmed using RNase-free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched using Oligo(dT) beads, and prokaryotic mRNA was enriched by removing rRNA using a Ribo-Zero™ Magnetic Kit (Epicentre, Madison, WI, United States). Then, the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse-transcribed into cDNA with random primers. Second-strand cDNA was synthesized using DNA polymerase I, RNase H, dNTPs, and buffer. The cDNA fragments were purified using a QiaQuick polymerase chain reaction (PCR) extraction kit (Qiagen, Venlo, The Netherlands) and end-repaired. Then, poly(A) was added and ligated to Illumina sequencing adapters. The ligation products were size-selected by agarose gel electrophoresis, amplified by PCR, and sequenced using Illumina HiSeq2500 by Gene Denovo Biotechnology Co. (Guangzhou, China). The genes with fold change ≥ 1.5 and P < 0.05 were considered significant differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses of the DEGs were conducted. To reduce sample variability, three replicates of harvested IEC-6 cells were pooled into one sample. Samples were prepared from the Sham, LPS, and LPS + ES groups (n = 3 per group).

Statistical analysis

All data were presented as the mean ± SE. The overall effect of different treatments was determined using ANOVA followed by multiple comparisons using the Newman-Keuls test. All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, United States) with a significant level of 0.05.

RESULTS
ES ameliorated histological destruction in the intestine of rats with LPS-induced sepsis

As presented in Figure 2, rats were pretreated with ES, 3-MA, or RAPA, followed by the induction of sepsis using LPS. Compared to rats in the Sham group (Figure 2A), intestinal tissue injury was more severe in rats in the LPS (Figure 2B) and LPS + 3MA groups (Figure 2D). However, in the LPS + ES (Figure 2C) and LPS + RAPA (Figure 2E) groups, edema, villus tipsloughing, and inflammatory cell infiltration into the mucosa were considerably attenuated at 12 hours and 24 hours compared to that in the LPS group.

Figure 2
Figure 2 Interstitial tissue pathological changes in sepsis model rats at 12 and 24 hours (n = 6). The interstitial tissues from rats in different groups were stained with hematoxylin-eosin (H&E). A: No significant tissue destruction or other symptoms were observed in the Sham group; B: Whereas significant pathological tissue changes were observed in the lipopolysaccharide group; C: After treatment with esmolol, the impairments were reduced; D: Significant pathological tissue changes were observed in rats treated with 3-methyladenine; E: The impairments were reduced in rats treated with rapamycin. H&E, × 100 magnification, scale bar = 50 μm; × 400 magnification, scale bar = 20 μm. 3-MA: 3-methyladenine; ES: Esmolol; NS: Normal saline; RAPA: Rapamycin; LPS: Lipopolysaccharide.

The interstitial villus length was shortened in response to LPS stimulation at 12 and 24 hours compared to that in the Sham group (Figure 3, P < 0.05). However, ES and RAPA treatment significantly mitigated this shortening vs that in the LPS group at both 12 and 24 hours (Figure 3, P < 0.05). There was no difference between the LPS and LPS + 3MA groups at both 12 hours and 24 hours (P > 0.05, Figure 3). These data robustly suggest that ES can inhibit intestinal damage induced by LPS.

Figure 3
Figure 3 Effects of esmolol on lipopolysaccharide-induced shortening of the interstitial villus (n = 6). A: The length of the interstitial villus in each group at 12 hours; B: The length of the interstitial villus in each group at 24 hours. Each bar presents the mean ± SE of the mean. n = 6 in each group. LPS: Lipopolysaccharide; ES: Esmolol; 3-MA: 3-methyladenine; RAPA: Rapamycin.
ES suppressed the levels of I-FABP, DAO, IL-1, and IL-6 and increased the levels of IL-10 in rats with LPS-induced sepsis

As markers of intestinal permeability, higher levels of I-FABP and DAO indicate more severe tissue damage. LPS and LPS + 3-MA considerably increased the production of I-FABP and DAO, in agreement with the observed pathologic changes in intestinal tissues in rats with sepsis, compared to the findings in the Sham group. Conversely, ES and RAPA significantly decreased the serum concentrations of I-FABP and DAO at all observed time points, indicating less severe injury (Figure 4A-D, P < 0.05). ES suppressed the levels of IL-1 and IL-6 and increased those of IL-10 in rats with LPS-induced sepsis at 12 hours (Figure 4E-G, P < 0.05).

