SARS-CoV-2 induces barrier damage and inflammatory responses in the human iPSC-derived intestinal epithelium.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread and led to global health crises. COVID-19 causes well-known respiratory failure and gastrointestinal symptoms, such as diarrhea, nausea, and vomiting. Thus, human gastrointestinal cell models are urgently needed for COVID-19 research; however, it is difficult to obtain primary human intestinal cells. In this study, we examined whether human induced pluripotent stem cell (iPSC)-derived small intestinal epithelial cells (iPSC-SIECs) could be used as a SARS-CoV-2 infection model. We observed that iPSC-SIECs, such as absorptive and Paneth cells, were infected with SARS-CoV-2, and remdesivir treatment decreased intracellular SARS-CoV-2 replication in iPSC-SIECs. SARS-CoV-2 infection decreased expression levels of tight junction markers, ZO-3 and CLDN1, and transepithelial electrical resistance (TEER), which evaluates the integrity of tight junction dynamics. In addition, SARS-CoV-2 infection increased expression levels of proinflammatory genes, which are elevated in patients with COVID-19. These findings suggest iPSC-SIECs as a useful in vitro model for elucidating COVID-19 pathology and drug development.

Potassium diformate alleviated inflammation of IPEC-J2 cells infected with EHEC.

Potassium diformate (KDF) is a kind of formate, which possesses the advantages of antimicrobial activity, growth promotion and preventing diarrhea in weaned piglets. However, the researches of KDF in animal production mostly focused on apparent indexes such as growth performance and the mechanisms of KDF on intestinal health have not been reported. Thus, porcine small intestinal epithelial cells (IPEC-J2) infected with Enterohemorrhagic Escherichia coli (EHEC) was used to investigate the role of KDF on alleviating intestinal inflammation in this study. The 0.125 mg/mL KDF treated IPEC-J2 cells for 6 h and IPEC-J2 cells challenged with 5 × 107 CFU/mL EHEC for 4 h were confirmed as the optimum concentration and time for the following experiment. The subsequent experiment was divided into four groups: control group (CON), EHEC group, KDF group, KDF+EHEC group. The results showed that KDF increased the cell viability and the gene expression levels of SGLT3 and TGF-ß, while decreased the content of IL-1ß compared with the CON group. The cell viability and the gene expressions of SGLT1, SGLT3, GLUT2, Claudin-1, Occludin and TGF-ß, and the protein expression of ZO-1 in EHEC group were lower than those in CON group, whereas the gene expressions of IL-1ß, TNF, IL-8 and TLR4, and the level of phosphorylation NF-кB protein were increased. Pretreatment with KDF reduced the content of IgM and IL-1ß, the gene expressions of IL-1ß, TNF, IL-8 and TLR4 and the level of phosphorylation NF-кB protein, and increased the gene expression of TGF-ß and the protein expression of Occludin in IPEC-J2 cells infected EHEC. In conclusion, 0.125 mg/mL KDF on IPEC-J2 cells for 6 h had the beneficial effects on ameliorating the intestinal inflammation because of reduced pro-inflammatory cytokines and enhanced anti-inflammatory cytokines through regulating NF-кB signaling pathway under the EHEC challenge.

The inflammatory injury of porcine small intestinal epithelial cells induced by deoxynivalenol is related to the decrease in glucose transport.

