• 대한의생명과학회지
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• 제18권4호
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• pp.362-368
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• 2012

Enterotoxigenic Bacteroides fragilis (ETBF) is an intestinal commensal bacterium implicated as a risk factor for colon cancer. The key virulence factor is a secreted toxin called B. fragilis toxin (BFT). In this study we used an in vitro bioassay to examine the prevalence of ETBF in colonic washings from patients with colorectal polyps and normal control patients. We found that 9.3% of polyp patients and 10.9% of non-polyp patients harbored ETBF, respectively. A total of nine ETBF clinical isolates were isolated and confirmed to be positive for the BFT gene by PCR analysis and the ability to induce IL-8 secretion in the colonic epithelial cell line HT29/c1. Two of the ETBF clinical strains were characterized further in vitro and in vivo. We found that the two ETBF clinical isolates induced E-cadherin cleavage in HT29/c1 cells and promoted colonic inflammation in C57BL/6 mice. Our results indicate that the prevalence of ETBF in polyp patients were similar in non-polyp patients suggesting that ETBF carriage does not positively correlate to polyp incidence.

• Archives of Pharmacal Research
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• 제26권1호
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• pp.70-75
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• 2003

The objective of the present study was to investigate the uptake process of 4-Phenylazobenzoxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-peptide), a hydrophilic and collagenase-labile pentapeptide, by isolated hepatocytes. For comparison, the uptake of Pz-peptide by Caco-2 cells and colonic cells, two known paracellular routes of Pz-peptide, was also evaluated. A simple and sensitive reversed-phase HPLC assay method using UV detection has been developed. The coefficient of variation for all the criteria of validation were less than 15%. The method was, therefore, considered to be sutable for measuring the concentration of Pz-peptide in the biological cells. Pz-peptide was extensively uptaked into hepatocytes. The initial velocity of Pz-peptide uptake assessed from the initial slope of the curve was plotted as Eadie-Hofstee plots. The maximum velocity ($V_{max}$) and the Michaelis constant ($K_m$) were 0.190$\pm$0.020 $nmol/min/10^6$ cells and 12.1$\pm$3.23 $\mu$M, respectively. The permeability-surface area product ($PS{influx}$) was calculated to be 0.0157 ml/min/10^6$ cells. $V_{max}$ and $K_m$ values for Caco-2 cells were calculated to be 6.22$\pm$0.930 pmol/min/10^6$ cells and 82.8$\pm$8.37 $\mu$M, respectively, being comparable with those of colonocytes (6.04$\pm$1.03 pmol/min/10^6$ cells and 87.8$\pm$13.2 $\mu$M, respectively). $PS_{influx}$ values for Caco-2 cells and colonocytes were calculated to be 0.0751 $\mu$l/min/10^6$ cells and 0.0688 $\mu$l/min/10^6$ cells, respectively. The more pronounced uptake of Pz-peptide by hepatocytes, when compared with Caco-2 cells and colonocytes, is probably due to its specific transporter. In conclusion, Pz-peptide, a paracellularly transported pentapeptide in the intestine and ocular epithelia, was uptaked into hepatocytes extensively. Although Pz-peptide is able to be uptaked into the Caco-2 cells and colonocytes, it is less pronounced when compared with hepatocytes. $PS_{influx}$ values of Caco-2 cells and colonocytes for unbound Pz-peptide under linear conditions were less than 0.4% when compared with that of hepatocytes.

• Journal of Microbiology and Biotechnology
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• 제27권4호
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• pp.694-700
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• 2017

