Histochemical study on the intestine goblet cells in cichlid and poecilid species (Teleostei)

Histochemical properties of intestine goblet cells in firemouth cichlid, zebra mbuna, freshwater angelfish and platyfish are described. Goblet cells occurred regularly in the epithelial cell layer throughout the entire intestine, they were strongly coloured by alcian blue at pH 2.5. This colour got gradually weaker when the pH was reduced, but still after alcian blue at pH 0.2 these cells displayed a distinct blue colour. When the goblet cells were treated with periodic acid-Schiff (PAS), they displayed a strong purple-magenta colour. The findings that a number of goblet cells displayed various colours between blue and purple-magenta when acidic alcian blue was followed by PAS, and between blue and red-brown when acidic alcian blue was followed by neutral red, may reflect different ages or stages of development and differentiation for these cells. However, such results may also suggest a true cellular heterogeneity in the present population of goblet cells, reflecting that the intestine mucus layer has a number of roles in teleosts like lubrication, protection, immunological defence, digestion and absorption.In the ferritin injected specimens of firemouth cichlid and platyfish, a number of macrophage-like cells in intestine wall displayed Prussian blue precipitations in tissue treated with acid ferrocyanide, suggesting that these cells play a cleansing role in the intestinal wall. No ferritin uptake was seen in the intestine goblet cells and eosinophilic granule cells.     

Blood cleansing in heart atrium of <Emphasis Type="Italic">Trichogaster leerii</Emphasis> (Bleeker, 1852), Anabantidae: Teleostei

The capability and capacity of endocardial tissue in heart of Trichogaster leerii (Pearl gourami) to take up intraperitoneally injected horse-spleen ferritin and the regular content of indigestible wastes in this tissue are described. The entire heart wall was spongy composed of muscle trabeculae enveloped by flat endocardial cells. In atrium these cells were filled by Prussian blue precipitations in sections treated with acid ferrocyanide, when the period of time between ferritin-injection and sacrifice was 6 h or more. The endocardial cell layers enveloping the muscle trabeculae in ventricle or covering the wall and valves of the ventricular apertures nearly lacked such precipitations. Those endocardial cell layers capable to endocytose much foreign ferritin, also reacted strongly with Schmorl’s solution in control hearts, i.e. these atrial cells are normally rich in indigestible wastes, probably lipofuscin. We suggest that the endocytic and lipofuscin-rich endocardial cells in heart atrium may play a role in the blood clearance in T. leerii and that this cleansing in heart is restricted to atrium only in this species.     

On the structure of intestine and presence of endocytic cells in the lamina propria of platy and black tetra (Teleostei)

The structure of the intestine in platy (Xiphophorus maculatus) and black tetra (Gymnocorymbus ternetzi) and the capability of cells within the intestinal wall to endocytose intraperitoneally injected horse-spleen ferritin, are described. The intestinal epithelial layer has about the same thickness in both species, but the width of the lamina propria and tunica muscularis in black tetra was only about 1/5 of that in platy. Ferritin was taken up by numerous cells within the lamina propria throughout the entire length of the platy intestine. The uptake was demonstrated as large and strongly coloured intracellular Prussian blue granules in sections treated with acid ferrocyanide. There was no such uptake by the lamina propria in black tetras. We suggest that the high numbers of endocytic cells within the intestinal lamina propria of platies provide a local defence against foreign cells and particles. Such a functional role may to some extent compensate for the lack of an HCl-based defence in the digestive system of this stomach-less species.     

Uptake of foreign ferritin in platy Xiphophorus maculatus (Poeciliidae: Teleostei)

The ability and capacity of various tissues in platy Xiphophorus maculatus L. to take up horse-spleen ferritin injected into the blood stream are described. Ferritin was injected intraperioneally, and the cellular uptake was demonstrated as Prussian blue precipitations in tissues treated with acid ferrocyanide solutions. Ferritin was detected within the heart endocardial cells and macrophages in the trunk kidney and spleen, 1/4 h after the injection, i.e. foreign ferritin was taken up very rapidly by these cells. When the time elapsed between the ferritin injection and sacrifice exceeded 6 h, these cells, and also macrophages in the gill and intestine, were almost completely filled with ferritin. At these stages, however, the amounts of Prussian blue precipitations per volume unit of the tissue were much larger in the heart than in the other organs studied in the present work, i.e. the endocardial tissue seems to play an important role in the clearance of the blood circulation in this species. We suggest that this tissue in platy is specialized to endocytose waste and foreign macromolecules, including pathogenic particles, from the blood stream. The eosinophilic and neutrophilic granulocytes do not appear to take up foreign ferritin, i.e. these cells may play no endocytotic role in the clearance of foreign macromolecules in platy.     