The present study aimed to investigate the effects of deoxynivalenol (DON) stimulation on inflammatory injury and the expression of the glucose transporters sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter protein 2 (GLU2) in porcine small intestinal epithelial cells (IPEC-J2). Additionally, the study aimed to provide initial insights into the connection between the expression of glucose transporters and the inflammatory injury of IPEC-J2 cells. DON concentration and DON treatment time were determined using the CCK­8 assay. Accordingly, 1.0 µg/mL DON and treatment for 24 h were chosen for subsequent experiments. Then IPEC-J2 cells were treated without DON (CON, N = 6) or with 1 µg/mL DON (DON, N = 6). Lactate dehydrogenase (LDH) content, apoptosis rate, and proinflammatory cytokines including interleukin (IL)-1ß, Il-6, and tumor necrosis factor α (TNF-α) were measured. Additionally, the expression of AMP-activated protein kinase α1 (AMPK-α1), the content of glucose, intestinal alkaline phosphatase (AKP), and sodium/potassium-transporting adenosine triphosphatase (Na+/K+-ATPase) activity, and the expression of SGLT1 and GLU2 of IPEC-J2 cells were also analyzed. The results showed that DON exposure significantly increased LDH release and apoptosis rate of IPEC-J2 cells. Stimulation with DON resulted in significant cellular inflammatory damage, as evidenced by a significant increase in proinflammatory cytokines (IL-1ß, IL-6, and TNF-α). Additionally, DON caused damage to the glucose absorption capacity of IPEC-J2 cells, indicated by decreased levels of glucose content, AKP activity, Na+/K+-ATPase activity, AMPK-α1 protein expression, and SGLT1 expression. Correlation analysis revealed that glucose absorption capacity was negatively correlated with cell inflammatory cytokines. Based on the findings of this study, it can be preliminarily concluded that the cell inflammatory damage caused by DON may be associated with decreased glucose absorption.

Glucose is one of the most basic nutrients necessary to sustain animal life and plays a crucial role in animal body composition and energy metabolism. Previous studies suggested a link between glucose absorption and inflammatory injury. In the present study, deoxynivalenol (DON) stimulation caused severe inflammatory injury and reduced the glucose absorption capacity of IPEC-J2 cells. Pearson's correlation analysis revealed a negative correlation between glucose absorption capacity and cell inflammatory cytokines. Ultimately, it can be speculated that the cellular inflammatory response triggered by DON may be related to the altered expression of glucose transporters.

Air-liquid interface culture and modified culture medium promote the differentiation of human induced pluripotent stem cells into intestinal epithelial cells.

An in vitro system that evaluates pharmacokinetics in the small intestine is crucial for the development of oral drugs. We produced human induced pluripotent stem cell-derived small intestinal epithelial cells (hiSIECs) with high drug metabolizing enzyme and drug transporter activities. However, the gene expression of our hiSIECs partially differed from that of the human small intestine, with low drug metabolizing enzyme activities. Therefore, we used air-liquid interface (ALI) culture and 5-aza-2'-deoxycytidine (5AZA)-free medium to generate hiSIECs (novel hiSIECs). Novel hiSIECs showed enhanced gene expression of drug metabolizing enzymes, such as cytochrome P450 (CYP)3A4, CYP2C9, CYP2C19, and carboxylesterase 2 that are highly expressed in the small intestine. In addition, the expression of genes involved in nutrient absorption-one of the major functions of the small intestine-also increased. The novel hiSIECs expressed ZO-1 and E-cadherin. Moreover, the novel hiSIECs exhibited a barrier function that allowed low lucifer yellow permeation. The novel hiSIECs showed high activities of CYP3A4, CYP2C9, and CYP2C19, which are abundantly expressed in the small intestine. In conclusion, the novel hiSIECs have great potential as an in vitro system to evaluate pharmacokinetics in the small intestine.

Porcine Epidemic Diarrhea Virus Replication in Human Intestinal Cells Reveals Potential Susceptibility to Cross-Species Infection.

Various coronaviruses have emerged as a result of cross-species transmission among humans and domestic animals. Porcine epidemic diarrhea virus (PEDV; family Coronaviridae, genus Alphacoronavirus) causes acute diarrhea, vomiting, dehydration, and high mortality in neonatal piglets. Porcine small intestinal epithelial cells (IPEC-J2 cells) can be used as target cells for PEDV infection. However, the origin of PEDV in pigs, the host range, and cross-species infection of PEDV remain unclear. To determine whether PEDV has the ability to infect human cells in vitro, human small intestinal epithelial cells (FHs 74 Int cells) were inoculated with PEDV LJX and PEDV CV777 strains. The results indicated that PEDV LJX, but not PEDV CV777, could infect FHs 74 Int cells. Furthermore, we observed M gene mRNA transcripts and N protein expression in infected FHs 74 Int cells. A one-step growth curve showed that the highest viral titer of PEDV occurred at 12 h post infection. Viral particles in vacuoles were observed in FHs 74 Int cells at 24 h post infection. The results proved that human small intestinal epithelial cells are susceptible to PEDV infection, suggesting the possibility of cross-species transmission of PEDV.

