Proteomics analysis of human intestinal organoids during hypoxia and reoxygenation as a model to study ischemia-reperfusion injury

Intestinal ischemia-reperfusion (IR) injury is associated with high mortality rates, which have not improved in the past decades despite advanced insight in its pathophysiology using in vivo animal and human models. The inability to translate previous findings to effective therapies emphasizes the need for a physiologically relevant in vitro model to thoroughly investigate mechanisms of IR-induced epithelial injury and test potential therapies. In this study, we demonstrate the use of human small intestinal organoids to model IR injury by exposing organoids to hypoxia and reoxygenation (HR). A mass-spectrometry-based proteomics approach was applied to characterize organoid differentiation and decipher protein dynamics and molecular mechanisms of IR injury in crypt-like and villus-like human intestinal organoids. We showed successful separation of organoids exhibiting a crypt-like proliferative phenotype, and organoids exhibiting a villus-like phenotype, enriched for enterocytes and goblet cells. Functional enrichment analysis of significantly changing proteins during HR revealed that processes related to mitochondrial metabolism and organization, other metabolic processes, and the immune response were altered in both organoid phenotypes. Changes in protein metabolism, as well as mitophagy pathway and protection against oxidative stress were more pronounced in crypt-like organoids, whereas cellular stress and cell death associated protein changes were more pronounced in villus-like organoids. Profile analysis highlighted several interesting proteins showing a consistent temporal profile during HR in organoids from different origin, such as NDRG1, SDF4 or DMBT1. This study demonstrates that the HR response in human intestinal organoids recapitulates properties of the in vivo IR response. Our findings provide a framework for further investigations to elucidate underlying mechanisms of IR injury in crypt and/or villus separately, and a model to test therapeutics to prevent IR injury.Subject terms: Mechanisms of disease, Proteomics     

Anti-inflammatory and tight junction protective activity of the herbal preparation STW 5-II on mouse intestinal organoids

BackgroundIrritable bowel syndrome (IBS) is a functional bowel disorder, in which recurrent abdominal pain is associated with defecation or a change in bowel habits. STW 5-II is a combination of six medicinal herbs with a clinically proven efficacy in managing IBS.AimThis study aims to establish an in vitro IBS model using mouse intestinal organoids and to explore the anti-inflammatory and tight junction protective activities of the multi-herbal preparation STW 5-II.MethodsIntestinal organoids were cultured in 1:1 Matrigel™ and medium domes. Inflammation and tight junction disruption were induced by a cocktail of cytokines (TNFα, IFNγ, IL-1β, IL-6) and bacterial proteins (LPS, flagellin). Organoids were treated with different concentrations of STW 5-II, and its multi-target activity was assessed using microarray analyses, RT-qPCR, immunofluorescence, western blot, immunohistochemistry, and a FITC permeability assay. In addition, we analyzed the expression of pNF-κB, pSTAT1, iNOS and ZO-1. In silico analyses were conducted to predict and identify the active components that may be responsible in mediating the multi-target anti-inflammatory activity of STW 5-II.ResultsAn organoid based IBS model was successfully established. STW 5-II effectively reduced the cytokines-induced overexpression of the pro-inflammatory mediators pNF-κB, pSTAT1 and iNOS. Moreover, STW 5-II attenuated cytokine-mediated downregulation of the tight junction protein, ZO-1. This finding was confirmed by a FITC permeability assay. In silico analyses revealed a promising inhibitory activity of some isolated compounds from STW 5-II against NF-κB, STAT1 and iNOS.ConclusionSTW 5-II possesses multiple anti-inflammatory as well as tight junction protective activities that could explain its clinically proven efficacy in managing IBS symptoms.     