Figure 4
Figure 4 Esmolol suppressed the levels of fatty acid-binding protein, diamine oxidase, interleukin-1, and interleukin-6 and increased the levels of interleukin-10 in rats with lipopolysaccharide-induced sepsis. A: Fatty acid-binding protein (I-FABP) concentration at 12 hours; B: I-FABP concentration at 24 hours; C: Diamine oxidase (DAO) concentration at 12 hours; D: DAO concentration at 24 hours; E: Interleukin (IL-1) level at 12 hours; F: IL-6 level at 12 hours; G: IL-10 level at 12 hours. Lipopolysaccharide (LPS) significantly increased the production of I-FABP and DAO, whereas esmolol (ES) and rapamycin markedly decreased their serum levels (A-D). LPS significantly increased the production of IL-1 and IL-6 and decreased that of IL-10 at 12 hours. ES suppressed the levels of IL-1 and IL-6 and increased those of IL-10 in rats with LPS-induced sepsis at 12 hours. n = 6 in each group. I-FABP: Fatty acid-binding protein; DAO: Diamine oxidase; ES: Esmolol; 3-MA: 3-methyladenine; IL: Interleukin; RAPA: Rapamycin.
LPS-induced sepsis contributed to changes in autophagy in the rat intestine

The initiation of autophagy in the LPS-induced sepsis model was detected at different timepoints, and the data revealed that the effects of LPS on autophagy changed overtime. An increase in LC3-II expression was observed in LPS-treated animals at 3 hours compared to that at baseline (P < 0.05). Notably, from 12 hours to 24 hours, there were significant decreases in the expression of LC3-II and Beclin-1 compared to the baseline levels (Figure 5, P < 0.05).

Figure 5
Figure 5 Effects of lipopolysaccharide on autophagy changed with time in rats with sepsis. A: The expression of Beclin-1 and LC3-II in the intestine was detected by western blotting; B: Quantitative analysis of Beclin-1 protein expression at different time points; C: Quantitative analysis of LC3-II protein expression at different time points. Lipopolysaccharide (10 mg/kg) induced LC3-II at 3 hours (A and C) but inhibited Beclin-1 and LC3-II at 12 hours and 24 hours (A, B, and C). Data in the Sham group were set to 1. n = 6 in each group.
Effects of ES on the LPS-induced downregulation of LC3-II, Beclin-1, p-AMPK, and p-ULK1 and upregulation of p-mTOR

In the sepsis model induced by the ip injection of LPS (10 mg/kg), the AMPK/mTOR/ULK1 autophagy pathway was markedly suppressed. We observed decreased Beclin-1, LC3-II (Figure 6A-F), p-AMPK, p-ULK1 expression and increased p-mTOR expression (Figure 6G-N) following LPS treatment. Treatment with ES for 12 or 24 hours effectively suppressed the LPS-induced decreases in Beclin-1, LC3-II (Figure 6A-F), p-AMPK and p-ULK1 expression and increase in p-mTOR expression (Figure 6G-N). These results demonstrated that ES significantly reversed the LPS-induced inhibition of autophagy. Similar outcomes were obtained with RAPA treatment at 12 and 24 hours, as it increased the expression of Beclin-1, LC3-II (Figure 6A-F), p-AMPK, and p-ULK1 and decreased that of p-mTOR (Figure 6G-N).