Glucagon- and glicentin-immunoreactive cells in the human digestive tract

Summary The distribution and cellular location of substances reacting with anti-glucagon or anti-glicentin sera, i.e., glucagon-like and glicentin-like immunoreactivities, were studied in the human digestive tract using the immunofluorescence and immunoperoxidase methods. Both types of immunoreactivity were (1) absent in the antrum, (2) abundant in cells located at the periphery of pancreatic islets, (3) unevenly present in cells scattered in the epithelium of the small intestinal mucosa, the glicentin-immunoreactive cells being particularly abundant in the ileum. In the pancreas, and, when simultaneously present, in the intestine, both glucagon and glicentin immunoreactivities were located in the same cells.The precise ultrastructural location of each immunoreactivity was readily made using colloidal gold and ferritin tracers on ultrathin sections of glutaraldehyde-osmium fixed and epoxy resin-embedded tissues. In the pancreas, both glucagon and glicentin immunoreactivities were found in the granules of the A-type cells; the glucagon immunoreactivity was only present in the core of the granule, whereas the glicentin immunoreactivity was found either in the peripheral halo only, or throughout the entire granule. In the small intestine, both immunoreactivities were located inside the granules of the L-type cells.Quantitative specificity tests suggested that the glucagon- and the glicentin-like substances of the pancreas differ from those found in the intestine.Work supported by INSERM, A.T.P. number: 167539     

Partial characterization of eosinophilic granule cells (EGCs) and identification of mast cells of the intestinal lamina propria in rainbow trout (Oncorhynchus mykiss). Biochemical and cytochemical study

Summary— The role of intestinal eosinophilic granule cells (EGCs) is still a subject of discussion. The aim of this study was to obtain additional functional data for a better characterization of these cells. Biochemical studies indicated the presence of small amounts of histamine, a characteristic and consistent marker of mast cells, in the posterior gut. On the other hand, histamine is always absent from homogenates of isolated EGCs. Using colorimetric assays, we were able to show aryl sulphatase B activity (18.5 ± 3.7 nM nitrocatechol/10⁶ cells) and detected peroxidase (1.86 ± 0.03 ng/10⁶ cells) in EGC homogenates. A cytochemical study enabled us to localize peroxidase in the granules of EGCs. These cells can also phagocytose latex beads. EGCs should thus be considered as homologous with mammalian eosinophils and not with mast cells. The screening for cells in the mucosae containing chondroitin sulphate revealed sparsely represented cells in the loose connective tissue in immediate proximity to blood capillaries. These cells could be mucosal mast cells.     

Characteristics of mast cells in Chediak-Higashi mice: light and electron microscopic studies of connective tissue and mucosal mast cells

The beige mouse, a homologue of the Chediak-Higashi syndrome in man, possesses abnormally large granules in many tissue cells. The granules in the mucosal mast cells (MMC) of the small intestine of beige and littermate C57BL/6J mice were examined after infecting the mice with the intestinal parasite, Nippostrongylus brasiliensis. MMC in both beige and littermate mice had irregular granules which contained paracrystalline substructures embedded in an amorphous matrix. Granules were not observed in fusion with the cell membrane. Instead, in late-stage mast cells, the granule membrane broke down, the granule contents were spread throughout the cytoplasm, and the cell organelles disintegrated. Unlike connective tissue mast cells, MMC were poorly demonstrated with formalin fixation and toluidine blue staining.     