Rotavirus alters paracellular permeability and energy metabolism in Caco-2 cells

Rotaviruses infect epithelial cells of the small intestine, but the pathophysiology of the resulting severe diarrhea is incompletely understood. Histological damage to intestinal epithelium is not a consistent feature, and in vitro studies showed that intestinal cells did not undergo rapid death and lysis during viral replication. We show that rotavirus infection of Caco-2 cells caused disruption of tight junctions and loss of transepithelial resistance (TER) in the absence of cell death. TER declined from 300 to 22 Omega. cm(2) between 8 and 24 h after infection and was accompanied by increased transepithelial permeability to macromolecules of 478 and 4,000 Da. Distribution of tight junction proteins claudin-1, occludin, and ZO-1 was significantly altered during infection. Claudin-1 redistribution was notably apparent at the onset of the decline in TER. Infection was associated with increased production of lactate, decreased mitochondrial oxygen consumption, and reduced cellular ATP (60% of control at 24 h after infection), conditions known to reduce the integrity of epithelial tight junctions. In conclusion, these data show that rotavirus infection of Caco-2 intestinal cells altered tight junction structure and function, which may be a response to metabolic dysfunction.     

In-Depth Comparison of Matrigel Dissolving Methods on Proteomic Profiling of Organoids

Patient-derived organoids recently emerged as promising ex vivo 3D culture models recapitulating histological and molecular characteristics of original tissues, thus proteomic profiling of organoids could be valuable for function investigation and clinical translation. However, organoids are usually cultured in murine Matrigel (served as scaffolds and matrix), which brings an issue to separate organoids from Matrigel. Because of the complex compositions of Matrigel and thousands of identical peptides shared between Matrigel and organoids, insufficiently dissolved Matrigel could influence proteomic analysis of organoids in multiple ways. Thus, how to dissolve Matrigel matrix and recovery organoid cells efficiently is vital for sample preparation. Here, we comprehensively compared three popular Matrigel dissolving methods (cell recovery solution, dispase, and PBS–EDTA buffer) and investigated the effect of undissolved Matrigel proteins on proteomic profiles of organoids. By integrative analysis of label-free proteomes of Matrigel and stable isotope labeling by amino acids in cell culture proteomes of organoids collected by three methods, respectively, we found that dispase showed an optimal efficiency, with the highest peptide yield and the highest incorporation ratio of stable isotope labeling by amino acids in cell culture labels (97.1%), as well as with the least potential Matrigel contaminants. To help analysis of proteomic profiles of organoids collected by the other two methods, we identified 312 high-confidence Matrigel contaminants, which could be filtered out to attenuate Matrigel interference with minimal loss of biological information. Together, our study identifies bioinformatics and experimental approaches to eliminate interference of Matrigel contaminants efficiently, which will be valuable for basic and translational proteomic research using organoid models.     

Harnessing cerebral organoids for Alzheimer's disease research

Alzheimer's disease (AD) is a devastating neurodegenerative disorder affecting the aging population. Despite many studies, there remains an urgent need to identify the root causes of AD, together with potential treatments. Cerebral organoid technology has made it possible to model human neurophysiology and disease with increasing accuracy in patient-derived tissue cultures. Here, we review the most recent advances in modeling AD in organoids and other engineered three-dimensional cell culture systems. Early studies demonstrated that familial AD patient-derived organoids robustly develop disease pathology. Ongoing work has expanded this focus to investigate the genetic and environmental causes of late-onset sporadic AD and harness organoids for high-throughput drug screens. Future organoid models will need to incorporate additional cell types and tissues implicated in disease pathogenesis, including microglia and vasculature. We anticipate the continuation of this rapid progress in developing cerebral organoid technology toward facilitating our understanding of and informing treatment strategies for AD.     

Myosin light chain kinase mediates intestinal barrier disruption following burn injury

Background

Severe burn injury results in the loss of intestinal barrier function, however, the underlying mechanism remains unclear. Myosin light chain (MLC) phosphorylation mediated by MLC kinase (MLCK) is critical to the pathophysiological regulation of intestinal barrier function. We hypothesized that the MLCK-dependent MLC phosphorylation mediates the regulation of intestinal barrier function following burn injury, and that MLCK inhibition attenuates the burn-induced intestinal barrier disfunction.