Figure 6
Figure 6 Western blotting was performed for Beclin-1, LC3-II, p-AMPK, p-ULK1, and p-mTOR in intestinal tissue homogenates in the Sham, lipopolysaccharide, lipopolysaccharide + esmolol, lipopolysaccharide + 3-methyladenine, and lipopolysaccharide + rapamycin treatment groups (after 12 and 24 hours of treatment). A-C: The 12-hour protein levels of Beclin-1, LC3-II and β-Actin were determined by western blotting of each group; D-F: The 24-hour protein levels of Beclin-1, LC3-II and β-Actin were determined by western blotting of each group; G-J: The 12-hour protein levels of p-AMPK, p-ULK1, p-mTOR and β-Actin were determined by western blotting of each group; K-N: The 24-hour protein levels of p-AMPK, p-ULK1,p-mTOR and β-Actin were determined by western blotting of each group.β-actin was used as a loading control and for normalization. Representative immunoblots and densitometry analyses are presented. Data in the Sham group were set to 1. n = 6 in each group. 3-MA: 3-methyladenine; ES: Esmolol; RAPA: Rapamycin; LPS: Lipopolysaccharide.

The autophagy inhibitor 3-MA had no effect on LPS-induced autophagy. However, treatment with 3-MA for 12 hours slightly reversed the LPS-induced decrease in p-ULK1 expression (Figure 6G and H), and increase in p-mTOR expression (Figure 6B and I). Additionally, treatment with 3-MA for 24 hours reversed the LPS-induced decrease in p-AMPK expression (Figure 6K and L).

ES increased the formation of autophagosomes in rats with LPS-induced sepsis

The number of autophagosomes was clearly lower in the LPS group than in the Sham group (Figure 7). Meanwhile, the number of autophagosomes was restored by ES and RAPA treatment, but it was not influenced by 3-MA.

Figure 7
Figure 7 Evaluation of autophagosome counts in the Sham, lipopolysaccharide, lipopolysaccharide + esmolol, lipopolysaccharide + rapamycin, and lipopolysaccharide + 3-methyladenine groups. A: Transmission electron microscopy was used to observe the number of autophagosomes formed in the intestinal tissue after the induction of sepsis. Blue and orange arrows denote autophagosomes. The autophagosome indicated by the orange arrow is magnified in the right panel. Magnification: × 125000; inset: × 500000; B: Autophagosome count based on transmission electron microscopy. Scale bar: 500 nm. n = 3 in each group. 3-MA: 3-methyladenine; ES: Esmolol; RAPA: Rapamycin; LPS: Lipopolysaccharide.
ES increased viability and autophagy in LPS-treated IEC-6 cells

To assess the impact of LPS on cell autophagy, IEC-6 cells were exposed to LPS at various concentrations (0, 1, 5, 10, 50, and 100 μg/mL) for 6 hours. The results demonstrated that LPS restrained cell autophagy in a concentration-dependent manner (Figure 8A-C). Furthermore, IEC-6 cells were treated with or without 100 μg/mL LPS for various intervals (0, 0.5, 1, 2, 6, 12, and 24 hours). As depicted in Figure 8D-F, LPS suppressed cell autophagy in a time-dependent manner.

Figure 8
Figure 8 Esmolol significantly improves cell viability and induces autophagy in IEC-6 cells following lipopolysaccharide treatment. A-C: The effect of different concentrations of lipopolysaccharide on the expression of autophagy markers Beclin-1 and LC3-II in IEC-6 cell; D-F: The effect of both different duration of lipopolysaccharide on the expression of autophagy markers Beclin-1 and LC3-II induced by lipopolysaccharide (LPS) in IEC-6 cells; G-I: The effect of different concentrations of ES on the expression of autophagy markers Beclin-1 and LC3-II in LPS-stimulated IEC-6 cells; J: Esmolol (1 × 10-4 g/mL) significantly increases cell viability in LPS-treated IEC-6 cells. n = 6 in each group. LPS: Lipopolysaccharide; ES: Esmolol.

To evaluate the protective effect of ES on IEC-6 cells, cells were pretreated with ES (1 × 10-5, 1 × 10-4, and 1 × 10-3 g/mL) for 30 minutes and then incubated with LPS (100 μg/mL) for 6 hours. ES treatment increased the expression of Beclin-1 and LC3-II in LPS-treated cells (Figure 8G-I). Meanwhile, ES (1 × 10-4 g/mL) increased the viability of LPS-treated IEC-6 cells; thus, the optimal ES concentration was 1 × 10-4 g/mL (Figure 8J).