Lipopolysaccharide promotes apoptosis and oxidative injury of porcine small intestinal epithelial cells by down-regulating the expression of glutamine transporter ASCT2.

The present study aimed to investigate the effects of lipopolysaccharide (LPS) stimulation on oxidative damage, apoptosis, and glutamine (Gln) transporter Alanine-Serine-Cysteine transporter 2 (ASCT2) expression in porcine small intestinal epithelial cells (IPEC-J2), and preliminarily elucidated the relationship between ASCT2 expression level and oxidative damage and apoptosis of IPEC-J2 cells. IPEC-J2 cells were treated without (control group, CON, N = 6) or with 1 µg/mL LPS (LPS group, LPS, N = 6). Cell viability, lactate dehydrogenase (LDH) content, malonaldehyde (MDA), anti-oxidant enzymes (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GSH-Px], and total anti-oxidant capacity [T-AOC]), apoptosis of IPEC-J2 cells, the expression of Caspase3, the expression of ASCT2 mRNA and ASCT2 protein was detected. The results showed that LPS stimulation of IPEC-J2 cells significantly reduced the cell viability, and anti-oxidant enzymes activity (SOD, CAT, and GSH-Px), and significantly increased LDH and MDA release. Flow cytometry results showed that LPS stimulation significantly increased the late apoptosis rate and the total apoptosis rate of IPEC-J2 cells. The immunofluorescence results showed that the fluorescence intensity of LPS stimulated IPEC-J2 cells was significantly enhanced. LPS stimulation significantly decreased the mRNA and protein expression of ASCT2 in IPEC-J2 cells. The correlation analysis showed that ASCT2 expression was negatively correlated with apoptosis, and positively correlated with the anti-oxidant capacity of IPEC-J2 cells. According to the results of this study, it can be preliminarily concluded that LPS promotes the apoptosis and oxidative injury of IPEC-J2 cells by down-regulating the expression of ASCT2.

Glutamine (Gln) is the main energy source for animal eukaryotic cells including intestinal epithelial cells (IECs), which is absorbed mainly mediated by Alanine-Serine-Cysteine transporter 2 (ASCT2). Previous studies have shown that lipopolysaccharide (LPS) stimulation can lead to oxidative damage, increased apoptosis, decreased glutamine absorption, and down-regulated ASCT2 mRNA and protein expression, suggesting that ASCT2 expression is involved in intestinal injury. However, the relationship between ASCT2 expression and cell apoptosis during cell injury has not been discussed in detail. The present study showed that ASCT2 expression was negatively correlated with apoptosis, and positively correlated with the anti-oxidant capacity of porcine small intestinal epithelial cells (IPEC-J2). According to the results of this study, it can be preliminarily concluded that LPS promotes the apoptosis and oxidative injury of IPEC-J2 cells by down-regulating the expression of ASCT2.

Dual RNA Sequencing Reveals Key Events When Different Giardia Life Cycle Stages Interact With Human Intestinal Epithelial Cells In Vitro.