Degranulation of eosinophilic granule cells induced by capsaicin and substance P in the intestine of the rainbow trout (Oncorhynchus mykiss Walbaum)

Summary Adult rainbow trout (Oncorhynchus mykiss) were injected intraperitoneally with capsaicin, substance P, serotonin, or a control of saline vehicle or bovine serum albumin (0.5 g/g body weight). Fish were sacrificed 30 min and 1,2 and 4 h post-injection, the gut was dissected out, and a small section of the upper intestine was processed for electron microscopy. A significant proportion of eosinophilic granule cells (EGCs) of the intestine were in close association with non-myelinated neuronal bundles in all fish (4 fish per treatment and time period), but there was no significant difference between treatment or time, suggesting that the association was unaffected by these factors. Close examination of EGC ultrastructure showed that fish treated with capsaicin and substance P exhibited limited degranulation of the EGCs in the stratum compactum and extensive crinophagic-like degranulation in the lamina propria. Cells of the lamina propria contained characteristic multivesicular-like bodies. The degranulation was reminiscent of both mast cell degranulation and endocrine cell crinophagy. EGCs of fish treated with serotonin or a control were unaffected, suggesting that the serotoninergic neurons, believed to be involved in gut motility, were not responsible for degranulation. It is apparent that EGCs of the trout intestine may be under nervous control, as has been demonstrated previously for mammalian mast cells.     

Scanning electron microscopy of goldfish, Carassius auratus, intestinal mucosa

Scanning electron microscopy was used to examine the intestinal bulb and the caudal portion of the intestine proper of the goldfish, Carassius auratus . The observations made correlated well with previous light microscope studies by other investigators. The mucosal surface of the entire intestine of the goldfish is thrown up into folds, which are oriented along the long axis of the digestive tract in the intestinal bulb, but run more or less transversely in the caudal intestine. Tops of the folds were observed to be rounded or flattened, and the folds themselves formed wavy or zigzagging patterns in the mucosal surface. Numerous mucus-secreting goblet cells were seen, which were apparently more numerous or more active in the caudal intestine than in the intestinal bulb. The goblet cells are not uniformly distributed throughout the mucosa: they are more evident on the sides of the folds, although occasionally they appear to be located in clusters on the tips.
The goblet cells were observed to contain varying amounts of mucous material, and/or to be of different sizes. Although no histochemical tests were performed, the possibility that digestive enzymes known to be present in the intestine may be elaborated by the goblet cells was considered, based on their variable appearance.     

Enteric glia inhibit intestinal epithelial cell proliferation partly through a TGF-beta1-dependent pathway

Although recent studies have shown that enteric neurons control intestinal barrier function, the role of enteric glial cells (EGCs) in this control remains unknown. Therefore, our goal was to characterize the role of EGCs in the control of intestinal epithelial cell proliferation using an in vivo transgenic and an in vitro coculture model. Assessment of intestinal epithelial cell proliferation after ablation of EGCs in transgenic mice demonstrated a significant increase in crypt cell hyperplasia. Furthermore, mucosal glial network (assessed by immunohistochemical detection of S-100beta) is altered in colon adenocarcinoma compared with control tissue. In an in vitro coculture model of subconfluent Caco-2 cells seeded onto Transwell filters with EGCs, Caco-2 cell density and [3H]thymidine incorporation were significantly lower than in control (Caco-2 cultured alone). Flow cytometry analysis showed that EGCs had no effect on Caco-2 cell viability. EGCs induced a significant increase in Caco-2 cell surface area without any sign of cellular hypertrophy. These effects by EGCs were also seen in various transformed or nontransformed intestinal epithelial cell lines. Furthermore, TGF-beta1 mRNA was expressed, and TGF-beta1 was secreted by EGCs. Exogenously added TGF-beta1 reproduced partly the EGC-mediated effects on cell density and surface area. In addition, EGC effects on Caco-2 cell density were significantly reduced by a neutralizing TGF-beta antibody. In conclusion, EGCs have profound antiproliferative effects on intestinal epithelial cells. Functional alterations in EGCs may therefore modify intestinal barrier functions and be involved in pathologies such as cancer or inflammatory bowel diseases.     

Uptake and fate of luminally administered horseradish peroxidase in resting and isoproterenol-stimulated rat parotid acinar cells

The formation and fate of apical endocytic vesicles in resting and isoproterenol-stimulated rat parotid acinar cells were studied using luminally administered horseradish peroxidase (HRP) to mark the vesicles. The tracer was taken up from the lumen by endocytosis in small, smooth-surfaces "c"- or ring-shaped vesicles. About 1 h after HRP administration the vesicles could be found adjacent to the Golgi apparatus. At later times HRP reaction product was localized in multivesicular bodies and lysosomes; in isoproterenol-stimulated cells it was also present in autophagic vacuoles. HRP reaction product was never localized in any structure associated with secretory granule formation. These results suggest that the apical endocytic vesicles play a role in membrane recovery, but that they are degraded and not reutilized directly in secretory granule formation. Additionally, it was found that when isoproterenol was injected before HRP administration, the apical junctional complexes became permeable to the tracer, allowing it to gain access to the lateral and basal intercellular spaces. This permeability may provide an additional route whereby substances in the extracellular fluid could reach the saliva.     

