KIOM-79에 의한 p65 단백질의 핵내 이동 및 iNOS 발현 억제

김진숙(Jin-Sook Kim),장대식(Dae-Sik Jang),전영진(Young-Jin Jeon),유호진(Ho-Jin You),박경한(Kyeong Han Park),김 명(Jin Ming),문형윤(Hyung-Yoon Moon),윤상필(Sang-Pil Yoon),장인엽(In-Youb Chang) 대한해부학회 2006 Anatomy & Cell Biology Vol.39 No.2

후박 (Magnolia officinalis), 갈근 (Pueraria lobata), 감초 (Glycyrrhiza uralensis) 및 대극(Euphorbia pekinensis)은 염증성질병을 포함한 다양한 질병을 치료하기 위해 사용되었다. 본 실험에서는 각각의 천연물의 복합추출물인 KIOM-79가 큰포식세포에서 inducible NO synthase (iNOS) 유전자의 발현을 억제시키는 기전을 밝혀내기 위하여 면역형광염색, RT-PCR, electrophoretic mobility shift assay (EMSA) 등을 실시하였다. 면역형광염색 소견을 보면 RAW 264.7 세포에 lipopolysaccharide (LPS)에 의해 유도된 iNOS 단백질의 발현이 KIOM-79에 의해 억제되는 것을 알 수 있다. Western blot과 RT-PCR 분석에 의하면 KIOM-79가 LPS에 의한 iNOS의 발현을 억제함을 확인 할 수 있다. LPS와 KIOM-79를 처리한 RAW 264.7 세포에서 nuclear extract를 추출하여 EMSA로 분석한 결과, LPS에 의해 유도된 NF-κB/Rel의 DNA 결합이 KIOM-79에 의해 억제됨을 확인하였다. 면역형광염색 소견을 보면 NF-κB/Rel의 구성단백질 중의 하나인 p65단백질은 세포질에서 발현되고 있으며, LPS를 처리하면 p65가 핵으로 이동함을 알 수 있다. 이때 KIOM-79를 처리하면 LPS에 의한 p65의 핵 내 이동이 억제됨을 확인할 수 있다. 결론적으로, KIOM-79는 큰포식세포에 작용해 iNOS 유전자의 발현을 억제하며, 이러한 큰포식세포의 활성억제는 p65 단백질의 핵 내 이동 억제를 통해서 유도되는 것으로 사료된다. We demonstrate that KIOM-79, combined extracts isolated from Magnolia officinalis, Pueraria lobata, Glycyrrhiza uralensis, and Euphorbia pekinensis, inhibits LPS-induced expression of iNOS gene in RAW 264.7 cells. Treatment of RAW 264.7 cells with KIOM-79 inhibited LPS-stimulated nitric oxide production in a doserelated manner. Immunohisto-chemical staining of iNOS and RT-PCR analysis showed that the decrease of NO was due to the inhibition of iNOS gene expression. Immunostaining of p65 and EMSA showed that KIOM-79 inhibited NF-κ/Rel nuclear translocation and DNA binding, respectively. Collectively, this series of experiments indicates that KIOM inhibits iNOS gene expression by blocking NF-κ/Rel. Due to the critical role that NO release plays in mediating inflammatory responses, the inhibitory effects of KIOM-79 on iNOS suggest that KIOM-79 may represent a useful anti-inflammatory agent.