Giardia intestinalis is a protozoan parasite causing diarrheal disease, giardiasis, after extracellular infection of humans and other mammals' intestinal epithelial cells (IECs) of the upper small intestine. The parasite has two main life cycle stages: replicative trophozoites and transmissive cysts. Differentiating parasites (encysting cells) and trophozoites have recently been shown to be present in the same regions of the upper small intestine, whereas most mature cysts are found further down in the intestinal system. To learn more about host-parasite interactions during Giardia infections, we used an in vitro model of the parasite's interaction with host IECs (differentiated Caco-2 cells) and Giardia WB trophozoites, early encysting cells (7 h), and cysts. Dual RNA sequencing (Dual RNAseq) was used to identify differentially expressed genes (DEGs) in both Giardia and the IECs, which might relate to establishing infection and disease induction. In the human cells, the largest gene expression changes were found in immune and MAPK signaling, transcriptional regulation, apoptosis, cholesterol metabolism and oxidative stress. The different life cycle stages of Giardia induced a core of similar DEGs but at different levels and there are many life cycle stage-specific DEGs. The metabolic protein PCK1, the transcription factors HES7, HEY1 and JUN, the peptide hormone CCK and the mucins MUC2 and MUC5A are up-regulated in the IECs by trophozoites but not cysts. Cysts specifically induce the chemokines CCL4L2, CCL5 and CXCL5, the signaling protein TRKA and the anti-bacterial protein WFDC12. The parasite, in turn, up-regulated a large number of hypothetical genes, high cysteine membrane proteins (HCMPs) and oxidative stress response genes. Early encysting cells have unique DEGs compared to trophozoites (e.g. several uniquely up-regulated HCMPs) and interaction of these cells with IECs affected the encystation process. Our data show that different life cycle stages of Giardia induce different gene expression responses in the host cells and that the IECs in turn differentially affect the gene expression in trophozoites and early encysting cells. This life cycle stage-specific host-parasite cross-talk is an important aspect to consider during further studies of Giardia's molecular pathogenesis.

Expression profiles of NOD-like receptors and regulation of NLRP3 inflammasome activation in Toxoplasma gondii-infected human small intestinal epithelial cells.

BACKGROUND: Toxoplasma gondii is a parasite that primarily infects through the oral route. Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) play crucial roles in the immune responses generated during parasitic infection and also drive the inflammatory response against invading parasites. However, little is known about the regulation of NLRs and inflammasome activation in T. gondii-infected human small intestinal epithelial (FHs 74 Int) cells. METHODS: FHs 74 Int cells infected with T. gondii were subsequently evaluated for morphological changes, cytotoxicity, expression profiles of NLRs, inflammasome components, caspase-cleaved interleukins (ILs), and the mechanisms of NLRP3 and NLRP6 inflammasome activation. Immunocytochemistry, lactate dehydrogenase assay, reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative RT-PCR, and western blotting techniques were utilized for analysis. RESULTS: Under normal and T. gondii-infected conditions, members of the NLRs, inflammasome components and caspase-cleaved ILs were expressed in the FHs Int 74 cells, except for NLRC3, NLRP5, and NLRP9. Among the NLRs, mRNA expression of NOD2, NLRP3, NLRP6, and NAIP1 was significantly increased in T. gondii-infected cells, whereas that of NLRP2, NLRP7, and CIITA mRNAs decreased significantly in a time-dependent manner. In addition, T. gondii infection induced NLRP3, NLRP6 and NLRC4 inflammasome activation and production of IL-1ß, IL-18, and IL-33 in FHs 74 Int cells. T. gondii-induced NLRP3 inflammasome activation was strongly associated with the phosphorylation of p38 MAPK; however, JNK1/2 had a weak effect. NLRP6 inflammasome activation was not related to the MAPK pathway in FHs 74 Int cells. CONCLUSIONS: This study highlighted the expression profiles of NLRs and unraveled the underlying mechanisms of NLRP3 inflammasome activation in T. gondii-infected FHs 74 Int cells. These findings may contribute to understanding of the mucosal and innate immune responses induced by the NLRs and inflammasomes during T. gondii infection in FHs 74 Int cells.

First Metabolic Insights into Ex Vivo Cryptosporidium parvum-Infected Bovine Small Intestinal Explants Studied under Physioxic Conditions.