Clostridium difficile, which causes pseudomembranous colitis, releases toxin A and toxin B. These toxins are considered to be the main causative agents for the disease pathogenesis, and their expression is associated with a marked increase of apoptosis in mucosal epithelial cells. Colonic epithelial cells are believed to form a physical barrier between the lumen and the submucosa, and abnormally increased mucosal epithelial cell apoptosis is considered to be an initial step in gut inflammation responses. Therefore, one approach to treating pseudomembranous colitis would be to develop agents that block the mucosal epithelial cell apoptosis caused by toxin A, thus restoring barrier function and curing inflammatory responses in the gut. We recently isolated an antimicrobial peptide, Periplanetasin-2 (Peri-2, YPCKLNLKLGKVPFH) from the American cockroach, whose extracts have shown great potential for clinical use. Here, we assessed whether Peri-2 could inhibit the cell toxicity and inflammation caused by C. difficile toxin A. Indeed, in human colonocyte HT29 cells, Peri-2 inhibited the toxin A-induced decrease in cell proliferation and ameliorated the cell apoptosis induced by this toxin. Moreover, in the toxin A-induced mouse enteritis model, Peri-2 blocked the mucosal disruption and inflammatory response caused by toxin A. These results suggest that the American cockroach peptide Peri-2 could be a possible drug candidate for addressing the pseudomembranous colitis caused by C. difficile toxin A.

• Asian Pacific Journal of Cancer Prevention
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• 제17권3호
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• pp.1023-1035
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• 2016

The purpose of this study was to investigate the role of colon cancer stem cells (CSCs) during chemically-induced rat multi-step colon carcinogenesis with or without the treatment with a specific cyclooxygenase-2 inhibitor drug (celecoxib). Two experiments were performed, the first, a short term 12 week colon carcinogenesis bioassay in which only surrogate markers for colon cancer, aberrant crypt foci (ACF) lesions, were formed. The other experiment was a medium term colon cancer rat assay in which tumors had developed after 32 weeks. Treatment with celecoxib lowered the numbers of ACF, as well as the tumor volumes and multiplicities after 32 weeks. Immunohistochemical proliferating cell nuclear antigen (PCNA) labeling indexes LI (%) were downregulated after treatment by celecoxib. Also different cell surface antigens known to associate with CSCs such as the epithelial cell adhesion molecule (EpCAM), CD44 and CD133 were compared between the two experiments and showed differential expression patterns depending on the stage of carcinogenesis and treatment with celecoxib. Flow cytometric analysis demonstrated that the numbers of CD133 cells were increased in the colonic epithelium after 12 weeks while those of CD44 but not CD133 cells were increased after 32 weeks. Moreover, aldehyde dehydrogenase-1 activity levels in the colonic epithelium (a known CSC marker) detected by ELISA assay were found down-regulated after 12 weeks, but were up-regulated after 32 weeks. The data have also shown that the protective effect of celecoxib on these specific markers and populations of CSCs and on other molecular processes such as apoptosis targeted by this drug may vary depending on the genetic and phenotypic stages of carcinogenesis. Therefore, uncovering these distinction roles of CSCs during different phases of carcinogenesis and during specific treatment could be useful for targeted therapy.

• Natural Product Sciences
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• 제19권4호
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• pp.290-296
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• 2013

In preliminary tests, we examined the effect of several fractions isolated from fermented pine needle extract on pacemaker potentials in cultured interstitial cells of Cajal (ICCs) from the mouse colon using a whole cell patch clamp technique. Among these fractions, Fraction 3 (F3) elicited the most powerful depolarization of membrane. Therefore, the aim of the present study was to investigate the effect of F3 obtained from fermented extract of Pinus densiflora needle on pacemaker potentials in ICCs and to establish its mechanism of action. Colonic ICCs generated spontaneous periodic pacemaker potentials in the current-clamp mode. F3 depolarized the membrane and decreased the frequency and amplitude of pacemaker potentials in a dose-dependent fashion. The F3-induced effects on pacemaker potentials were blocked by methoctramine, a muscarinic $M_2$ receptor antagonist, and by glycopyrrolate, a muscarinic $M_3$ receptor antagonist. The F3-induced effects on pacemaker potentials were blocked by external $Na^+$-free solution and by flufenamic acid, a non-selective cation channel blocker, as well as by the removal of external $Ca^{2+}$ and in the presence of thapsigargin, a $Ca^{2+}$-ATPase inhibitor in the endoplasmic reticulum. Taken together, these results suggest that F3 of pine needle extract modulates the pacemaker activity of colonic ICCs by the activation of non-selective cation channels via muscarinic $M_2$ and $M_3$ receptors. And external $Ca^{2+}$ influx and intracellular $Ca^{2+}$ release are involved in F3 actions on ICCs.

