Sepsis-induced Intestinal Barrier Dysfunction Research Articles

Intestinal Piezo1 aggravates intestinal barrier dysfunction during sepsis by mediating Ca2+ influx

IntroductionIntestinal barrier dysfunction is a pivotal factor in sepsis progression. The mechanosensitive ion channel Piezo1 is associated with barrier function; however, its role in sepsis-induced intestinal barrier dysfunction remains poorly understood.MethodsThe application of cecal ligation and puncture (CLP) modeling was performed on both mice of the wild-type (WT) variety and those with Villin-Piezo1flox/flox genetic makeup to assess the barrier function using in vivo FITC-dextran permeability measurements and immunofluorescence microscopy analysis of tight junctions (TJs) and apoptosis levels. In vitro, Caco-2 monolayers were subjected to TNF-α incubation. Moreover, to modulate Piezo1 activation, GsMTx4 was applied to inhibit Piezo1 activation. The barrier function, intracellular calcium levels, and mitochondrial function were monitored using calcium imaging and immunofluorescence techniques.ResultsIn the intestinal tissues of CLP-induced septic mice, Piezo1 protein levels were notably elevated compared with those in normal mice. Piezo1 has been implicated in the sepsis-mediated disruption of TJs, apoptosis of intestinal epithelial cells, elevated intestinal mucosal permeability, and systemic inflammation in WT mice, whereas these effects were absent in Villin-Piezo1flox/flox CLP mice. In Caco-2 cells, TNF-α prompted calcium influx, an effect reversed by GsMTx4 treatment. Elevated calcium concentrations are correlated with increased accumulation of reactive oxygen species, diminished mitochondrial membrane potential, and TJ disruption.ConclusionsThus, Piezo1 is a potential contributor to sepsis-induced intestinal barrier dysfunction, influencing apoptosis and TJ modification through calcium influx-mediated mitochondrial dysfunction.

Nano Parthenolide Improves Intestinal Barrier Function of Sepsis by Inhibiting Apoptosis and ROS via 5-HTR2A.

Intestinal barrier dysfunction is an important complication of sepsis, while the treatment is limited. Recently, parthenolide (PTL) has attracted much attention as a strategy of sepsis, but whether nano parthenolide (Nano PTL) is therapeutic in sepsis-induced intestinal barrier dysfunction is obscured. In this study, cecal ligation and puncture (CLP)-induced sepsis rats and lipopolysaccharide (LPS)-stimulated intestinal epithelial cells (IECs) were used to investigate the effect of PTL on intestinal barrier dysfunction. Meanwhile, we synthesized Nano PTL and compared the protective effect of Nano PTL with ordinary PTL on intestinal barrier function in septic rats and IECs. Network pharmacology and serotonin 2A (5-HTR2A) inhibitor were used to explore the mechanism of PTL on the intestinal barrier function of sepsis. The encapsulation rate of Nano PTL was 95±1.5%, the drug loading rate was 11±0.5%, and the average uptake rate of intestinal epithelial cells was 94%. Ordinary PTL and Nano PTL improved the survival rate and survival time of septic rats, reduced the mean arterial pressure and the serum level of inflammatory cytokines, and protected the liver and kidney functions in vivo, and increased the value of transmembrane resistance (TEER) reduced the reactive oxygen species (ROS) and apoptosis in IECs in vitro through 5-HTR2A. Nano PTL had better effect than ordinary PTL. Ordinary PTL and Nano PTL can protect the intestinal barrier function of septic rats by inhibiting apoptosis and ROS through up-regulating 5-HTR2A, Nano PTL is better than ordinary PTL.

