Intestinal barrier dysfunction occurs in critical illnesses and involves the inflammatory and hypoxic injury of intestinal epithelial cells. Researchers are still defining the underlying mechanisms and evaluating therapeutic strategies for restoring intestinal barrier function. The anti-inflammatory drug, emodin, has been shown to exert a protective effect on intestinal barrier function; however, its mechanisms of action remain unknown. In this study, we investigated the protective effects of emodin on intestinal barrier function and the underlying mechanisms in intestinal epithelial cells challenged with lipopolysaccharide (LPS) and hypoxia/reoxygenation (HR). To induce barrier dysfunction, Caco-2 monolayers were subjected to HR with or without LPS treatment. Transepithelial electrical resistance and paracellular permeability were measured to evaluate barrier function. The expression of the tight junction (TJ) proteins, zonula occludens (ZO)-1, occludin, and claudin-1, as well as that of hypoxia-inducible factor (HIF)-1α, phospho-IκB-α, phospho-nuclear factor (NF)-κB p65 and cyclooxygenase (COX)-2 was determined by western blot analysis. The results revealed that emodin markedly attenuated the decrease in transepithelial electrical resistance and the increase in paracellular permeability in the Caco-2 monolayers treated with LPS and subjected to HR. Emodin also markedly alleviated the damage caused by LPS and HR (manifested by a decrease in the expression of the TJ protein, ZO-1), and inhibited the expression of HIF-1α, IκB-α, NF-κB and COX-2 in a dose-dependent manner. In conclusion, our data suggest that emodin attenuates LPS- and HR-induced intestinal epithelial barrier dysfunction by inhibiting the HIF-1α and NF-κB signaling pathways and preventing the damage caused to the TJ barrier (shown by the decrease in the expression of ZO-1).
A number of critical illnesses, such as shock, trauma and burns, as well as cardiac and abdominal surgery and small intestinal transplantation, can lead to intestinal ischemia/reperfusion (I/R) injury, which destroys intestinal barrier function (
The hypoxia-inducible factor (HIF)-1 heterodimer consists of oxygen-labile HIF-1α and constitutively expressed HIF-1β. The transcription factor, HIF-1, mediates a wide spectrum of physiological and cellular adaptive responses, such as angiogenesis, metabolic adaption, erythropoiesis and vascular tone (
NF-κB is an important regulator of inflammatory signaling, containing p65 (RelA), RelB, c-Rel, p50 (NF-κB1) and p52 (NF-κB2). There are two primary activation pathways for NF-κB. The canonical signaling pathway is dependent on IKK-β activation. LPS, TNF-α or IL-1 activate each respective receptor. This leads to an activation of IKK-β in the IKK complex, which can then phosphorylate IκB-α, which in turn results in the degradation of IκB-α. NF-κB can then translocate to the nucleus. The non-canonical signaling pathway is dependent on IKK-α activation. Through a variety of adapter proteins and signaling kinases, this leads to the translocation of NF-κB (
The NF-κB and HIF pathways are intimately associated and there is a significant level of crosstalk between these pathways at a number of levels (
Emodin is a natural anthraquinone compound that is isolated from the traditional Chinese medicine,
In the present study, we hypothesized that emodin modulates intestinal barrier injury which is induced by LPS and hypoxia/reoxygenation (HR) in intestinal cells
Caco-2 cells (a human colonic cell line) obtained from the American Type Culture Collection (Manassas, VA, USA) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 15% fetal bovine serum, 4.0 mM l-glutamine, 1% non-essential amino acids, 100 U/ml penicillin and 100 U/ml streptomycin (all from Invitrogen Life Technologies, Carlsbad, CA, USA). The cells were maintained in a humidified 37°C, 5% CO2 incubator, and passaged by partial digestion with 0.25% trypsin and 0.53 mM EDTA (Biochrom AG, Berlin, Germany) in Hank’s balanced salt solution (HBSS; Invitrogen Life Technologies) without Ca2+ and Mg2+.
Cytotoxic properties were assessed by MTT assay. The Caco-2 cells were seeded at a density of 105 cells/well in 96-well plates for 24 h to promote attachment. The medium was replaced with fresh medium containing various concentrations of emodin (Tianjin Institute for Drug Control, Tianjin, China; 20, 40, 60 or 80 μM) for 72 h. Following treatment, the cells were incubated in the dark with MTT solution (Invitrogen Life Technologies) for 4 h at 37°C. The solution was aspirated and the formazan crystal product was solubilized in 100 μl DMSO. The absorbance was measured at 570 nm using a microplate ELISA reader. All experiments were carried out in triplicate and the results are presented as the means ± SEM.