Methodology/Principal Findings

Male balb/c mice were assigned randomly to either sham burn (control) or 30% total body surface area (TBSA) full thickness burn without or with intraperitoneal injection of ML-9 (2 mg/kg), an MLCK inhibitor. In vivo intestinal permeability to fluorescein isothiocyanate (FITC)-dextran was measured. Intestinal mucosa injury was assessed histologically. Tight junction proteins ZO-1, occludin and claudin-1 was analyzed by immunofluorescent assay. Expression of MLCK and phosphorylated MLC in ileal mucosa was assessed by Western blot. Intestinal permeability was increased significantly after burn injury, which was accompanied by mucosa injury, tight junction protein alterations, and increase of both MLCK and MLC phosphorylation. Treatment with ML-9 attenuated the burn-caused increase of intestinal permeability, mucosa injury, tight junction protein alterations, and decreased MLC phosphorylation, but not MLCK expression.

Conclusions/Significance

The MLCK-dependent MLC phosphorylation mediates intestinal epithelial barrier dysfunction after severe burn injury. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury.     

Evaluation of the radiation response and regenerative effects of mesenchymal stem cell-conditioned medium in an intestinal organoid system

Intestinal organoids have recently emerged as an in vitro model relevant to the gut system owing to their recapitulation of the native intestinal epithelium with crypt–villus architecture. However, it is unclear whether intestinal organoids reflect the physiology of the in vivo stress response. Here, we systemically investigated the radiation response in organoids and animal models using mesenchymal stem cell-conditioned medium (MSC-CM), which contains secreted paracrine factors. Irradiated organoids exhibited sequential induction of viability loss and regrowth after irradiation (within 12 days), similar to the response of the native intestinal epithelium. Notably, treatment with MSC-CM facilitated the reproliferation of intestinal stem cells (ISCs) and restoration of damaged crypt-villus structures in both models. Furthermore, Wnt/Notch signaling pathways were commonly upregulated by MSC-CM, but not radiation, and pharmacologically selective inhibition of Wnt or Notch signaling attenuated the enhanced recovery of irradiated organoids, with increases in ISCs, following MSC-CM treatment. Interestingly, the expression of Wnt4, Wnt7a, and active β-catenin was increased, but not notch family members, in MSC-CM-treated organoid after irradiation. Treatment of recombinant mouse Wnt4 and Wnt7a after irradiation improved to some extent intestinal epithelial regeneration both in vitro and in vivo. Overall, these results suggested that intestinal organoids recapitulated the physiological stress response of the intestinal epithelium in vivo. Thus, our findings provided important insights into the physiology of intestinal organoids and may contribute to the development of strategies to enhance the functional maturation of engineered organoids.     

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.     

Dissolved oxygen concentration regulates human hepatic organoid formation from pluripotent stem cells in a fully controlled bioreactor

Developing technologies for scalable production of human organoids has gained increased attention for “organoid medicine” and drug discovery. We developed a scalable and integrated differentiation process for generation of hepatic organoid from human pluripotent stem cells (hPSCs) in a fully controlled stirred tank bioreactor with 150 ml working volume by application of physiological oxygen concentrations in different liver tissue zones. We found that the 20–40% dissolved oxygen concentration [DO] (corresponded to 30–60 mmHg pO₂ within the liver tissue) significantly influences the process outcome via regulating the differentiation fate of hPSC aggregates by enhancing mesoderm induction. Regulation of the [DO] at 30% DO resulted in efficient generation of human fetal-like hepatic organoids that had a uniform size distribution and were comprised of red blood cells and functional hepatocytes, which exhibited improved liver-specific marker gene expressions, key liver metabolic functions, and, more important, higher inducible cytochrome P450 activity compared to the other trials. These hepatic organoids were successfully engrafted in an acute liver injury mouse model and produced albumin after implantation. These results demonstrated the significant impact of the dissolved oxygen concentration on hPSC hepatic differentiation fate and differentiation efficacy that should be considered ascritical translational aspect of established scalable liver organoid generation protocols for potential clinical and drug discovery applications.     