The Polo-Like Kinase 1–Mammalian Target of Rapamycin Axis Regulates Autophagy to Prevent Intestinal Barrier Dysfunction During Sepsis

The intestines play a crucial role in the development of sepsis. The balance between autophagy and apoptosis in intestinal epithelial cells is dynamic and determines intestinal permeability. The present study focused on the potential role of autophagy in sepsis-induced intestinal barrier dysfunction and explored the mechanisms invivo and invitro. Excessive apoptosis in intestinal epithelia and a disrupted intestinal barrier were observed in septic mice. Promoting autophagy with rapamycin reduced intestinal epithelial apoptosis and restored intestinal barrier function, presenting as decreased serum diamine oxidase (DAO) and fluorescein isothiocyanate-dextran 40 (FD40) levels and increased expression of zonula occludens-1 (ZO-1) and Occludin. Polo-like kinase 1 (PLK1) knockdown in mice ameliorated intestinal epithelial apoptosis and the intestinal barrier during sepsis, whereas these effects were reduced with chloroquine and enhanced with rapamycin. PLK1 also promoted cell autophagy and improved lipopolysaccharide-induced apoptosis and high permeability invitro. Moreover, PLK1 physically interacted with mammalian target of rapamycin (mTOR) and participated in reciprocal regulatory crosstalk in intestinal epithelial cells during sepsis. This study provides novel insight into the role of autophagy in sepsis-induced intestinal barrier dysfunction and indicates that the PLK1-mTOR axis may be a promising therapeutic target for sepsis.

PLK1 protects intestinal barrier function during sepsis by targeting mitochondrial dynamics through TANK-NF-κB signalling

BackgroundIntestinal barrier integrity in the pathogenesis of sepsis is critical. Despite an abundance of evidence, the molecular mechanism of the intestinal barrier in sepsis pathology remains unclear. Here, we report a protective role of polo-like kinase 1 (PLK1) in intestinal barrier integrity during sepsis.MethodsMice with PLK1 overexpression (CAG-PLK1 mice) or PLK1 inhibition (BI2536-treated mice) underwent caecal ligation and puncture (CLP) to establish a sepsis model. The intestinal barrier function, apoptosis in the intestinal epithelium, mitochondrial function and NF-κB signalling activity were evaluated. To suppress the activation of NF-κB signalling, the NF-κB inhibitor PDTC, was administered. The Caco-2 cell line was chosen to establish an intestinal epithelial injury model in vitro.ResultsSepsis destroyed intestinal barrier function, induced excessive apoptosis in the intestinal epithelium, and disrupted the balance of mitochondrial dynamics in wild-type mice. PLK1 overexpression alleviated sepsis-induced damage to the intestinal epithelium by inhibiting the activation of NF-κB signalling. PLK1 colocalized and interacted with TANK in Caco-2 cells. Transfecting Caco-2 cells with TANK-SiRNA suppressed NF-κB signalling and ameliorated mitochondrial dysfunction, apoptosis and the high permeability of cells induced by lipopolysaccharide (LPS). Furthermore, TANK overexpression impaired the protective effect of PLK1 on LPS-induced injuries in Caco-2 cells.ConclusionOur findings reveal that the PLK1/TANK/NF-κB axis plays a crucial role in sepsis-induced intestinal barrier dysfunction by regulating mitochondrial dynamics and apoptosis in the intestinal epithelium and might be a potential therapeutic target in the clinic.

Toll-like receptor 4 deficiency alleviates lipopolysaccharide-induced intestinal barrier dysfunction

BackgroundThe intestinal tract is considered the body’s “engine” and the most impacted organ during sepsis. In this study, we explored toll-like receptor 4 (TLR4) functions in sepsis-induced intestinal barrier dysfunction. MethodsWild-type and TLR4-knockout (KO) mice were used to establish a sepsis-induced dysfunctional intestinal barrier model via the intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg). Hematoxylin and eosin staining, Transmission electron microscope, enzyme linked immunosorbent assay, western blot, quantitative real-time polymerase chain reaction, TdT-mediated dUTP nick end labeling staining, 16 S rRNA gene sequencing were used to explore differences in inflammatory cytokines, apoptosis, tight junction (TJ) protein expression, and intestinal flora diversity between groups. ResultsTLR4-deficiency reduced procalcitonin and C-reactive protein to prevent sepsis, and also inhibited inflammatory response by decreasing interleukin (IL)− 1β, IL-6 and tumor necrosis factor-α levels. Also, BAX/Bcl2 and cleaved-caspase 3 expressions were decreased in TLR4-KO mice to suppress the intestinal mucosal cell apoptosis. TJ proteins, including zonula occludens protein, Occludin and Claudin-5 were significantly increased and intestinal fatty acid binding protein, myosin light chain and myosin light chain kinase were reduced in TLR4-KO mice. Additionally, 16 S rRNA gene sequencing indicated that TLR4-deficiency improved flora diversity and altered normal and abnormal bacterial proportions. ConclusionsTLR4 deficiency alleviated LPS-induced intestinal barrier dysfunction by reducing inflammatory responses and apoptosis, impairing intestinal damage, and regulating intestinal flora disturbance.