Paneth cells in intestinal homeostasis and tissue injury

Adult stem cell niches are often co-inhabited by cycling and quiescent stem cells. In the intestine, lineage tracing has identified Lgr5(+) cells as frequently cycling stem cells, whereas Bmi1(+), mTert(+), Hopx(+) and Lrig1(+) cells appear to be more quiescent. Here, we have applied a non-mutagenic and cell cycle independent approach to isolate and characterize small intestinal label-retaining cells (LRCs) persisting in the lower third of the crypt of Lieberkühn for up to 100 days. LRCs do not express markers of proliferation and of enterocyte, goblet or enteroendocrine differentiation, but are positive for Paneth cell markers. While during homeostasis, LR/Paneth cells appear to play a supportive role for Lgr5(+) stem cells as previously shown, upon tissue injury they switch to a proliferating state and in the process activate Bmi1 expression while silencing Paneth-specific genes. Hence, they are likely to contribute to the regenerative process following tissue insults such as chronic inflammation.     

The effects of cytoskeletal inhibitors on intestinal iron absorption in the rat

An established and validated method using loops of intestine in vivo in rats was used to study the effects of cytoskeletal inhibitors on iron absorption. Radioactive iron instilled into the loop of intestine pretreated with test substance was monitored in the blood and, after death, ferritin loading with radioactive iron was measured on density gradients of mucosal cell homogenates and absorbed iron in the carcass was determined. Colchicine, vincristine and cytochalasin B all caused dose- and time-dependent inhibition of iron absorption, and the effects of cytochalasin B were reversible within 1 h. It is not known which cellular component is the vehicle for the transcellular movement of iron from the intestinal lumen onto plasma transferrin; however, this study showed that the uptake of iron by ferritin in an iron-absorbing loop of intestine paralleled the actual absorption of iron into the carcass. This phenomenon did not occur in non-iron-absorbing intestinal and was inhibited by the action of the cytoskeletal inhibitors in the iron-absorbing region. Previously we had shown that iron uptake into cells and onto cellular transferrin was virtually the same throughout the small intestine, irrespective of the iron-absorbing capacity of the region. The results of this study therefore suggest that iron absorption depends on an intact cytoskeletal system and that ferritin in the iron-absorbing cell is able to load from the pool of iron committed to transcellular movement onto plasma transferrin.     

CC chemokine ligands 25 and 28 play essential roles in intestinal extravasation of IgA antibody-secreting cells

CCL25 (also known as thymus-expressed chemokine) and CCL28 (also known as mucosae-associated epithelial chemokine) play important roles in mucosal immunity by recruiting IgA Ab-secreting cells (ASCs) into mucosal lamina propria. However, their exact roles in vivo still remain to be defined. In this study, we first demonstrated in mice that IgA ASCs in small intestine expressed CCR9, CCR10, and CXCR4 on the cell surface and migrated to their respective ligands CCL25, CCL28, and CXCL12 (also known as stromal cell-derived factor 1), whereas IgA ASCs in colon mainly expressed CCR10 and CXCR4 and migrated to CCL28 and CXCL12. Reciprocally, the epithelial cells of small intestine were immunologically positive for CCL25 and CCL28, whereas those of colon were positive for CCL28 and CXCL12. Furthermore, the venular endothelial cells in small intestine were positive for CCL25 and CCL28, whereas those in colon were positive for CCL28, suggesting their direct roles in extravasation of IgA ASCs. Consistently, in mice orally immunized with cholera toxin (CT), anti-CCL25 suppressed homing of CT-specific IgA ASCs into small intestine, whereas anti-CCL28 suppressed homing of CT-specific IgA ASCs into both small intestine and colon. Reciprocally, CT-specific ASCs and IgA titers in the blood were increased in mice treated with anti-CCL25 or anti-CCL28. Anti-CXCL12 had no such effects. Finally, both CCL25 and CCL28 were capable of enhancing alpha4 integrin-dependent adhesion of IgA ASCs to mucosal addressin cell adhesion molecule-1 and VCAM-1. Collectively, CCL25 and CCL28 play essential roles in intestinal homing of IgA ASCs primarily by mediating their extravasation into intestinal lamina propria.     