Effects of ES on the LPS-induced decreases in LC3-II, Beclin-1, p-AMPK, and p-ULK1 expression and increase in p-mTOR expression in vitro

In IEC-6 cell cells treated with LPS (100 μg/mL), the AMPK/mTOR/ULK1 autophagy pathway was significantly suppressed. We observed decreased Beclin-1 and LC3-II expression and increased p-mTOR expression (Figure 9A-D). Treatment with ES for 6 hours completely suppressed the LPS-induced decreases in Beclin-1, LC3-II and increase in p-mTOR expression (Figure 9A-D). ES also completely suppressed the LPS-induced decreases p-AMPK and p-ULK1 (Figure 9E-G). These results revealed that ES markedly reversed the LPS-induced inhibition of autophagy. Similarly, RAPA increased the expression of Beclin-1, LC3-II, p-AMPK and p-ULK1 expression and decreased p-mTOR expression. The autophagy inhibitor 3-MA had no effect on LPS-induced autophagy. However, the LPS-induced increases in p-AMPK and p-ULK1 expression were slightly reversed by treatment with 3-MA (Figure 9E-G).

Figure 9
Figure 9 Western blot analysis was performed for Beclin-1, LC3-II, p-AMPK, p-ULK1, and p-mTOR in IEC-6 cells in the Sham, lipopolysaccharide, lipopolysaccharide + esmolol, lipopolysaccharide + 3-methyladenine, and lipopolysaccharide + rapamycin treatment groups (after 6 hours of treatment). A-D: Protein levels of Beclin-1, LC3-II, p-mTOR and β-Actin were determined by western blotting. The representative blots of Beclin-1, LC3-II, p-AMPK, p-ULK1, and p-mTOR and β-actin after treatment. β-actin was used as loading control and for normalization; E-G: Protein levels of p-AMPK, p-ULK1 and βActin were determined by western blotting. Representative immunoblots and densitometry analyses are presented. Data in the Sham group were set to 1 n = 6 in each group. 3-MA: 3-methyladenine; ES: Esmolol; RAPA: Rapamycin; LPS: Lipopolysaccharide.
Characterization of ES-treated sepsis via RNA sequencing Analyses

To further characterize the molecular targets of ES, transcriptome sequencing was performed using IEC-6cells from the Sham, LPS, and LPS + ES groups. Figure 10A displays a heatmap of the clustering analysis of differentially expressed mRNAs between the Sham, LPS, and LPS + ES groups. Figure 10B illustrates the identification of 495 DEG sin the LPS group relative to the Sham group. Among these, 304 genes were upregulated, and 191 were downregulated. By contrast, the LPS + ES group featured 36 DEGs vs the LPS group, including 19 upregulated and 17downregulated genes (Figure 10C). KEEG functional enrichment analysis (Figure 10D) revealed that, relative to the LPS group, the DEGs in the LPS + ES group were primarily associated with the TNF, IL-17, and NF-κB signaling pathways. GO functional enrichment analysis between the LPS and LPS + ES groups identified that ES primarily affected cytokine activity, response to chemical, positive regulation of inflammatory response, signaling receptor binding, and autophagy (Figure 10E).

Figure 10
Figure 10  Esmolol regulates the expression of genes related to sepsis. A: Heatmap for hierarchical cluster analysis of differentially expressed genes (DEGs) between the groups. Red denotes high expression, and blue denotes low expression; B: Volcano plots of upregulated and downregulated DEGs between the lipopolysaccharide (LPS) and Sham groups; C: Volcano plots of upregulated and downregulated DEGs between the LPS + esmolol and LPS groups; D: Kyoto Encyclopedia of Genes and Genomes functional enrichment analysis; E: Gene Ontology functional enrichment analysis. n = 3 in each group. ES: Esmolol; LPS: Lipopolysaccharide.
ES modulates inflammatory factors via AMPK in vitro