The cells were plated at a density of 5×104/cm2 on collagen-coated permeable polycarbonate membrane Transwell supports with 0.4 μm pores (Corning, Corning, NY, USA) and were grown as monolayers prior to the experiments. In the normoxia control experiments, the cells were cultured at 37°C in a humidified incubator containing 95% air and 5% CO2, using pre-equilibrated normoxic medium. In the LPS experiments, the cell monolayers were treated with various concentrations of LPS from
TEER values, an indicator of TJ permeability to ionic solutes, were measured using a Millicell-ERS voltohmmeter (Millipore, Bedford, MA, USA). Each measurement was calculated by subtracting the resistance value of the filters and fluids, and expressed as a percentage of the initial values.
To investigate intestinal permeability, we used the flux of FITC-conjugated dextran (molecular weight, 4 kDa) (Sigma) as a probe. The movement of FITC-dextran across the monolayers represented the apical-to-basal paracellular permeability of the intestinal barrier. The cells were treated with LPS (1 μg/ml), H3 hR3 h + LPS or H3 hR3 h + LPS + emodin (60 μmol/l) for 6 h. The apical chamber was filled with 100 μl of the different solutions with 1 mg/ml FITC-dextran in HBSS and the basolateral chamber was filled with 600 μl of growth medium followed by incubation at 37°C. Basolateral samples (100 μl) were taken every 1 h over the last 3 h, replenishing with fresh medium at each sample time point. Fluorescence was determined using a fluorescence microplate plate reader (Fluorescence Spectrophotometer F-7000; Hitachi High Technologies America, Inc., Schaumburg, IL, USA). Apparent permeability co-efficients (
where dQ/dt is the transport rate (mg/sec), A is the cell monolayer surface area (cm2) and C0 is the initial concentration in the donor compartment (mg/ml).
The Caco-2 monolayers were washed twice with ice-cold PBS, and then lysed in RIPA buffer with a cocktail of protease inhibitors on ice. Cell debris was separated by centrifugation at 15,000 × g for 20 min at 4°C. The quantity of protein in the supernatants was determined using a BCA protein assay kit (BioTeke Corporation, Beijing, China). Equal amounts of protein samples were separated by SDS-PAGE and were transferred onto PVDF membranes (Millipore). After blocking with 5% non-fat milk for 1 h, the membranes were incubated with primary antibodies specific for ZO-1 (Cat. no. 61–7300, dilution 1:1,000), occludin (Cat. no. 33–1500, dilution 1:1,000), claudin-1 (Cat. no. 37–4900, dilution 1:1,000; all from Invitrogen Life Technologies), HIF-1α (Cat. no. 610959, dilution 1:1,000; BD Biosciences, Franklin Lakes, NJ, USA), COX-2 (Cat. no. 35–8200, dilution 1:1,000; Invitrogen Life Technologies), phospho-NF-κB p65 (Ser536) (Cat. no. 3033, dilution 1:1,000), phospho-IκB-α (Ser32) (Cat. no. 2859, dilution 1:1,000) and β-tubulin (Cat. no. 2128, dilution 1:1,000; all from Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. After 3 washes, the membranes were incubated with peroxidase-conjugated secondary antibodies for 1 h. Proteins were detected using the enhanced chemiluminescence detection kit (Millipore). Band intensities were quantified using Quantity One software (Bio-Rad, Hercules, CA, USA). Relative expression was normalized to β-tubulin.
The differences among multiple groups were assessed by one-way ANOVA using SPSS software version 13.0. All the data are presented as the means ± SEM. All reported significance levels represent two-tailed P-values. A value of P<0.05 was considered to indicate a statistically significant difference.
