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Inhibition of PFKFB Preserves Intestinal Barrier Function in Sepsis by Inhibiting NLRP3/GSDMD.

ABSTRACT: Intestinal barrier dysfunction is associated with the occurrence and development of sepsis. Further, aerobic glycolysis plays an essential role in inflammation and cell death. This study is aimed at investigating the protective effect and mechanism of PFKFB3 inhibition on intestinal barrier dysfunction in sepsis mice. Sepsis mouse models were established by cecal ligation and puncture (CLP) in wild-type mice and Gsdmd^-/- mice. The results showed that the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in the small intestines was significantly upregulated in sepsis. 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), the specific inhibitor of PFKFB3, and Gsdmd gene knockout significantly inhibited the inflammatory response and cell death caused by sepsis, thus alleviating intestinal damage and barrier dysfunction. 3PO was also shown to significantly inhibit oxidative stress and NLRP3/caspase-1/GSDMD-dependent cell pyroptosis in the small intestines. The in vitro studies revealed that 3PO reduced NLRP3/caspase-1/GSDMD-dependent cell pyroptosis by inhibiting ROS. Taken together, our results suggest that PFKFB3 is involved in inflammation, oxidative stress, and pyroptosis during sepsis and enhances intestinal damage, which may provide important clues about the potential targets to be exploited in this highly lethal disease.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC9803577 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Inhibition of PFKFB Preserves Intestinal Barrier Function in Sepsis by Inhibiting NLRP3/GSDMD.

Zhang Yongsheng Y Liu Yukun Y Xie Zhenxing Z Liu Qinxin Q Zhuang Yangfan Y Xie Weiming W Wang Xiang X Gao Wei W Yang Fan F Li Zhanfei Z Bai Xiangjun X Wang Yuchang Y

Oxidative medicine and cellular longevity 20221223

Intestinal barrier dysfunction is associated with the occurrence and development of sepsis. Further, aerobic glycolysis plays an essential role in inflammation and cell death. This study is aimed at investigating the protective effect and mechanism of PFKFB3 inhibition on intestinal barrier dysfunction in sepsis mice. Sepsis mouse models were established by cecal ligation and puncture (CLP) in wild-type mice and Gsdmd<sup>-/-</sup> mice. The results showed that the expression of 6-phosphofructo- ...[more]

PMID: 36589684

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Project description:OBJECTIVES:The 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. DESIGN:To 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. RESULTS:The 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. CONCLUSIONS:Endogenous 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.

Project description:Intestinal barrier function defects and dysregulation of intestinal immune responses are two key contributory factors in the pathogenesis of ulcerative colitis (UC). Phenazine biosynthesis-like domain-containing protein (PBLD) was recently identified as a tumor suppressor in gastric cancer, hepatocellular carcinoma, and breast cancer; however, its role in UC remains unclear. Therefore, we analyzed colonic tissue samples from patients with UC and constructed specific intestinal epithelial PBLD-deficient (PBLDIEC-/-) mice to investigate the role of this protein in UC pathogenesis. We found that epithelial PBLD was decreased in patients with UC and was correlated with levels of tight junction (TJ) and inflammatory proteins. PBLDIEC-/- mice were more susceptible to dextran sulfate sodium (DSS)- and 2,4,6-trinitrobenzene sulfonic acid-induced colitis compared with wild-type (WT) mice. In DSS-induced colitis, PBLDIEC-/- mice had impaired intestinal barrier function and greater immune cell infiltration in colonic tissue than WT mice. Furthermore, TJ proteins were markedly reduced in PBLDIEC-/- mice compared with WT mice with colitis. Nuclear factor (NF)-κB activation was markedly elevated and resulted in higher expression levels of downstream effectors (C-C motif chemokine ligand 20, interleukin [IL]-1β, IL-6, and tumor necrosis factor [TNF]-α) in colonic epithelial cells isolated from PBLDIEC-/- mice than WT mice with colitis. PBLD overexpression in intestinal epithelial cells (IECs) consistently inhibited TNF-α/interferon-γ-induced intestinal barrier disruption and TNF-α-induced inflammatory responses via the suppression of NF-κB. In addition, IKK inhibition (IKK-16) rescued excessive inflammatory responses induced by TNF-α in PBLD knockdown FHC cells. Co-immunoprecipitation assays showed that PBLD may interact with IKKα and IKKβ, thus inhibiting NF-κB signaling, decreasing inflammatory mediator production, attenuating colonic inflammation, and improving intestinal barrier function. Modulating PBLD expression may provide a novel approach for treatment in patients with UC.

Project description:BackgroundIntestinal barrier dysfunction, which is associated with reactive enteric glia cells (EGCs), is not only a result of early sepsis but also a cause of multiple organ dysfunction syndrome. Inhibition of platelet activation has been proposed as a potential treatment for septic patients because of its efficacy in ameliorating the organ damage and barrier dysfunction. During platelet activation, CD40L is translocated from α granules to the platelet surface, serving as a biomarker of platelet activation a reliable predictor of sepsis prognosis. Given that more than 95% of the circulating CD40L originate from activated platelets, the present study aimed to investigate if inhibiting platelet activation mitigates intestinal barrier dysfunction is associated with suppressing reactive EGCs and its underlying mechanism.MethodsCecal ligation and puncture (CLP) was performed to establish the sepsis model. 24 h after CLP, the proportion of activated platelets, the level of sCD40L, the expression of tight-junction proteins, the intestinal barrier function and histological damage of septic mice were analyzed. In vitro, primary cultured EGCs were stimulated by CD40L and LPS for 24 h and EGCs-conditioned medium were collected for Caco-2 cells treatment. The expression of tight-junction proteins and transepithelial electrical resistance of Caco-2 cell were evaluated.ResultsIn vivo, inhibiting platelet activation with cilostazol mitigated the intestinal barrier dysfunction, increased the expression of ZO-1 and occludin and improved the survival rate of septic mice. The efficacy was associated with reduced CD40L+ platelets proportion, decreased sCD40L concentration, and suppressed the activation of EGCs. Comparable results were observed upon treatment with compound 6877002, a blocker of CD40L-CD40-TRAF6 signaling pathway. Also, S-nitrosoglutathione supplement reduced intestinal damage both in vivo and in vitro. In addition, CD40L increased release of TNF-α and IL-1β while suppressed the release of S-nitrosoglutathione from EGCs. These EGCs-conditioned medium reduced the expression of ZO-1 and occludin on Caco-2 cells and their transepithelial electrical resistance, which could be reversed by CD40-siRNA and TRAF6-siRNA transfection on EGCs.ConclusionsThe inhibition of platelet activation is related to the suppression of CD40L-CD40-TRAF6 signaling pathway and the reduction of EGCs activation, which promotes intestinal barrier function and survival in sepsis mice. These results might provide a potential therapeutic strategy and a promising target for sepsis.

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Inhibition of PFKFB Preserves Intestinal Barrier Function in Sepsis by Inhibiting NLRP3/GSDMD.

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Inhibition of PFKFB Preserves Intestinal Barrier Function in Sepsis by Inhibiting NLRP3/GSDMD.

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