ELISA was employed to assess alterations in inflammatory factors within IEC-6 cells 6 hours after drug administration in each group. The findings revealed that, in comparison to the Sham group, the LPS and LPS + CC groups exhibited marked elevation of IL-1, IL-6, TNF-α, IL-17, and IL-10 expression. Following ES treatment, the pro-inflammatory cytokines IL-1, IL-6, TNF-α, and IL-17 were suppressed, whereas the anti-inflammatory cytokineIL-10 was upregulated. When comparing the LPS + ES + CC and LPS + ES groups, the former exhibited upregulation of the pro-inflammatory cytokines IL-1, IL-6, TNF-α, and IL-17 alongside downregulation of the anti-inflammatory cytokine IL-10 (Figure 11).

Figure 11
Figure 11  Enzyme-linked immunosorbent assay was employed to assess alterations in inflammatory factors within IEC-6 cells after 6 hours of treatment in each group. A: Interleukin (IL)-1 level at 6 hours; B: IL-6 level at 6 hours; C: IL-10 level at 6 hours; D: Tumor necrosis factor-α level at 6 hours; E: IL-17 level at 6 hours. ES: Esmolol; LPS: Lipopolysaccharide; IL: Interleukin; TNF-α: Tumor necrosis factor-α; CC: Compound C.
ES ameliorates LPS-induced intestinal injury via the AMPK/mTOR/ULK1 autophagy pathway

To investigate the role of the AMPK/mTOR/ULK1 autophagy pathway in the protective effects of ES on LPS-induced intestinal injury, IEC-6 cells were challenged with LPS to mimic the in vitro environment. The protective effect of ES was neutralized by the AMPK inhibitor CC (Figure 12A; P < 0.05). Compared with the findings in the Sham group, cell viability was decreased in the LPS and LPS + CC groups after 6h of stimulation. After 6 hours of ES treatment, cell viability was increased significantly. However, the combination of LPS + ES + CC reversed this trend.

Figure 12
Figure 12  Esmolol ameliorates lipopolysaccharide-induced intestinal injury via the AMPK/mTOR/ULK1 autophagy pathway. A: The MTT assay was used to detect cell viability; B-D: Western blotting analysis was performed to analyze Beclin-1, LC3-II expression in IEC-6 cells in the Sham, lipopolysaccharide (LPS), LPS + esmolol (ES), LPS + Compound C (CC), and LPS + ES + CC treatment groups (after 6 hours of treatment); E-H: Western blotting analysis was performed to analyze p-AMPK, p-ULK1, and p-mTOR expression in IEC-6 cells after 6 hours of treatment in each group. β-actin was used as a loading control and for normalization. Representative immunoblots and densitometry analyses are presented. Data in the Sham group were set to 1. n = 6 in each group. ES: Esmolol; CC: Compound C; LPS: Lipopolysaccharide.

Western blotting revealed that the expression of Beclin-1 and activated LC3-II was reduced in the LPS group (Figure 12B-D), suggesting impaired autophagy during LPS-induced intestinal injury. Additionally, downregulation in p-AMPK and p-ULK1, along with upregulation in p-mTOR, was observed (Figure 12E-H), indicating inactivation of the AMPK/mTOR/ULK1 signaling pathway. ES pretreatment significantly restored the impairment of autophagy and the AMPK/mTOR/ULK1 pathway after stimulation at 6 hours with LPS. However, the ameliorating effect of ES was counteracted by CC (Figure 12).

DISCUSSION

The findings of this study demonstrated that ES administration reduced intestinal damage in rats and cells with LPS-induced sepsis, and the effects of ES were associated with the regulation of inflammatory factors and activation of autophagy via modulation of the AMPK/mTOR/ULK1 signaling pathway. The potential molecular mechanism of these effects is presented in Figure 13. Based on the current data, ES could be used as a therapeutic drug to improve intestinal function in patients with sepsis. High morbidity and mortality rates are common in sepsis[20], and the gastrointestinal tract is one of the organs most severely affected by sepsis[21]. The disorder can induce a chain of events that lead to intestinal mucosa degradation, which makes it easier for pathogens and poisons to enter the bloodstream[22,23]. Thus, protecting the mucosal lining of the gastrointestinal tract is a critical goal in both the prevention and management of sepsis[24,25].