Emodin (<80 μM) did not exert any growth inhibitory or general cytotoxic effects on the Caco-2 cells (
It has been demonstrated that LPS or hypoxia causes intestinal barrier dysfunction (
Consistent with the changes in TEER,
It has been demonstrated that alterations in the expression of TJ proteins are involved in intestinal barrier disruption induced by pro-inflammatory cytokines or burn injury (
Different types of non-hypoxic cell stimulation have been shown to increase HIF-1α expression in macrophages (
In time course experiments, the cells were stimulated with 10−3 mg/ml LPS for different periods of time. The maximal induction of HIF-1α was attained after 30 min in the presence of LPS; after 30 min, HIF-1α levels decreased, and decreased to the minimum levels at 4 h; after 4 h HIF-1α expression increased again (
Both HIF-1α and NF-κB are the key oxygen-sensitive transcriptional regulators in inflammatory and hypoxic conditions. A recent study revealed a high degree of interdependence between the HIF and NF-κB signaling pathways, as well as a correlation between inflammation and hypoxia (
In the cells exposed to HR, the expression of IκB, NF-κB and COX-2 showed a similar trend, first increasing and then decreasing as the duration of reoxygenation increased (
Having demonstrated the protective effects of emodin on intestinal barrier function
It is well known that the gut is the ‘motor’ of critical illness and the origin of sepsis in a number of intensive care patients. The balance between the intestinal epithelium, immune system and endogenous microflora of the gut breaks down, leading to the development of gut-origin systemic diseases (
It has been demonstrated that LPS or HR disrupts intestinal epithelial barrier function both
The intestinal epithelial barrier plays a significant role in preventing antigens and pathogens from entering the intestinal mucosa and encountering the immune system (
Under normal oxygen conditions, HIF-α is rapidly destroyed through the proteasomal degradation pathway in the cytoplasm. By contrast, hypoxia or inflammation is associated with the stabilization of HIF-α. When HIF-α is stabilized, it can translocate to the nucleus and form a heterodimer with the HIF-1β subunit, allowing for transcriptional activity. HIF-α has 3 isoforms, HIF-1α, HIF-2α and HIF-3α. HIF-1α and HIF-2α have common and distinguishing characteristics in hypoxia and inflammation (
As a transcription factor, NF-κB plays an essential role in inflammation and innate immunity. It is interesting to note that both the HIF-1α and NF-κB pathways are regulated by inflammatory mediators, as well as by hypoxia (
Hypoxia and inflammation share an interdependent relationship. Both animal and human studies have indicated that hypoxia elicits tissue inflammation, so-called hypoxia-elicited inflammation (
COX-2 and other key proinflammatory genes are transcriptional in a manner that is both HIF-1 and NF-κB dependent (
In this study, we demonstrated that emodin attenuated intestinal epithelial barrier dysfunction caused by LPS and HR
In conclusion, the present study demonstrates that emodin attenuates intestinal barrier dysfunction elicited by LPS and HR by inhibiting the NF-κB and HIF-1α signaling pathways. This may be one of the molecular mechanisms responsible for the protective effects of emodin against intestinal epithelial barrier dysfunction triggered by inflammation and hypoxia. Thus, targeting the restoration of intestinal barrier function is a worthwhile therapeutic strategy for sepsis and other critical illnesses. Hopefully, the results of the present study may lead to the development of novel therapies that may improve clinical outcomes and prognosis of patients with gut-derived sepsis.
The present study was supported by the National Basic Research Program of China (973 program, 2009CB522703). The authors thank Professor Feng Wang for providing comments on the manuscript and helpful discussions.
Effects of emodin on cell viability. (A) Structure of emodin. (B) Cells were treated with various concentrations of emodin for 6 h, prior to cytotoxicity being measured.
Emodin attenuates barrier dysfunction induced by hypoxia/reoxygenation (HR) and lipopolysaccharide (LPS) in Caco-2 monolayers. (A) Caco-2 monolayers were exposed to H3 hR3 h and LPS (LPS 1 μg/ml, 1% O2) and emodin (E; 60 μmol/l) was added to the basal chamber for 6 h. The designated conditions were control (medium only), H3 hR3 h + LPS, H3 hR3 h + LPS + emodin. Emodin significantly inhibited the decrease in transepithelial electrical resistance (TEER) induced by simultaneous exposure to HR and LPS. (B) Caco-2 monolayers were treated as described for (A). Apparent permeability co-efficients (
Emodin prevents the decrease in the expression of zonula occludens (ZO)-1 induced by lipopolysaccharide (LPS) and hypoxia/reoxygenation (HR) injury. Caco-2 monolayers were treated as described in
Treatment with lipopolysaccharide (LPS) induced hypoxia-inducible factor (HIF)-1α and cyclooxygenase (COX)-2 expression in Caco-2 cells in a dose- and time-dependent manner. (A–C) The cells were treated with LPS at different doses (10−6-1 mg/ml) for 6 h. *P<0.05, compared with 10−6 mg/ml group. (D–F) The cells were treated with LPS (10−3 mg/ml) for different periods of time (0.5–24 h). *P<0.05, compared with 0.5 h group.