Neutrophil extracellular traps impair intestinal barrier functions in sepsis by regulating TLR9-mediated endoplasmic reticulum stress pathway

Increased neutrophil extracellular traps (NETs) formation has been found to be associated with intestinal inflammation, and it has been reported that NETs may drive the progression of gut dysregulation in sepsis. However, the biological function and regulation of NETs in sepsis-induced intestinal barrier dysfunction are not yet fully understood. First, we found that both circulating biomarkers of NETs and local NETs infiltration in the intestine were significantly increased and had positive correlations with markers of enterocyte injury in abdominal sepsis patients. Moreover, the levels of local citrullinated histone 3 (Cit H3) expression were associated with the levels of BIP expression. To further confirm the role of NETs in sepsis-induced intestinal injury, we compared peptidylarginine deiminase 4 (PAD4)-deficient mice and wild-type (WT) mice in a lethal septic shock model. In WT mice, the Cit H3-DNA complex was markedly increased, and elevated intestinal inflammation and endoplasmic reticulum (ER) stress activation were also found. Furthermore, PAD4 deficiency alleviated intestinal barrier disruption and decreased ER stress activation. Notably, NETs treatment induced intestinal epithelial monolayer barrier disruption and ER stress activation in a dose-dependent manner in vitro, and ER stress inhibition markedly attenuated intestinal apoptosis and tight junction injury. Finally, TLR9 antagonist administration significantly abrogated NETs-induced intestinal epithelial cell death through ER stress inhibition. Our results indicated that NETs could contribute to sepsis-induced intestinal barrier dysfunction by promoting inflammation and apoptosis. Suppression of the TLR9–ER stress signaling pathway can ameliorate NETs-induced intestinal epithelial cell death.

Mesenchymal stem cell-derived microvesicles improve intestinal barrier function by restoring mitochondrial dynamic balance in sepsis rats

BackgroundSepsis is a major cause of death in ICU, and intestinal barrier dysfunction is its important complication, while the treatment is limited. Recently, mesenchymal stem cell-derived microvesicles (MMVs) attract much attention as a strategy of cell-free treatment; whether MMVs are therapeutic in sepsis induced-intestinal barrier dysfunction is obscure.MethodsIn this study, cecal ligation and puncture-induced sepsis rats and lipopolysaccharide-stimulated intestinal epithelial cells to investigate the effect of MMVs on intestinal barrier dysfunction. MMVs were harvested from mesenchymal stem cells and were injected into sepsis rats, and the intestinal barrier function was measured. Afterward, MMVs were incubated with intestinal epithelial cells, and the effect of MMVs on mitochondrial dynamic balance was measured. Then the expression of mfn1, mfn2, OPA1, and PGC-1α in MMVs were measured by western blot. By upregulation and downregulation of mfn2 and PGC-1α, the role of MMVs in mitochondrial dynamic balance was investigated. Finally, the role of MMV-carried mitochondria in mitochondrial dynamic balance was investigated.ResultsMMVs restored the intestinal barrier function by improving mitochondrial dynamic balance and metabolism of mitochondria. Further study revealed that MMVs delivered mfn2 and PGC-1α to intestinal epithelial cells, and promoted mitochondrial fusion and biogenesis, thereby improving mitochondrial dynamic balance. Furthermore, MMVs delivered functional mitochondria to intestinal epithelial cells and enhanced energy metabolism directly.ConclusionMMVs can deliver mfn2, PGC-1α, and functional mitochondria to intestinal epithelial cells, synergistically improve mitochondrial dynamic balance of target cells after sepsis, and restore the mitochondrial function and intestinal barrier function. The study illustrated that MMVs might be a promising strategy for the treatment of sepsis.