Existence of serotonin and neuropeptides-immunoreactive endocrine cells in the small and large intestines of the mole-rats (Spalax leucodon)

The present study was conducted to clarify the regional distribution and relative frequency of endocrine cells secreting serotonin, substance P (SP), cholecystokinin-8 (CCK-8), vasoactive intestinal polypeptide (VIP) and neurotensin in the small and large intestine of the mole-rats (Spalax leucodon), by specific immunohistochemical methods. In the small and large intestine of mole-rats (Spalax leucodon), serotonin, SP and VIP were identified with various frequencies, but CCK-8 and neurotensin were not observed. Most of the IR cells in the small and large intestine were located in the intestinal crypt and epithelium however, they were more frequency in the intestinal crypt. Serotonin-IR cells were detected throughout the whole intestinal tract, predominantly in the duodenum and colon. SP-IR cells were demonstrated throughout the whole intestinal tract except for the ileum and rectum with highest frequencies in the cecum. VIP-IR cells were found in all parts of the small intestine except for the large intestine.In conclusion, the general distribution patterns and relative frequency of intestinal endocrine cells of the mole-rats (Spalax leucodon) was similar to those of some rodent species. However, some species-dependent unique distributions and frequencies characteristics of endocrine cells were also observed in the present study.     

Eosinophilic granule cells in Carassius auratus scale epidermis

Eosinophilic granule cells (EGCs) were characterized in Carassius auratus scale epidermis in situ and in explants. Live EGCs were readily identified by the presence of numerous large cytoplasmic granules observed with DIC microscopy. Histochemical staining with toluidine blue and alcian blue yielded granule metachromasia and pale blue granules, respectively, both consistent with mammalian mast cell staining. However, EGCs also share some features with mammalian basophils as neutral red dye was selectively incorporated into EGC granules. EGCs within scale epidermis were actively motile, displaying average speeds of 16 μm/min and maximum speeds of greater than 40 μm/min and showing morphological plasticity during migration. The predominant motile phenotype was elongate with a well‐developed leading lamella, while a broader body motile morphology was observed to a lesser extent. A trailing, relatively unchanged uropod was associated with every motile EGC and invariably contained one or a few granules. A rounded EGC shape without a leading‐edge or trailing uropod was also observed and was generally associated with static cells. Individual cells readily switched between the three major shapes during motility; static cells could abruptly develop a polarized morphology, and actively motile cells switched between elongate and broad‐bodied shapes or the static, rounded shape.     

A Highlights from MBoC Selection: Rab21 in enterocytes participates in intestinal epithelium maintenance

Membrane trafficking is defined as the vesicular transport of proteins into, out of, and throughout the cell. In intestinal enterocytes, defects in endocytic/recycling pathways result in impaired function and are linked to diseases. However, how these trafficking pathways regulate intestinal tissue homeostasis is poorly understood. Using the Drosophila intestine as an in vivo system, we investigated enterocyte-specific functions for the early endosomal machinery. We focused on Rab21, which regulates specific steps in early endosomal trafficking. Depletion of Rab21 in enterocytes led to abnormalities in intestinal morphology, with deregulated cellular equilibrium associated with a gain in mitotic cells and increased cell death. Increases in apoptosis and Yorkie signaling were responsible for compensatory proliferation and tissue inflammation. Using an RNA interference screen, we identified regulators of autophagy and membrane trafficking that phenocopied Rab21 knockdown. We further showed that Rab21 knockdown-induced hyperplasia was rescued by inhibition of epidermal growth factor receptor signaling. Moreover, quantitative proteomics identified proteins affected by Rab21 depletion. Of these, we validated changes in apolipoprotein ApoLpp and the trehalose transporter Tret1-1, indicating roles for enterocyte Rab21 in lipid and carbohydrate homeostasis, respectively. Our data shed light on an important role for early endosomal trafficking, and Rab21, in enterocyte-mediated intestinal epithelium maintenance.     