The apicomplexan Cryptosporidium parvum causes thousands of human deaths yearly. Since bovines represent the most important reservoir of C. parvum, the analysis of infected bovine small intestinal (BSI) explants cultured under physioxia offers a realistic model to study C. parvum-host cell-microbiome interactions. Here, C. parvum-infected BSI explants and primary bovine small intestinal epithelial cells were analysed for parasite development and metabolic reactions. Metabolic conversion rates in supernatants of BSI explants were measured after infection, documenting an immediate parasite-driven metabolic interference. Given that oxygen concentrations affect cellular metabolism, measurements were performed at both 5% O2 (physiological intestinal conditions) and 21% O2 (commonly used, hyperoxic lab conditions). Overall, analyses of C. parvum-infected BSI explants revealed a downregulation of conversion rates of key metabolites-such as glucose, lactate, pyruvate, alanine, and aspartate-at 3 hpi, followed by a rapid increase in the same conversion rates at 6 hpi. Moreover, PCA revealed physioxia as a driving factor of metabolic responses in C. parvum-infected BSI explants. Overall, the ex vivo model described here may allow scientists to address pending questions as to how host cell-microbiome alliances influence intestinal epithelial integrity and support the development of protective intestinal immune reactions against C. parvum infections in a realistic scenario under physioxic conditions.

Pharmacokinetic functions of human induced pluripotent stem cell-derived small intestinal epithelial cells.

To develop a novel intestinal drug absorption system using intestinal epithelial cells derived from human induced pluripotent stem (iPS) cells, the cells must possess sufficient pharmacokinetic functions. However, the CYP3A4/5 activities of human iPS cell-derived small intestinal epithelial cells prepared using conventional differentiation methods is low. Further, studies of the CYP3A4/5 activities of human iPS-derived and primary small intestinal cells are not available. To fill this gap in our knowledge, here we used forskolin to develop a new differentiation protocol that activates adenosine monophosphate signaling. mRNA expressions of human iPS cell-derived small intestinal epithelial cells, such as small intestine markers, drug-metabolizing enzymes, and drug transporters, were comparable to or greater than those of the adult small intestine. The activities of CYP3A4/5 in the differentiated cells were equal to those of human primary small intestinal cells. The differentiated cells had P-glycoprotein and PEPT1 activities equivalent to those of Caco-2 cells. Differentiated cells were superior to Caco-2 cells for predicting the membrane permeability of drugs that were absorbed through a paracellular pathway and via drug transporters. In summary, here we produced human iPS cell-derived small intestinal epithelial cells with CYP3A4/5 activities equivalent to those of human primary small intestinal cells.

Development and characterization of 2-dimensional culture for buffalo intestinal cells.

Small intestinal epithelial cells (IEC) play a major role in the absorption of nutrients and toxins. Due to the similarity of genome-wide single copy protein orthologues between cattle and human, establishment of ruminant's primary small IEC culture could be a valuable tool for toxicity studies. Therefore, the current study focused on the development and characterization of buffalo IEC culture, as cattle slaughter is banned in India. The buffalo jejunum fragments were washed consecutively several times in saline, warm phosphate buffered saline (PBS), PBS with 5 mM dithiothreitol, digesting solution and 2% sorbitol in PBS. The cells were cultured on 17 µg/cm2 collagen coated plates and transwell plates with serum (2% Fetal bovine serum (FBS) and 10% FBS) and serum-free culture conditions. The cells were differentiated into typical epithelial cobblestone morphology from day 5 onwards in 50% successful cultures. The cultured IEC were characterized by gene expression of epithelial cell markers, cytokeratin and vimentin, and enterocyte markers like villin, zonula occluden (ZO1), fatty acid binding protein 2 (FABP2) and small intestinal peptidase (IP). Based on the morphology and gene expression profile, 10% FBS has been recommended for culturing primary buffalo IEC on collagen coated plates for 10 days. However, 50% of the successful cultures could not show epithelial phenotype on 10% FBS culture conditions even on collagen coated plates. Interestingly, undifferentiated IEC showed an increasing expression of FABP2, IP and ZO1 transcripts compared to differentiated intestinal cells with 10% FBS on collagen plates. Therefore, future studies are needed to understand the role of FABP2, IP and ZO1 in differentiation of buffalo IEC.