氧化应激对肠屏障功能障碍发病的影响

机体累积过量的活性氧自由基所导致的氧化应激是多种肠道疾病发生的共同病理生理基础。肠上皮细胞间的紧密连接是维持肠屏障功能的重要结构基础之一。近年来研究表明,氧化应激能通过多种途径破坏肠上皮细胞间的紧密连接,导致肠上皮屏障功能障碍。本文对蛋白激酶C、丝裂原活化蛋白激酶、蛋白质的修饰以及缺氧诱导因子-1(HIF-1)在肠屏障功能障碍中的作用机制进行简要概述,旨在为肠屏障功能障碍的治疗和预后提供新的思路。     

An internal region of the peroxisomal membrane protein PMP47 is essential for sorting to peroxisomes

Targeting sequences on peroxisomal membrane proteins have not yet been identified. We have attempted to find such a sequence within PMP47, a protein of the methylotrophic yeast, Candida boidinii. This protein of 423 amino acids shows sequence similarity with proteins in the family of mitochondrial carrier proteins. As such, it is predicted to have six membrane-spanning domains. Protease susceptibility experiments are consistent with a six-membrane-spanning model for PMP47, although the topology for the peroxisomal protein is inverted compared with the mitochondrial carrier proteins. PMP47 contains two potential peroxisomal targeting sequences (PTS1), an internal SKL (residues 320- 322) and a carboxy terminal AKE (residues 421-423). Using a heterologous in vivo sorting system, we show that efficient sorting occurs in the absence of both sequences. Analysis of PMP47- dihydrofolate reductase (DHFR) fusion proteins revealed that amino acids 1-199 of PMP47, which contain the first three putative membrane spans, do not contain the necessary targeting information, whereas a fusion with amino acids 1-267, which contains five spans, is fully competent for sorting to peroxisomes. Similarly, a DHFR fusion construct containing residues 268-423 did not target to peroxisomes while residues 203-420 appeared to sort to that organelle, albeit at lower efficiency than the 1-267 construct. However, DHFR constructs containing only amino acids 185-267 or 203-267 of PMP47 were not found to be associated with peroxisomes. We conclude that amino acids 199-267 are necessary for peroxisomal targeting, although additional sequences may be required for efficient sorting to, or retention by, the organelles.     

小鼠肠道类器官三种不同条件培养基的比较研究

摘要 目的：比较三种不同条件培养基对小鼠类器官形态和增殖速度的影响。方法：取C57BL/6小鼠的小肠和结肠,EDTA法分离隐窝,以基质胶包埋,加入不同小鼠肠道类器官培养基培养7 天,使用光学显微镜记录和比较类器官形成率和出芽情况。随后进行二代类器官培养,使用TrypLE将类器官消化为单细胞,重新包埋和培养,使用光学显微镜记录和比较不同类器官培养基对二代类器官的培养效率。采用荧光定量PCR比较不同条件培养类器官中干细胞标志物Lgr5和分化标志物MUC2的表达。使用免疫荧光法检测类器官中ki-67的表达。结果：对于小肠类器官的培养,使用条件培养基1、IntestiCult条件培养基和L-WRN培养基培养结肠类器官的形成率分别为（18.2±4.5） %、（63.8±4.0） %和（82.1±8.4） %。其中使用IntestiCult条件培养基培养类器官的出芽率更高。对于结肠类器官的培养,使用条件培养基1、IntestiCult条件培养基和L-WRN培养基培养结肠类器官的形成率分别为（17.3±7.3） %、（58.0±6.1） %和（46.3±7.4） %。对于二代类器官的培养,IntestiCult条件培养基和L-WRN培养基都能够支持消化为单细胞后的二代类器官培养。干细胞标志物Lgr5和分化细胞（杯状细胞）标志物MUC2的表达无明显差异。使用L-WRN培养基的类器官ki-67阳性比例更高,增殖速度更快。结论：本研究比较了三种不同条件培养基对小鼠类器官形态和增殖速度的影响。经过对比,L-WRN培养基更有利于小鼠肠道类器官的形成和增殖速度。     

The science and engineering of stem cell-derived organoids-examples from hepatic,biliary, and pancreatic tissues