Inflammatory cells of teleostean fish: a review focusing on mast cells/eosinophilic granule cells and rodlet cells

In contrast to the roles played by monocytes/macrophages, neutrophils and lymphocytes, the presence and functions of basophils, mast cells/eosinophilic granule cells, eosinophils and rodlet cells in teleosts are areas of controversy. The tissue distribution of mast cells/eosinophilic granule cells in species from a certain genus shows a characteristic pattern, and this pattern is usually also present at the family level. Functionally, the mast cells/eosinophilic granule cells of teleosts show close similarity to the mast cells of mammals. Acute tissue damage is causing mast cell/eosinophilic granule cell degranulation and release of mediators of inflammation, whereas an increase in the number of these cells is often found in chronically inflamed tissues. The mast cells/eosinophilic granule cells of teleosts show marked diversity in their staining properties, with both basophilic and acidophilic components in their granules. In some fish families, e.g. the labrids, the eosinophilic component is dominating, whereas in the pike the granules are strongly basophilic and show the metachromatic staining characteristics found in the granules of mast cells, but being more akin to the granules of the mucosal than to those of the connective tissue type of mast cells of mammals. With respect to rodlet cells, a cell type hitherto clearly demonstrated only in teleosts, a characteristic distribution pattern seems to be established in certain families. In other families rodlet cells are absent in some individuals and present in different tissues in others. However, there is a close relation between the presence of helminths or other noxious agents and the presence of rodlet cells. Massive aggregations of such cells can be seen in affected epithelia of gills or the intestinal tract, and in individuals of species from some fish families they also occur in association with mesothelial and endothelial tissues. The rodlet cell may represent a type of eosinophilic granulocyte that populates the tissues at its immature stage and mature in response to the appropriate stimuli, in a way similar to that of mast cell precursors. Present evidence points to a functional role for the rodlet cells of teleosts in host defence against parasites.     

Ultrastructural study on route of gut bacterial translocation in a rat after spinal cord injury

Objective: To observe the ultrastructural change of the route of gut bacterial translocation in a rat with spinal cord injury(SCI).Methods: Forty Wistar rats were divided into the following groups: control group and 3 SCI groups(10 in each group). The rats in the SCI groups were established SCI model at 24 h, 48 h, and 72 h after SCI. Small intestine mucous membrane tissue was identified and assayed by transmission electron microscope, scanning electron microscope and immunofluorescence microscopy. Results: Small intestine mucous membrane tissue in control group was not damaged significantly, but those in SCI groups were damaged significantly. Proliferation bacteria in gut lumen attached on microvilli. The extracellular bacteria torn the intestinal barrier and perforated into the small intestinal mucosal epithelial cell. The bacteria and a lot of particles of the seriously damaged region penetrated into the lymphatic system and the blood system directly. Some bacteria were internalized into the goblet cell through the apical granule. Some bacteria and particles perforated into the submucosa of the M cell running the long axis of M cells through the tight junctions. In the microcirculation of mucosa, the bacteria that had already broken through the microvilli into blood circulation swim accompanying with erythrocytes. Conclusion: The routes of bacterial translocation interact and format a vicious circle. At early step, the transcellular pathway of bacterial translocation is major. Following with the destroyed small intestine mucous, the routes of bacterial translocation through the lymphatic system and the blood system become direct pathways. The goblet cell-dendritic cell and M cell pathway also play an important role in the bacterial translocation.     

NOX4 inhibition protects enteric glial cells against Clostridium difficile toxin B toxicity via attenuating oxidative and Endoplasmic reticulum stresses

Enteric glial cells (EGCs), one main cell population of the enteric nervous system (ENS), play a major role in regulating intestinal barrier function. Clostridium difficile toxin B (TcdB) is the major virulence factor produced by C. difficile and estimated to be toxic to EGCs by inducing cell death, cell cycle arrest, and inflammatory cytokine production; however, the detailed mechanism for such effect is still unclear. In this study, we further evaluated the toxic effect of TcdB on EGCs and the involvement of NADPH oxidases in such process using the rat-transformed EGCs (CRL-2690). The results showed that NOX4 was activated by TcdB in EGCs and functioned as the major factor causing cytotoxicity and cell apoptosis. Mechanically, NOX4-generated H₂O₂ was the inducer of oxidative stress, Ca²⁺ homeostasis disorder, and ER stress in EGCs upon TcdB treatment, and NOX4 inhibition protected EGCs against TcdB toxicity via attenuating these dysfunctions. These findings contribute to our understanding of the mechanism by which TcdB affects EGCs and suggest the potential value of NOX4 inhibition for treatment against C. difficile infection.