Figure 13
Figure 13  Possible molecular mechanism by which esmolol influences signaling crosstalk in lipopolysaccharide-induced intestinal injury. Esmolol, as a selective β1-adrenoceptor antagonist, alleviates intestinal injury in rats with sepsis by augmenting autophagy, downregulating interleukin (IL-1), IL-17, TNF-α, and IL-6, and upregulating IL-10 through the AMPK/mTOR/ULK1 pathway. IL: Interleukin; LPS: Lipopolysaccharide.

Nevertheless, there is little clinical evidence to support the use of ES to treat intestinal damage caused by sepsis, and it is not considered a frontline treatment[5]. The effects of ES on sepsis have long been debated. For instance, some studies indicated that heart rate could be safely controlled by selective β1-blockers during septic shock[26]. However, the mechanism by which ES promotes intestinal epithelial cell survival in sepsis is unknown.

Under physiological conditions, only trace levels of I-FABP are present in the bloodstream[27]. Thus, circulating I-FABP has been suggested as a biomarker of intestinal permeability[28], and it may represent translocation from the gut into the circulation. Another intracellular enzyme found in the intestinal epithelium is DAO. These two enzymes can readily cross the intestinal mucosa and enter the peripheral circulation when intestinal permeability becomes abnormally high because of mucosal disruption[28]. In the current study, we found higher serum levels of DAO and I-FABP in the LPS group than in the Sham group, suggesting increased permeability in LPS-treated rats. However, ES significantly lowered the serum concentrations of DAO and I-FABP, implying that ES attenuated the intestinal damage induced by LPS. Meanwhile, ES suppressed the levels of IL-1 and IL-6 and increased those of IL-10 in rats with LPS-induced sepsis. The complex relationship between intestinal function and autophagy is highlighted by a substantial body of evidence. Autophagy plays a significant role in several aspects of intestinal physiology, including metabolic regulation, preservation of the intestinal epithelial structure, functioning of specialized intestinal epithelial groups, control of inflammatory pathways, and prevention of infection[29]. Reduced resistance to enteric infections might arise from intestinal epithelial cells lacking autophagy genes[30-32]. Previous research demonstrated an increase in autophagic activity during the hyperdynamic phase of sepsis, which is subsequently followed by a decrease in autophagy[33]. The expression of marker proteins associated with autophagy, including LC3 and Beclin-1, increased for up to 8 hours after CLP cecal ligation puncture and then returned to baseline by 12 hours[34], and ghrelin activated autophagy in intestinal epithelial cells, protecting the small intestinal epithelium against sepsis-related damage[34]. In another study of the mechanism underlying the effects of sodium tanshinone IIA sulfonate against LPS-induced small intestinal injuries, the recovery of the intestine was associated with the suppression of inflammatory factors and amplification of autophagy[35]. An improved survival rate was also observed in septic animals administered an autophagy inhibitor following endotoxemia[36]. However, the effects of autophagy modulation can yield different outcomes at different intervals. In the current study, we recorded a surge in LC3-II expression 3 hours post-LPS exposure, whereas Beclin-1 and LC3-II expressions were suppressed at 12 and 24 hours, which indicates a time-dependent control of LPS-induced autophagy.