Protective effect of ethyl pyruvate on gut barrier function through regulations of ROS-related NETs formation during sepsis

BackgroundSepsis impairs the function of the intestinal barrier through neutrophil extracellular traps (NETs). Reactive oxygen species (ROS)-induced activation of mitogen-activated protein kinase (MAPK) is involved in NET formation. Ethyl pyruvate (EP), a potent and effective ROS scavenger, ameliorates sepsis-associated intestinal barrier dysfunction, but the detailed mechanism is unknown. The current study aimed to explore the eff ;ects of EP on sepsis-induced intestinal barrier dysfunction and whether ROS and NETs were involved. MethodsA sepsis model was induced in mice by intraperitoneal injection of LPS (10 mg/kg). The mice were divided into 4 groups: (1) sham group; (2) LPS group; (3) DNase-1 + LPS group; and (4) EP + LPS group. EP or DNase-1 was intraperitoneally injected after the LPS model was established. After 24 h, the small intestine and blood were collected for analysis. Human neutrophils were harvested and incubated with phorbol-12-myristate-13-acetate (PMA) or PMA + EP, and ROS and NET generation was measured. ResultsEP significantly decreased proinflammatory cytokines and MPO-DNA in the LPS model. In addition, EP suppressed NET formation in the intestines of endotoxemic mice. The decrease in NETs induced by EP or DNase-1 alleviated histopathological damage, intestinal cell apoptosis and increased tight junction expression. In vitro, the treatment of EP abolished PMA-induced ROS production and NET formation which could be reversed by H2O2 treatment. Meanwhile, EP also abolished MAPK ERK1/2 and p38 activation during PMA-induced NET formation. ConclusionThis study was the first to demonstrate that EP alleviated NET formation and sepsis-induced intestinal damage through blockage of ROS mediated MAPK ERK1/2 and p38 activation.

Protective Role of Somatostatin in Sepsis-Induced Intestinal Barrier Dysfunction through Inhibiting the Activation of NF-κB Pathway.

Somatostatin (SST) has a protective role in intestinal injury, inflammatory response, and intestinal mucosal barrier in rats with acute pancreatitis. However, its function in sepsis-induced intestinal barrier dysfunction remains largely unknown. A mouse sepsis model was constructed, and SST was injected into the tail vein. Then, hematoxylin and eosin staining (HE) was used to detect the intestinal barrier dysfunction. Enzyme-linked immunosorbent assay was used to detect the level of tumor necrosis factor α- (TNF-) α, interleukin- (IL-) 6, and interleukin- (IL-) 10 in the ileum. Expressions of tight junction proteins, zonula occludens- (ZO-) 1 and Claudin-1, and NF-κB p65 in the ileum were detected using western blot and immunohistochemistry as needed. Furthermore, JSH-23 as an inhibitor of the NF-κB pathway was injected into sepsis mice with SST or not. Mice with sepsis showed an obvious intestinal barrier dysfunction with decreasing specific somatostatin receptor subtype (SSTRs), and increasing TNF-α, IL-6, and IL-10 in the ileum. SST could relieve the injury, the decrease of SSTRs, and the increase of TNF-α and IL-6 induced by sepsis and also further enhanced the expression of IL-10. Further analysis showed that ZO-1 and Claudin-1 were reduced in the ileum by sepsis but enhanced by SST. NF-κB p65 was promoted in the ileum by sepsis but inhibited by SST. Further experiments confirmed that NF-κB inhibitor JSH-23 could repair the intestinal barrier dysfunction and enhance the protective effect of SST on the intestinal barrier. SST, with a protective effect on intestinal barrier dysfunction through suppression of NF-κB, could be a potential therapeutic drug for sepsis-induced intestinal barrier dysfunction.