• 대한수의학회지
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• 제33권3호
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• pp.429-442
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• 1993

Serpulina(S) hyodysenteriae B 204 strain이 생산한 세포독소에 의한 장점막의 변화를 알아보기 위하여 일반사육조건에서 사육된 8주령, 수컷 잡종돼지 2마리의 결장을 외과적으로 결찰한 계제(loop)를 이용하여 실험하였다. Serpulina균을 trypticase soy broth(TSB)에 배양하여 결찰한 결장계제 4곳에 각각 멸균한 Serpulina균 TSB, 여과한 Serpulina균 TSB, 세척한 Serpulina균, 무처치 Serpulina균 TSB를 접종하였다. 점막조직을 접종(p.i.) 24, 48시간 후에 채취한 후 처리하여 주사전자현미경과 투과전자현미경으로 관찰하였다. 여과한 Serpulina균 TSB를 접종한 경우는 변화가 관찰되지 않았으나 세척한 Serpulina균을 접종한 경우는 무처치 Serpulina균 TSB를 접종한 부위에서 나타난 것과 유사한 초기변화가 관찰되었다. 본 실험의 결과는 Serpulina균의 세포독소가 실험적 감염시 초기병변형성에 별로 기여하지 않음을 시사하였다. Serpulina균의 독소가 병변을 야기하는데 관여하는 기전은 밝혀지지 않았다.

• 대한한의학방제학회지
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• 제27권4호
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• pp.237-244
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• 2019

Objectives : The purpose of this study was to investigate the effects of Carthami flos on pacemaker potentials of small intestinal and colonic Interstitial Cells of Cajal (ICC). Methods : To dissociate the ICC, we used enzymatic digestions from the small intestine and colon in mice. In the ICC, the electrophysiological whole-cell patch-clamp configuration was used to record pacemaker potentials in the cultured ICC. Results : 1. The ICC generated pacemaker potentials in the murine small intestine and colon. 2. Pretreatment with a Ca²⁺ free solution and thapsigargin, a Ca²⁺-ATPase inhibitor in the endoplasmic reticulum, stopped the pacemaker potentials. In the case of Ca²⁺-free solutions, Carthami flos did not induce membrane depolarizations in the murine small intestine and colon. However, when thapsigargin in a bath solution was applied, Carthami flos induced membrane depolarizations only in the murine colon. 3. Pretreatment with 2-APB (transient receptor potential melastatin (TRPM) channel inhibitor) abolished the pacemaker potentials and suppressed Carthami flos-induced effects in the murine small intestine and colon. 4. However, pretreatment with T16Ainh-AO1 (Ca²⁺ activated Cl- channel; anoctamin 1 (ANO1) inhibitor) did not affect the pacemaker potentials and induced Carthami flos-induced effects only in the murine small intestine. Conclusions : These results suggest that Carthami flos can modulate the pacemaker activity of ICC and the mechanisms underlying pacemaking in ICC might be different in the small intestine and the colon.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• 제12권2호
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• pp.177-182
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• 2009

목 적: 소아에서 다양한 원인에 의해 발생한 대장염에서 점막의 형태학적 변화와 세포 접합에 다양한 변화가 있으리라 예상되어 세포 간의 결합을 유지하는 E-cadherin의 변화를 살펴보았다. 방 법: 1998년 1월부터 2003년 8월까지 부산대학교병원 소아청소년과에서 하부 위장관 내시경술과 대장점막 조직 검사를 통해 대장염으로 진단된 39명을 대상으로 하였다. 파라핀 블록에서 면역조직화학염색법을 이용하여 E-cadherin의 세포 내 발현을 조사하였다. 주변의 정상 조직과 비교하여 E-cadherin의 발현이 동일한 강도와 양상을 가진 세포가 50% 이상인 경우를 정상으로 판정하였고, 발현이 정상인 세포가 50% 미만이거나 염색 분포의 이상이 있거나 전혀 염색되지 않은 경우를 이상으로 판정하였다. 결 과: 1) 본 연구에서 비특이성 대장염 15예(38.5%), 크론병 7예(17.9%), 감염성 대장염 5예(12.8%), 음식 단백 과민성 직결장염 5예(12.8%), 궤양성 대장염 3예(7.7%), Henoch-Schonlein purpura 대장염 2예(5.1%), 그외 베체트병, 허혈성 대장염 1예가 포함되었다. 2) 모든 종류의 대장염에서 상피세포 E-cadherin 발현 감소가 관찰되었으며, 77%의 대상 표본에서 E-cadherin 발현감소가 있었다. 3) 활동성 염증이 심한 부위에서 Ecadherin 발현 감소가 현저하였으며 병변부에서 떨어진 상피세포에서는 정상 발현을, 궤양 주위나 재생 상피가 있는 부위는 심한 발현 감소를 보였다. 결 론: 모든 종류의 염증성 대장 질환에서 E-cadherin 발현 감소가 있었다. 이러한 변화는 염증과 궤양이 있는 부위에서 상피세포 접합을 느슨하게 함으로써 상피세포의 재생을 위한 세포의 이동을 용이하게 하기 위한 작용이라고 판단된다.