Fish Skin Gelatin Hydrolysate Production by Ginger Powder Induces Glutathione Synthesis To Prevent Hydrogen Peroxide Induced Intestinal Oxidative Stress via the Pept1-p62-Nrf2 Cascade.

Gelatin hydrolysate was reported to contain large amounts of biologically active peptides with excellent antioxidant properties. However, its inducement of antioxidant response within cells and the underlying molecular mechanism are far from clear. Here, gelatin from Nile tilapia skin was hydrolyzed by ginger protease to produce antioxidant hydrolysate, and three fish skin gelatin hydrolysate fractions (FSGHFs) were obtained by ultrafiltration. Fractionation of the hydrolysate increased the free radical scavenging capacity of the FSGHFs, particularly FSGHF3, which showed the lowest molecular weight (below 1000 Da). Furthermore, FSGHF3 treatment prior to H2O2 exposure increased cell viability and membrane integrity in IPEC-J2 cells. H2O2-induced ROS production and epithelial barrier damage were suppressed by FSGHF3 pretreatment. FSGHF3 stimulated the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), along with increases in the mRNA and protein expression of catalytic and modulatory subunits of γ-glutamylcysteine ligase as well as in the level of glutathione. Silencing of Nrf2 or p62 (an upstream regulator of Nrf2) suppressed FSGHF3-induced Nrf2 activation and its protection against H2O2-induced oxidative stress. Moreover, oligopeptides in FSGHF3 may mediate the cytoprotective effect against oxidative stress, which was confirmed by the result that FSGHF3 failed to inhibit the ROS production in H2O2-exposed cells with the knockdown of Pept1 (an oligopeptide transporter). Therefore, FSGHF3 can induce glutathione synthesis and prevent oxidative stress through the Pept1-p62-Nrf2 cascade and thus may be a functional food for gastrointestinal dysfunction.

P2X7 receptor mediates NLRP3-dependent IL-1ß secretion and parasite proliferation in Toxoplasma gondii-infected human small intestinal epithelial cells.

BACKGROUND: Toxoplasma gondii can invade and replicate in all nucleated cells in a wide range of host species, and infection induces IL-1ß production. IL-1ß plays central roles in the stimulation of the innate immune system and inflammation. However, little is known of the innate immune responses in human fetal small intestinal epithelial cells (FHs 74 Int cells) after T. gondii infection. METHODS: FHs 74 Int cells were infected with the T. gondii GFP-RH strain. Then, IL-1ß production and its mechanisms of action were evaluated using ELISA, MTT cell viability assays, Western blotting, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), and gene-specific small interfering RNA (siRNA) transfection. RESULTS: Infection of FHs 74 Int cells by T. gondii triggered significant time- and dose-dependent IL-1ß production. Although T. gondii activated NLRP1, NLRP3, NLRC4 and AIM2 inflammasomes in FHs 74 Int cells, NLRP3 levels were consistently and significantly time-dependently increased, while the other inflammasomes were not. Transfection with siRNA targeting NLRP3, cleaved caspase-1 (Casp-1) or ASC significantly reduced T. gondii-induced IL-1ß production, whereas T. gondii proliferation was markedly increased. Toxoplasma gondii infection activated P2X7 receptor (P2X7R) levels in FHs 74 Int cells in a time-dependent manner; however, transfection with siRNA targeting P2X7R significantly reduced T. gondii-induced IL-1ß secretion and substantially increased T. gondii proliferation, which is mediated by decreased protein expression levels of NLRP3, cleaved Casp-1 and ASC. Collectively, NLRP3-dependent IL-1ß secretion is mediated by P2X7R in small intestinal epithelial cells in response to T. gondii infection, thereby controlling parasite proliferation. CONCLUSIONS: This study revealed that the P2X7R/NLRP3 pathway plays important roles in IL-1ß secretion and inhibition of T. gondii proliferation in small intestinal epithelial cells. These results not only contribute to our understanding of the mucosal immune mechanisms of T. gondii infection but also offer new insight into the identification of innate resistance in the gut epithelium.