Transcriptomics is the scientific study of transcripts and their expression levels in cells or tissues. Using RNA sequencing, we extensively characterized gene expression profiles and functional changes following ES administration in IEC-6 cells. KEGG and GO functional enrichment analyses revealed that ES primarily affects the TNF, IL-17, and NF-κB signaling pathways; cytokine activity; response to chemical; positive regulation of inflammatory response; signaling receptor binding; and autophagy. During sepsis, immune cells release a large amount of inflammatory mediators such as IL-1β, IL-2, IL-6, TNF-α, chemokines, and cytokines. The pro-inflammatory cytokines IL-1β, IL-6, and HMGB1 mediate the inflammatory response of immune cells and promote systemic inflammatory response syndrome[37]. IL-10 can downregulate pro-inflammatory cytokines and increase the survival rate of mice with sepsis[38]. In a sepsis rat model, it was found that intestinal barrier function can be protected by suppressing pro-inflammatory mediators, including IL-1β, IL-6, IL-17, and TNF-α, or promoting the secretion of anti-inflammatory mediators including IL-10[39,40]. IL-17, produced by Th17 cells, is a pro-inflammatory mediator with a key role in modulating immune and inflammatory pathways[41]. IL-17 interacts with the IL-17 receptor, triggering downstream NF-κB and MAPK signaling pathways, thus promoting the release of additional pro-inflammatory cytokines, including IL-6 and IL-8[42]. Our study indicated that these cytokines (IL-6, IL-1, TNF-α, IL-17, and IL-10) were significantly upregulated in IEC-6 cells with LPS-induced sepsis. Post-ES treatment, IL-6, IL-1, TNF-α, and IL-17 were markedly suppressed, and IL-10 was significantly upregulated. These results suggested that ES alleviates sepsis-induced inflammatory infections by modulating the levels of these inflammatory cytokines. CC treatment reversed these changes, resulting in an increase in pro-inflammatory factor levels and a decrease in anti-inflammatory factor levels. Therefore, ES might regulate inflammatory factors by activating AMPK.

To further explain the pathways involved in the anti-sepsis function of ES, the current study explored the AMPK/mTOR/ULK1 pathway. Recent research revealed that puerarin protects against cerebral ischemia/reperfusion injury by modifying the AMPK/mTOR/ULK1 signaling pathway in rats[43]. In this pathway, the mTOR complex can be controlled by AMPK[44], and it contributes to the activation of ULK1[15]. However, whether this signaling transduction is involved in the effects of ES on LPS-induced intestinal injuries has not been documented. Our data illustrated that both ES and RAPA (an autophagy activator) entirely reversed LPS-induced changes in the expression of LC3-II, Beclin-1, p-AMPK, p-ULK1, and p-mTOR, along with an increased number of autophagosomes in the intestinal tissues of the sepsis model, ascertaining the involvement of AMPK/mTOR/ULK1-mediated autophagy in the effects of ES. In contrast, 3-MA (an autophagy inhibitor)[29] showed no impact on LPS-induced autophagy. The protective effect of ES was neutralized by the AMPK inhibitor. CC was found to reverse the induction of autophagy and AMPK/ULK1/mTOR pathway activity by ES.

CONCLUSION

Collectively, the findings outlined in the current study highlight that ES can alleviate intestinal injury in rats and cells with sepsis by augmenting autophagy, regulating inflammatory factors, and the effects were associated with the AMPK/mTOR/ULK1 signaling pathway. However, the data in the current study only revealed a relationship between the function of ES and changes in this pathway. Whether the activation of AMPK/mTOR/ULK1 signaling is indispensable for the anti-sepsis effects of ES requires more comprehensive validation, which will provide valuable information for the future treatment of septic intestinal damage.

ACKNOWLEDGEMENTS

We are highly appreciative of Professor Guo-Xing Zhang for sponsoring reagents and equipment.

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Footnotes

Peer review: Externally peer reviewed.

Peer-review model: Single blind

Specialty type: Gastroenterology and hepatology

Country of origin: China

Peer-review report’s classification

Scientific quality: Grade B, Grade B, Grade B

Novelty: Grade B, Grade B, Grade C

Creativity or innovation: Grade B, Grade B, Grade C

Scientific significance: Grade B, Grade B, Grade B

P-Reviewer: Biswas MS, PhD, Associate Professor, Postdoc, Bangladesh; Xu B, MD, PhD, Director, Head, Professor, China S-Editor: Qu XL L-Editor: A P-Editor: Wang WB

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