• 대한임상검사과학회지
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• 제55권1호
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• pp.37-44
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• 2023

Enterotoxigenic Bacteroides fragilis (ETBF)는 염증성장 질환과 대장암을 유발하며 아연 의존성 metalloprotease인 B. fragilis toxin (BFT)를 분비한다. BFT는 epithelial cell의 E-cadherin을 80 kDa ectodomain과 33 kDa intracellular domain으로 분절을 유도한다. 생성된 E-cadherin intracellular domain은 순차적으로 γ-secretase에 의해 분절되어 28 kDa E-cadherin intracellular fragment은 아직까지 밝혀지지 않는 기작으로 분해된다. 본 연구에서는 BFT 유도 E-cadherin 분절로 인해 생성된 28 kDa E-cadherin intracellular fragment는 proteasome에 의해서 분해된다는 것을 확인하였다. 또한 BFT 유도 E-cadherin 분절 기작이 대장암 세포가 아닌 인간 유방암 세포주 BT-474 세포에서도 동일한 기작으로 일어남을 확인하였다. 마지막으로 staurosporine은 인간 유방암 세포주 MCF7 세포에서 E-cadherin의 분절을 유도하고 γ-secretase에 의한 E-cadherin intracellular domain의 분절이 일어났으나 proteasome에 의한 분해는 일어나지 않았다. 이러한 결과는 ETBF가 서식하는 대장이 아닌 유방에서도 BFT에 의한 E-cadherin 분절이 일어날 수 있으며 ETBF가 대장암 이외의 다른 암에도 관여할 수 있음을 시사한다.

• 생명과학회지
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• 제26권5호
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• pp.531-536
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• 2016

본 연구는 최근 연구자 등이 보고한 NQO1 knockout (결핍) 생쥐의 점막기능 감소 및 장염유발 현상을 인간 대장상피세포에서 재확인하는 것이다. 이를 위해, 사람 대장상피세포주인 HT29 세포에 NQO1 억제제인 dicumarol을 처치한 다음 밀착연접 조절인자의 변화를 평가하였다. HT29 세포에 10 μM dicumarol을 처치하여 NQO1을 억제하면 인간 대장상피세포의 밀착연접이 유의하게 줄고 구성인자들(ZO1, occludin)의 단백질 양도 감소함을 확인하였다. 생쥐 대장 내강에 dicumarol (10 μM)을 직접 처치한 결과 점막 투과율이 현저하게 증가함도 확인하였다(장벽기능 감소). 이는 인간 대장상피세포의 밀착연접 형성과정이 NQO1에 의존적임을 보여준다. 그러나 dicumarol 처치는 ZO1과 occludin의 전사량을 억제하지 않았다. NOQ1 결핍 생쥐에서 ZO1과 occludin의 전사량이 크게 감소한다는 이전 보고를 감안하면, NQO1에 의한 밀착연접 단백질의 양적 조절 과정이 전사를 포함하는 다양한 경로와 연관되어 있음을 시사한다. 즉, NQO1에 의한 ZO1과 occludin 조절 과정에 프로테오좀 의존-단백질 변성과 같은 단백질 파괴 경로가 이용될 수 있다고 사료된다.