Cathelicidin preserves intestinal barrier function in polymicrobial sepsis

ObjectivesThe intestinal epithelium compartmentalizes the sterile bloodstream and the commensal bacteria in the gut. Accumulating evidence suggests that this barrier is impaired in sepsis, aggravating systemic inflammation. Previous studies reported that cathelicidin is differentially expressed in various tissues in sepsis. However, its role in sepsis-induced intestinal barrier dysfunction has not been investigated.DesignTo examine the role of cathelicidin in polymicrobial sepsis, cathelicidin wild-(Cnlp+/+) and knockout (Cnlp−/−) mice underwent cecal-ligation and puncture (CLP) followed by the assessment of septic mortality and morbidity as well as histological, biochemical, immunological, and transcriptomic analyses in the ileal tissues. We also evaluated the prophylactic and therapeutic efficacies of vitamin D3 (an inducer of endogenous cathelicidin) in the CLP-induced murine polymicrobial sepsis model.ResultsThe ileal expression of cathelicidin was increased by three-fold after CLP, peaking at 4 h. Knockout of Cnlp significantly increased 7-day mortality and was associated with a higher murine sepsis score. Alcian-blue staining revealed a reduced number of mucin-positive goblet cells, accompanied by reduced mucin expression. Increased number of apoptotic cells and cleavage of caspase-3 were observed. Cnlp deletion increased intestinal permeability to 4kD fluorescein-labeled dextran and reduced the expression of tight junction proteins claudin-1 and occludin. Notably, circulating bacterial DNA load increased more than two-fold. Transcriptome analysis revealed upregulation of cytokine/inflammatory pathway. Depletion of Cnlp induced more M1 macrophages and neutrophils compared with the wild-type mice after CLP. Mice pre-treated with cholecalciferol (an inactive form of vitamin D3) or treated with 1alpha, 25-dihydroxyvitamin D3 (an active form of VD3) had decreased 7-day mortality and significantly less severe symptoms. Intriguingly, the administration of cholecalciferol after CLP led to worsened 7-day mortality and the associated symptoms.ConclusionsEndogenous cathelicidin promotes intestinal barrier integrity accompanied by modulating the infiltration of neutrophils and macrophages in polymicrobial sepsis. Our data suggested that 1alpha, 25-dihydroxyvitamin D3 but not cholecalciferol is a potential therapeutic agent for treating sepsis.

PLK1 protects against sepsis-induced intestinal barrier dysfunction

Sepsis and sepsis-associated intestinal barrier dysfunction are common in intensive care units, with high mortality. The aim of this study is to investigate whether Polo-like kinase 1 (PLK1) ameliorates sepsis-induced intestinal barrier dysfunction in the intestinal epithelium. The mouse intestinal barrier was disrupted after Lipopolysaccharide (LPS) injection due to intestinal epithelial cell apoptosis and proliferation inhibition, accompanied by decreased PLK1. In HT-29 intestinal epithelial cells, LPS stimulation induced cell apoptosis and inhibited cell proliferation. Overexpression of PLK1 partly rescued the apoptosis and proliferation inhibition in HT29 cells caused by LPS. Finally, LPS stimulation promoted the reduction of PLK1, resulting in apoptosis and proliferation inhibition in intestinal epithelial cells, disrupting the intestinal epithelial barrier. These findings indicate that PLK1 might be a potential therapeutic target for the treatment of sepsis-induced intestinal barrier dysfunction.

Systemic blockade of P2X7 receptor protects against sepsis-induced intestinal barrier disruption

Sepsis, during which the intestinal epithelial barrier is frequently disrupted, remains a challenging and life-threatening problem in clinical practice. The P2X7 receptor (P2X7R) is a non-selective adenosine triphosphate-gated cation channel present in macrophages that is involved in inflammatory responses. However, little is known about the role of P2X7R in macrophages during sepsis-induced intestinal barrier disruption. In this study, mice were treated with the P2X7R antagonist A740003 or the agonist BzATP by intra-peritoneal injection after the induction of gut-origin sepsis. The survival rates, inflammatory responses, intestinal barrier integrity, macrophage marker expression, and ERK and NF-κB activities were evaluated. Intestinal macrophages were also isolated and studied after exposure to Brilliant Blue G or BzATP. We found that a systemic P2X7R blockade downregulated sepsis-induced inflammatory responses and attenuated intestinal barrier dysfunction based on the evidence that mice in the A740003-treated group exhibited alleviated pro-inflammatory cytokine synthesis, intestinal hyperpermeability, epithelial apoptosis rates and tight junction damage compared with the septic mice. These changes were partly mediated by the inhibition of M1 macrophages activation via ERK/NF-κB pathways. Our data presented herein show that a P2X7R blockade could be a potential therapeutic target for the treatment of sepsis-induced intestinal barrier dysfunction.