Lipopolysaccharide Upregulated Intestinal Epithelial Cell Expression of Fn14 and Activation of Fn14 Signaling Amplify Intestinal TLR4-Mediated Inflammation.

TLR4 in intestinal epithelial cells has been shown both inflammatory and homeostatic roles following binding of its cognate ligand lipopolysaccharide (LPS). TWEAK-Fn14 axis plays an important role in pathologies caused by excessive or abnormal inflammatory responses. This study aimed to evaluate potential cross-talk between TLR4 and TWEAK/Fn14 system in porcine small intestinal epithelial cells. Our in vivo results showed that, compared with the age-matched normal control piglets, increased expression of Fn14 in epithelium and decreased TWEAK expression in lamina propria were detected in the small intestinal of piglets stimulated with LPS. Consistent with this finding, treatment with LPS increased the expression of Fn14 and TLR4 while decreased TWEAK expression in porcine small intestinal epithelial cell lines SIEC02. Interestingly, modulating Fn14 activation using agonistic anti-Fn14 decreased TLR4-mediated TNF-α production by SIEC02. In addition, pretreatment of LPS-stimulated SIEC02 with recombinant TWEAK protein suppresses the expression of Fn14 and TNF-α and inhibits the negative impact of LPS on the tight junctional protein occludin expression. In conclusion, this study demonstrates that the TWEAK-independent Fn14 activation augments TLR4-mediated inflammatory responses in the intestine of piglets. Furthermore, the TWEAK-dependent suppression of Fn14 signaling may play a role in intestinal homeostasis.

Epidermal growth factor upregulates serotonin transporter and its association with visceral hypersensitivity in irritable bowel syndrome.

AIM: To investigate the role of epidermal growth factor (EGF) in visceral hypersensitivity and its effect on the serotonin transporter (SERT). METHODS: A rat model for visceral hypersensitivity was established by intra-colonic infusion of 0.5% acetic acid in 10-d-old Sprague-Dawley rats. The visceral sensitivity was assessed by observing the abdominal withdrawal reflex and recording electromyographic activity of the external oblique muscle in response to colorectal distension. An enzyme-linked immunosorbent assay was used to measure the EGF levels in plasma and colonic tissues. SERT mRNA expression was detected by real-time PCR while protein level was determined by Western blot. The correlation between EGF and SERT levels in colon tissues was analyzed by Pearson's correlation analysis. SERT function was examined by tritiated serotonin (5-HT) uptake experiments. Rat intestinal epithelial cells (IEC-6) were used to examine the EGF regulatory effect on SERT expression and function via the EGF receptor (EGFR). RESULTS: EGF levels were significantly lower in the rats with visceral hypersensitivity as measured in plasma (2.639 ± 0.107 ng/mL vs 4.066 ± 0.573 ng/mL, P < 0.01) and in colonic tissue (3.244 ± 0.135 ng/100 mg vs 3.582 ± 0.197 ng/100 mg colon tissue, P < 0.01) compared with controls. Moreover, the EGF levels were positively correlated with SERT levels (r = 0.820, P < 0.01). EGF displayed dose- and time-dependent increased SERT gene expressions in IEC-6 cells. An EGFR kinase inhibitor inhibited the effect of EGF on SERT gene upregulation. SERT activity was enhanced following treatment with EGF (592.908 ± 31.515 fmol/min per milligram vs 316.789 ± 85.652 fmol/min per milligram protein, P < 0.05) and blocked by the EGFR kinase inhibitor in IEC-6 cells (590.274 ± 25.954 fmol/min per milligram vs 367.834 ± 120.307 fmol/min per milligram protein, P < 0.05). CONCLUSION: A decrease in EGF levels may contribute to the formation of visceral hypersensitivity through downregulation of SERT-mediated 5-HT uptake into enterocytes.