• Journal of Animal Reproduction and Biotechnology
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• v.39 no.2
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• pp.105-113
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• 2024

Background: Aflatoxin B1 (AFB1) is a toxic metabolite generated by Aspergillus species and is commonly detected during the processing and storage of food; it is considered a group I carcinogen. The hepatotoxic effects, diseases, and mechanisms induced by AFB1 owing to chronic or acute exposure are well documented; however, there is a lack of research on its effects on the intestine, which is a crucial organ in the digestive process. Dogs are often susceptible to chronic AFB1 exposure owing to lack of variation in their diet, unlike humans, thereby rendering them prone to its effects. Therefore, we investigated the effects of AFB1 on canine small intestinal epithelial primary cells (CSIc). Methods: We treated CSIc with various concentrations of AFB1 (0, 1.25, 2.5, 5, 10, 20, 40, and 80 μM) for 24 h and analyzed cell viability and transepithelial-transendothelial electrical resistance (TEER) value. Additionally, we analyzed the mRNA expression of tight junction-related genes (OCLN, CLDN3, TJP1, and MUC2), antioxidant-related genes (CAT and GPX1), and apoptosis-related genes (BCL2, Bax, and TP53). Results: We found a significant decrease in CSIc viability and TEER values after treatment with AFB1 at concentrations of 20 μM or higher. Quantitative polymerase chain reaction analysis indicated a downregulation of OCLN, CLDN3, and TJP1 in CSIc treated with 20 μM or higher concentrations of AFB1. Additionally, AFB1 treatment downregulated CAT, GPX1, and BCL2. Conclusions: Acute exposure of CSIc to AFB1 induces toxicity, and exposure to AFB1 above a certain threshold compromises the barrier integrity of CSIc.

• Biomedical Science Letters
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• v.8 no.3
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• pp.107-113
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• 2002

Enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 have been shown to colonize the intestinal epithelial cell by the attaching and effacing (AE) mechanism. The AE lesion is mediated by an intimin, of which production and expression are controlled by a 3-Kb eaeA gene located EHEC chromosomal DNA. If the eaeA gene is mutated, EHEC O157:H7 strains lose capacity of adhesion to intestinal epithelial cells. In this study, a 891 bp of the 3'-end region of a gamma intimin was amplified by polymerase chain reaction (PCR). The PCR product was inserted into pSTBlue-1 cloning vector and transformed into DE3 (BL21) competent cell. After plasmid mini-preparation and restriction enzyme digestion of eaeA/891-pSTBlue-1 vector, target eaeA gene was re-inserted into pET-28a expression vector and was transformed. Then the expression of recombinant eaeA/891 (891 bp) gene was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG). The expression of the 40-KDa recombinant protein was identified in SDS-PAGE and confirmed by immunoblotting using the His.Tag$^{\circledR}$ and T$_{7}$.Tag$^{\circledR}$ monoclonal antibody. This recombinant protein expressed by eaeA gene could be applied in further studies on the mechanisms of E. coli O157:H7 infection and the development of recombinant vaccine.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.57-63
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• 1997

Animal and clinical investigations have shown that fluoroquinolones, new quinolone antibacterial agents (NQs), are well absorbed across the intestinal tract, with a bioavailability of 60-90% after oral administration. Although some types of carrier-mediated intestinal transport mechanisms have been reported for enoxacin (ENX), ofloxacin (OFLX) and sparfloxacin (SPFX), recent results using a human intestinal epithelial cell line, Caco-2, indicated a passive or nonsaturable transport of SPFX, one of the most hydrophobic NQs. The mechanism underlying the intestinal absorption of NQs is still largely unknown. The distribution of NQs into peripheral tissues including erythrocytes is very rapid and their tissue-to-plasma concentration ratios (Kp) are considerably larger than those of inulin (an extracellular fluid space marker), in spite of almost complete ionization of NQs at the physiological pH. Our findings suggest that OFLX and lomefloxacin (LFLX) are taken up by rat erythrocytes via a transport system common to that of a water-soluble vitamin, nicotinic acid.

• Asian-Australasian Journal of Animal Sciences
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• v.6 no.2
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• pp.281-290
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• 1993

This experiment was designed to evaluate the effect of degree of unsaturation (Experiment 1) and the chain length of constituent fatty acids of dietary fats (Experiment 2) on-3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activities in the liver and small intestine of chicks. Chicks were fed experimental diets for 10 days and then killed for the determination of the HMG-CoA reductase activities in the intestinal epithelial cell and hepatic microsomes. The hepatic HMG-CoA reductase activity showed the highest value in chicks fed the tallow-containing diet. Chicks fed diets containing safflower or coconut oil resulted in a significantly lower intestinal HMG-CoA reductase activity in comparison with those fed the olive oil-containing diet. The hepatic HMG-CoA reductase activity was significantly higher when fat-free and trilaurin were fed than when any other triglycerides were fed. This activity showed the lowest value in the chicks fed the diet containing tristearin. The HMG-CoA reductase activities in the jejunum and ileum were significantly or tended to be higher when trilaurin was fed than when any other triglycerides were fed. Except when trilaurin was fed, the presence of saturated fat in the diet did not have a significant effect on the intestinal HMG-CoA reductase activity, unlike the effect shown when a highly unsaturated fat was added to the diet. There was no significant correlation between the HMG-CoA reductase activities of the liver and intestinal, and the HMG-CoA reductase activity and cholesterol content of the intestinal epithelial cells.