Exposure to hypoxia (H) or hypoxia + lipopolysaccharide (LPS) activated the hypoxia-inducible factor (HIF)-1α and nuclear factor (NF)-κB signaling pathways in Caco-2 cells. (A) Western blot analysis of protein expression. (B–E) In the cells exposed to hypoxia, the expression of IκB, NF-κB and COX-2 showed similar trends, which decreased as the duration of hypoxia increased, and the minimum induction was observed at H3–4 h. The change in HIF-1α expression was a small increase and a decrease thereafter. The maximal induction of HIF-1α expression was at H3 h. In the cells exposed to hypoxia + LPS, the trends for IκB, NF-κB, COX-2 and HIF-1α were similar. There was an increase at H1 h; maximal induction was attained at H3 h; and there was a decrease thereafter. *P<0.05, compared with controls.
Hypoxia (H)/reoxygenation (R) (HR) or HR + lipopolysaccharide (LPS) activated the hypoxia-inducible factor (HIF)-1α and nuclear factor (NF)-κB signaling pathways. (A) Western blot analysis of protein expression. (B–E) In the cells exposed to HR, the expression of IκB, NF-κB and COX-2 showed a similar trend, an increase first and then a decrease with reoxygenation. The expression of HIF-1α decreased with reoxygenation, and the minimum induction was observed at H3 hR1–2 h. In the cells exposed to HR + LPS, the trends for all 4 proteins were similar. An increase was observed at H1 h; maximal induction was attained at H3 hR2–3 h; and a decrease was observed thereafter. *P<0.05, compared with controls.
(A–E) Effects of emodin on the expression of hypoxia-inducible factor (HIF)-1α, cyclooxygenase (COX)-2, phospho-IκB-α and phospho-nuclear factor (NF)-κB p65 induced by exposure to lipopolysaccharide (LPS) and hypoxia/reoxygenation (HR). The cells were treated with LPS + H3 hR2 h and different concentrations of emodin. Emodin inhibited the HIF-1 and NF-κB signaling pathways in a dose-dependent manner. The minimum induction of protein expression was observed following treatment with emodin at the concentration of 80 μmol/l. However, emodin is insoluble in water which is the main components of the medium. Emodin will be not soluble if too much is added to medium; 80 μM of emodin is already a rather high concentration. We tried 100 μM, the crystal was observed at the bottom of the culture flask. *P<0.05, compared with LPS + H3 hR2 h.
Emodin protects intestinal barrier function by blocking the hypoxia and inflammatory signaling pathways. Lipopolysaccharide (LPS) activates Toll-like receptors and CD14. This response activates IKK, which can then phosphorylate IκB-α, which in turn results in the degradation of IκB-α. Nuclear factor (NF)-κB translocates to the nucleus and regulates the downstream target gene, cyclooxygenase (COX)-2. In the presence of oxygen, prolyl hydroxylase domain protein (PHD) hydroxylates hypoxia-inducible factor (HIF)-1α proline residues and a VHL ubiquitin-protein ligase complex binds, leading to ubiquitination and subsequent proteasomal degradation. Under conditions of hypoxia or inflammation, hydroxylation is inhibited, HIF-1α accumulates, translates to the nucleus and dimerizes with HIF-1β. HIF-1 can regulate transcription of target gene COX-2. All inflammatory factors may destroy the intestinal barrier dysfunction by damaging the tight junction (TJ). Loss of intestinal barrier may worsen the intestinal and systemic disease, lead to SIRS, gut-origin sepsis and multiple organ dysfunction syndrome (MODS). As we known, the critical illness cause decrease in blood flow of gut and increase in intestinal bacteria and their product translocation. A feed-forward loop probably exists between intestinal barrier dysfunction and systemic disease. Emodin can inhibit HIF-1 and NF-κB signaling pathways and protect intestinal barrier functionby preserving TJ.