The field of organoid engineering promises to revolutionize medicine with wide-ranging applications of scientific, engineering, and clinical interest, including precision and personalized medicine, gene editing, drug development, disease modelling, cellular therapy, and human development. Organoids are a three-dimensional (3D) miniature representation of a target organ, are initiated with stem/progenitor cells, and are extremely promising tools with which to model organ function. The biological basis for organoids is that they foster stem cell self-renewal, differentiation, and self-organization, recapitulating 3D tissue structure or function better than two-dimensional (2D) systems. In this review, we first discuss the importance of epithelial organs and the general properties of epithelial cells to provide a context and rationale for organoids of the liver, pancreas, and gall bladder. Next, we develop a general framework to understand self-organization, tissue hierarchy, and organoid cultivation. For each of these areas, we provide a historical context, and review a wide range of both biological and mathematical perspectives that enhance understanding of organoids. Next, we review existing techniques and progress in hepatobiliary and pancreatic organoid engineering. To do this, we review organoids from primary tissues, cell lines, and stem cells, and introduce engineering studies when applicable. We discuss non-invasive assessment of organoids, which can reveal the underlying biological mechanisms and enable improved assays for growth, metabolism, and function. Applications of organoids in cell therapy are also discussed. Taken together, we establish a broad scientific foundation for organoids and provide an in-depth review of hepatic, biliary and pancreatic organoids.     

Interleukin-2 receptor beta subunit-dependent and -independent regulation of intestinal epithelial tight junctions

Interleukin (IL)-15 is able to regulate tight junction formation in intestinal epithelial cells. However, the mechanisms that regulate the intestinal barrier function in response to IL-15 and the involved subunits of the IL-15 ligand-receptor system are unknown. We determined the IL-2Rbeta subunit and IL-15-dependent regulation of tight junction-associated proteins in the human intestinal epithelial cell line T-84. The IL-2Rbeta subunit was expressed and induced signal transduction in caveolin enriched rafts in intestinal epithelial cells. IL-15-mediated tightening of intestinal epithelial monolayers correlated with the enhanced recruitment of tight junction proteins into Triton X-100-insoluble protein fractions. IL-15-mediated up-regulation of ZO-1 and ZO-2 expression was independent of the IL-2Rbeta subunit, whereas the phosphorylation of occludin and enhanced membrane association of claudin-1 and claudin-2 by IL-15 required the presence of the IL-2Rbeta subunit. Recruitment of claudins and hyperphosphorylated occludin into tight junctions resulted in a more marked induction of tight junction formation in intestinal epithelial cells than the up-regulation of ZO-1 and ZO-2 by itself. The regulation of the intestinal epithelial barrier function by IL-15 involves IL-2Rbeta-dependent and -independent signaling pathways leading to the recruitment of claudins, hyperphosphorylated occludin, ZO-1, and ZO-2 into the tight junctional protein complex.     

基于小分子筛选的嗅上皮类器官培养体系的建立

嗅上皮接收和传导气味信号是嗅觉系统的重要组成部分。嗅上皮的损伤在通常情况下可自发恢复,但特定疾病或衰老造成的嗅上皮损伤会引起嗅觉功能减退和嗅觉障碍。嗅上皮主要由基底细胞、支持细胞以及嗅感觉神经元组成。为了在体外建立包含多种细胞类型的嗅上皮类器官,本研究采用3D细胞培养技术,通过筛选小分子药物,构建了包含多种细胞类型的嗅上皮类器官模型,包含水平基底样细胞、球形基底样细胞、支持样细胞和嗅感觉神经元样细胞多种细胞类型。类器官培养体系中多种生长因子和小分子化合物在细胞增殖速度、细胞组成以及不同细胞类型标志基因的表达水平等方面对类器官产生影响。Wnt信号通路激活剂CHIR-99021能够提高嗅上皮类器官的成克隆率和增殖速度且有利于提高嗅上皮类器官中嗅感觉神经元样细胞标志基因的表达水平;培养体系的任一因子均能提高类器官中cKit阳性的球形基底样细胞克隆比例;表皮生长因子(epidermal growth factor,EGF)和维生素C均有利于类器官中水平基底样细胞标志基因的表达。本研究建立的嗅上皮类器官系统模拟了嗅上皮干细胞分化产生多种嗅上皮细胞类型的过程,为研究嗅上皮组织损伤再生、嗅觉障碍病理...     