• Journal of Animal Science and Technology
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• v.63 no.1
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• pp.114-124
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• 2021

The objective of this study was to characterize the enzymatic hydrolysis of lipopolysaccharide (LPS) by wheat phytase and to investigate the effects of wheat phytase-treated LPS on in vitro toxicity, cell viability and release of a pro-inflammatory cytokine, interleukin (IL)-8 by target cells compared with the intact LPS. The phosphatase activity of wheat phytase towards LPS was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine. In vitro toxicity of LPS hydrolyzed with wheat phytase in comparison to intact LPS was assessed. Cell viability in human aortic endothelial (HAE) cells exposed to LPS treated with wheat phytase in comparison to intact LPS was measured. The release of IL-8 in human intestinal epithelial cell line, HT-29 cells applied to LPS treated with wheat phytase in comparison to intact LPS was assayed. Wheat phytase hydrolyzed LPS, resulting in a significant release of inorganic phosphate for 1 h (p < 0.05). Furthermore, the degradation of LPS by wheat phytase was nearly unaffected by the addition of L-phenylalanine, the inhibitor of tissue-specific alkaline phosphatase or L-homoarginine, the inhibitor of tissue-non-specific alkaline phosphatase. Wheat phytase effectively reduced the in vitro toxicity of LPS, resulting in a retention of 63% and 54% of its initial toxicity after 1-3 h of the enzyme reaction, respectively (p < 0.05). Intact LPS decreased the cell viability of HAE cells. However, LPS dephosphorylated by wheat phytase counteracted the inhibitory effect on cell viability. LPS treated with wheat phytase decreased IL-8 secretion from intestinal epithelial cell line, HT-29 cell to 14% (p < 0.05) when compared with intact LPS. In conclusion, wheat phytase is a potential therapeutic candidate and prophylactic agent for control of infections induced by pathogenic Gram-negative bacteria and associated LPS-mediated inflammatory diseases in animal husbandry.

• Food Science of Animal Resources
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• v.24 no.1
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• pp.86-91
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• 2004

The ability of probiotics containing Lactobacillus acidophilus to adhere to the intestinal epithelium may play an important role in colonization of the gastrointestinal tract and preventing enteric pathogen such as enterohemorrhagic E. coli(EHEC O157:H7. In the study, we investigated the adhesion to human intestinal epithelial cells(HT-29) of strains of L. acidophilus(3 from human, 2 from pig, and 1 from calf). All of the tested strains of L. acidophilus were highly observed adhesion ability(from 10$\^$6/ to 10$\^$7/ cfu/mL), compared to L. rhamnosus GG as control. Also, adhered strains of L. acidophilus were significantly preserved in serial wash-out steps. However, no correlation could be observed between cell surface hydrophobicity and adhesion abilities of the tested strains of L. acidophilus. Inhibition of adhesion of EHEC O157:H7 was also examined, a 2 log cycle reduction was observed by all of the tested strains of L. acidophilus. These results suggest that the strains of L. acidophilus with high adhesion ability are resistant to wash-out and adhesion ability inhibition by selected strains of L. acidophilus helps to prevent adhesion of EHEC O157:H7 to intestinal epithelial cells.

• Anatomy and Cell Biology
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• v.56 no.1
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• pp.25-31
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• 2023

Johannes Nathanael Lieberkühn was a prodigious anatomist whose meticulous experiments and precise detailing helped in comprehending the microscopic anatomy of digestive system during early part of eighteenth century. Notably, his inventions in the field of microscopy aptly complemented his quest for anatomical knowledge at microscopic level. He designed a reflector (Lieberkühn reflector) which enhanced the amount of focussed light leading to bright illumination of tissue specimen. He invented the solar microscope which provided excellent resolution of minute anatomical details. Lieberkühn discovered the digestive juice secreting tubular glands (glands of Lieberkühn) present at the base of intestinal villi producing epithelial invaginations (crypts of Lieberkühn). He also described the intricate juxtaposition of blood vessels in relation to a single intestinal villi. Moreover, through empirically designed experimental set up, Lieberkühn was able to demonstrate the flow of lymph from intestinal villi to collecting lymphatic vessels. Also, his grandiose collection of laboratory specimens involving vascular anatomy are a testimony of his untiring efforts in academia. His contributions were seminal in comprehending the anatomy of digestive system and paved the way for future revelations. His work unveiled the enormous scope of microanatomy in medical science and catalysed the advent of histological staining methods a century later.