A Novel Role for Programmed Cell Death Receptor Ligand-1 (PD-L1) in Sepsis-Induced Intestinal Dysfunction.

Studies imply that intestinal barrier dysfunction is a key contributor to morbid events associated with sepsis. Recently, co-inhibitory molecule, programmed death-ligand1 (PD-L1) has been shown to be involved in the regulation of intestinal immune tolerance and/or inflammation. Our previous studies showed that PD-L1 gene deficiency reduced sepsis-induced intestinal injury morphologically. However, it isn't known how PD-L1 expression impacts intestinal barrier dysfunction during sepsis. Here we tested the hypothesis that PD-L1 expressed on intestinal epithelial cells (IECs) has a role in sepsis-induced intestinal barrier dysfunction. To address this, C57BL/6 or PD-L1 gene knockout mice were subjected to experimental sepsis and PD-L1 expression, intestinal permeability, tissue cytokine levels were assessed. Subsequently, septic or non-septic patient colonic samples (assigned by pathology report) were immunohistochemically stained for PD-L1 I a blinded fashion. Finally, human Caco2 cells were used for in vitro studies. The results demonstrated that PD-L1 was constitutively expressed and sepsis significantly up-regulates PD-L1 in IECs from C57BL/6 mice. Concurrently, we observed an increased PD-L1 expression in colon tissue samples from septic patients. PD-L1 gene deficiency reduced ileal permeability, tissue levels of IL-6, TNF-α and MCP-1, and prevented ileal tight junction protein loss compared to WT after sepsis. Comparatively, while Caco2 cell monolayers responded to inflammatory cytokine stimulation also with elevated PD-L1 expression, increased monolayer permeability and altering/decreasing monolayer tight junction protein morphology/expression; these changes were reversed by PD-L1 blocking antibody. Together these data indicate that ligation of ICE PD-L1 plays a novel role in mediating the pathophysiology of sepsis-induced intestinal barrier dysfunction.

TNF is a key mediator in sepsis‐induced intestinal barrier dysfunction but is independent of enterocyte NFκB

Inhibition of enterocyte NFκB augments sepsis‐induced intestinal hyperpermeability, gut apoptosis, and TNF expression. We hypothesized that TNF is a key mediator of gut barrier dysfunction and apoptosis in sepsis.Mice lacking enterocyte IKKβ (resultant NFκB inhibition) were created via Cre/lox recombination under control of the villin promoter. Wildtype (WT) and Vil‐Cre/IkkβF/Ä (IKKβ−/−) mice were injected 3 hr pre‐operatively with monoclonal anti‐TNF antibody or isotype control followed by cecal ligation and puncture (n=5–12/group). At 24 hr, intestinal permeability was assessed in vivo using FD4. Apoptosis was evaluated in crypts by active caspase‐3 staining. Blood was harvested for bacterial cultures.Anti‐TNF decreased permeability in septic WT (1832±321 vs. 4678±421 ng/ml; p<0.001) and septic IKKβ −/− mice (2528±446 vs. 6650±951 ng/ml; p<0.01). Sepsis‐induced apoptosis was decreased in both anti‐TNF treated septic WT (13±1 vs. 4±2; p<0.05) and septic IKKβ −/− mice (20±2 vs. 4±1; p<0.001). Anti‐TNF decreased bacteremia in septic WT (4×103±2×103 vs. 4×105±3×105 CFU/ml; p<0.05) and septic IKKβ −/− mice (500±500 vs. 3×106±2×106 CFU/ml; p<0.01). There were no differences between anti‐TNF treated groups for any parameter.TNF appears to be a key mediator in sepsis‐induced intestinal barrier dysfunction. However, this mechanism is independent of enterocyte NFκB. NIH GM66202 & GM082008.