A three-dimensional tissue culture model for the study of attach and efface lesion formation by enteropathogenic and enterohaemorrhagic Escherichia coli

We sought to develop a practical and representative model to study the interactions of enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) with human intestinal tissue. For this purpose, human intestinal epithelial HCT-8 cells were cultured under low-shear microgravity conditions in a rotating cell culture system. After 10 days, layered cell aggregates, or 'organoids', developed. Three lines of evidence indicated that these organoids exhibited traits characteristic of normal tissue. First, the organoids expressed normal intestinal tissue markers in patterns that suggested greater cellular differentiation in the organoids than conventionally grown monolayers. Second, the organoids produced higher levels of intestinally expressed disaccharidases and alkaline phosphatase on a cell basis than did conventionally cultured monolayers. Third, HCT-8 organoid tissue developed microvilli and desmosomes characteristic of normal tissue, as revealed by electron microscopy. Because the low-shear microgravity condition is proposed by modelling studies to more closely approximate conditions in the intestinal microvilli, we also tested the impact of microgravity of bacterial growth and virulence gene expression. No influence on growth rates was observed but intimin expression by EHEC was elevated during culture in microgravity as compared with normal gravity. That the responses of HCT-8 organoids to infection with wild-type EPEC or EHEC under microgravitational conditions approximated infection of normal tissue was demonstrated by the classical appearance of the resultant attaching and effacing lesions. We concluded that the low shear microgravity environment promoted growth of intestinal cell organoids with greater differentiation than was seen in HCT-8 cells maintained in conventional tissue culture and provided a reduced gravity environment for study of bacterial-host cell interactions.     

Gruffi: an algorithm for computational removal of stressed cells from brain organoid transcriptomic datasets

Organoids enable in vitro modeling of complex developmental processes and disease pathologies. Like most 3D cultures, organoids lack sufficient oxygen supply and therefore experience cellular stress. These negative effects are particularly prominent in complex models, such as brain organoids, and can affect lineage commitment. Here, we analyze brain organoid and fetal single‐cell RNA sequencing (scRNAseq) data from published and new datasets, totaling about 190,000 cells. We identify a unique stress signature in the data from all organoid samples, but not in fetal samples. We demonstrate that cell stress is limited to a defined subpopulation of cells that is unique to organoids and does not affect neuronal specification or maturation. We have developed a computational algorithm, Gruffi, which uses granular functional filtering to identify and remove stressed cells from any organoid scRNAseq dataset in an unbiased manner. We validated our method using six additional datasets from different organoid protocols and early brains, and show its usefulness to other organoid systems including retinal organoids. Our data show that the adverse effects of cell stress can be corrected by bioinformatic analysis for improved delineation of developmental trajectories and resemblance to in vivo data.     

Metformin protects against intestinal barrier dysfunction via AMPKα1‐dependent inhibition of JNK signalling activation

Disruption of the intestinal epithelial barrier, that involves the activation of C‐Jun N‐terminal kinase (JNK), contributes to initiate and accelerate inflammation in inflammatory bowel disease. Metformin has unexpected beneficial effects other than glucose‐lowering effects. Here, we provided evidence that metformin can protect against intestinal barrier dysfunction in colitis. We showed that metformin alleviated dextran sodium sulphate (DSS)‐induced decreases in transepithelial electrical resistance, FITC‐dextran hyperpermeability, loss of the tight junction (TJ) proteins occludin and ZO‐1 and bacterial translocation in Caco‐2 cell monolayers or in colitis mice models. Metformin also improved TJ proteins expression in ulcerative colitis patients with type 2 diabetes mellitus. We found that metformin ameliorated the induction of colitis and reduced the levels of pro‐inflammatory cytokines IL‐6, TNF‐a and IL‐1β. In addition, metformin suppressed DSS‐induced JNK activation, an effect dependent on AMP‐activated protein kinase α1 (AMPKα1) activation. Consistent with this finding, metformin could not maintain the barrier function of AMPKα1‐silenced cell monolayers after DSS administration. These findings highlight metformin protects against intestinal barrier dysfunction. The potential mechanism may involve in the inhibition of JNK activation via an AMPKα1‐dependent signalling pathway.