• Korean Journal of Clinical Laboratory Science
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• v.43 no.4
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• pp.161-170
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• 2011

Quercetin is one of the most distributed flavonoids in the plant kingdom and occurs naturally in a wide range of fruits and vegetables. This study was undertaken to determine whether quercetin exerts beneficial effect against necrotic and apoptotic cell death induced by hydrogen peroxide ($H_2O2$) in intestinal cells using the human-derived cultured T84 colonic epithelial cell line. Necrotic cell death was induced by exposing cells to 0.5 mM $H_2O_2$ for 2 h and apoptosis was induced by incubating cells in normal culture medium for 18 h following exposure of cells to 0.5 mM $H_2O2$ for 2 h. Cell viability was evaluated by the trypan blue exclusion assay and apoptosis was assessed by Hoechst 33258 staining and flow cytometry. $H_2O_2$ induced necrotic cell death in a time and dose-dependent fashion. Both necrotic and apoptotic cell deaths were not prevented by the antioxidants N,N'-diphenyl-p-phenylenediamine(DPPD) and Trolox, whereas both cell deaths induced by the organic hydroperoxide t-butylhydroperoxide (tBHP) were prevented by DPPD, suggesting that $H_2O_2$ induces cell death through a lipid peroxidation-independent mechanism. $H_2O2$-induced necrotic death was prevented by deferoxamine and 3-aminobenzamide, while the apoptotic cell death was not affected by these agents. Quercetin prevented both necrotic and apoptotic cell deaths induced by $H_2O_2$ in a dose-dependent manner. $H_2O_2$ caused activation of poly (ADP-ribose) polmerase (PARP), which was inhibited by deferoxamine, 3-aminobenzamide, and quercetin, but not DPPD. These results indicate that quercetin inhibits both necroticand apoptotic deaths of T84 cells. The anti-necrotic effect of quercetin may be attributed to its iron chelator activity rather than a direct $H_2O_2$ scavenging capacity and antioxidant. The present study suggests that quercetin may play a therapeutic role in the treatment of human gastrointestinal diseases mediated by oxidants.

• Journal of Microbiology and Biotechnology
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• v.25 no.11
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• pp.1849-1855
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• 2015

Staphylococcus aureus plays an important role in sepsis, septic shock, pneumonia, and wound infections. Here, we demonstrate that Lactobacillus plantarum extracts inhibited S. aureus-induced cell death of a human epithelial cell line, HT-29. In particular, we have shown that S. aureus-induced cell death was abolished by neutralization of α-toxin, indicating that α-toxin is the major mediator of S. aureus-induced cell death. DNA fragmentation experiment and caspase assay revealed that the S. aureus-induced cell death was apoptosis. L. plantarum extracts inhibited the generation of effector caspase-3 and the initiator caspase-9 in S. aureus- or α-toxin-induced cell death. Moreover, expression of Bcl-2, an anti-apoptotic protein, was activated in L. plantarum extract-treated cells as compared with the S. aureus- or α-toxin-treated only cells. Furthermore, S. aureus-induced apoptosis was efficiently inhibited by lipoteichoic acid and peptidoglycan of L. plantarum. Together, our results suggest that L. plantarum extracts can inhibit the S. aureus-mediated apoptosis, which is associated with S. aureus spreading, in intestinal epithelial cells, and may provide a new therapeutic reagent to treat bacterial infections.

• Journal of Microbiology and Biotechnology
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• v.28 no.1
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• pp.59-64
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• 2018

Listeria monocytogenes can asymptomatically inhabit the human intestine as a commensal bacterium. However, the mechanism by which L. monocytogenes is able to inhabit the intestine without pathogenic symptoms remains unclear. We compared the invasion efficiency of L. monocytogenes strains with the 268- and 385-bp-long actA gene. Clinical strains SMFM-CI-3 and SMFM-CI-6 with 268-bp actA isolated from patients with listeriosis, and strains SMFM-SI-1 and SMFM-SI-2 with the 385-bp gene isolated from carcasses, were used for inoculum preparation. The invasion efficiency of these strains was evaluated using Caco-2 cells (intestinal epithelial cell line), prepared as normal and healthy cells with tightened tight junctions and senescent cells with loose tight junctions that were loosened by adriamycin treatment. The invasion efficiency of L. monocytogenes strains with the 268-bp-long actA gene was 1.1-2.6-times lower than that of the strains with the 385-bp-long gene in normal and healthy cells. However, the invasion efficiency of both types of strains did not differ in senescent cells. Thus, L. monocytogenes strains with the 268-bp-long actA gene can inhabit the intestine asymptomatically as a commensal bacterium, but they may invade the intestinal epithelial cells and cause listeriosis in senescent cells.