위 평활근에서 비교감성 비콜린성 이완반응과 Substance P에 의한 조절

허광식 ( Gwang Sik Heo ),장성종 ( Sung Jong Chang ),박찬국 ( Chan Guk Park ),김만우 ( Man Woo Kim ),장인엽 ( In Youb Jang ),김진호 ( Jin Ho Kim ),신무경 ( Moo Kyoung Shin ),염철호 ( Cheol Ho Yeum ),윤평진 ( Pyung Jin Yoon ),전제열 대한소화기학회 2003 대한소화기학회지 Vol.41 No.5

Background/Aims: In a gastric fundic strip, electrical field stimulation (EFS) evokes TTX-sensitive biphasic responses, consisting firstly of cholinergic contraction followed by a transient relaxation. It is well known that nonadrenergic noncholinergic (NANC) inhibitory nerve mediates a transient relaxation. This study was performed to investigate the characterization of relaxation and its modulation by substance P. Methods: Using Guinea-pig gastric fundic smooth muscle tissues, we recorded mechanical contractions induced by EFS in the organ bath with platinum. Results: NG-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, reduced EFS-induced relaxation and these effects were reversed by L-arginine, a precursor of nitric oxide (NO). Sodium nitroprusside, a NO-donor, suppressed the fundic basal tension. Cell permeable 8-bromo-cGMP inhibited noradrenaline-induced contraction. The application of substance P increased basal tension and EFS-induced contraction and relaxation. NK-1 receptor antagonist ([D-Pro9-(spiro--lactam)9,10-Trp11]-Substance P) inhibited substance P-induced effects. TEA and apamin, K+ channel blockers, increased basal tension and EFS-induced relaxation. Conclusions: These results indicate that NANC inhibitory responses are mainly mediated by NO in the guinea-pig fundus and the release of NO is modulated by substance P through NK-1 receptor and by prejunctional K+ channels. (Korean J Gastroenterol 2003;41:358-365)

소화관 기계적 폐쇄에 따른 Interstitial Cells of Cajal (ICC) 및 위장관운동의 변화

전제열(Jae-Yeoul Jun),정춘해(Choon-Hae Chung),유호진(Ho-Jin You),김경희(Kyung-Hee Kim),김장만(Jang-Man Kim),김기훈(Kee-Hune Kim),박도영(Do-Young Park),장인엽(In-Youb Chang) 대한해부학회 2002 Anatomy & Cell Biology Vol.35 No.5

Interstitial cells of Cajal (ICC)는 내장 민무늬근세포와 소화관 신경종말들과 밀접한 해부학적 연관성을 가지고 있으며, ICC가 소화관 연동운동에 있어서 소화관 연동운동의 향도잡이 역할을 할 뿐만 아니라, 소화관신경으로부터 민무늬근세포로 전해지는 신경전달을 매개, 조절한다고 알려져 있다. 최근 여러 소화관운동장애 질환에서 ICC의 감소 및 소실이 병인과 연관성이 있다는 주장들이 제기되는 실정이다. 이에 따라 저자들은 수술적으로 생쥐 작은창자를 부분폐쇄를 시킨 다음, 작은창자 근육층의 비대증을 유도한 후 소화관 운동의 변화 및 ICC의 형태학적 변화를 밝혀내고자 ICC에 대한 면역형광염색과 전기생리학적 실험을 병행하여 실시하였다. 작은창자를 부분폐쇄시키고 2주 후, 폐쇄부위보다 입쪽의 작은창자부위에서 육안적으로 창자의 직경이 증가하고, 광학현미경적으로 근육층의 비대가 나타나는 것을 관찰할 수 있었다. ICC에 대한 면역형광염색(c-kit staining) 결과, ICC 그물망이 폐쇄부위보다 입쪽으로 25 mm 정도까지 소실 또는 뚜렷하게 감소되는 것을 관찰할 수 있었으며, 이러한 형태학적 결과와 병행하여 전기생리학적으로도 서파의 소실 또는 감소가 나타남을 관찰할 수 있었다. 그러나 폐쇄부위보다 항문쪽에서는 ICC와 서파의 이상소견은 관찰되지 않았다. 본 실험 결과 소화관의 폐쇄에 따라 입쪽부위 ICC의 소실과 서파의 소실을 관찰할 수 있었다. 따라서 앞으로 ICC 구조를 변화하게 하는 분자생물학적 그리고 유전학적 신호체계에 대해 면밀한 분석을 할 경우 소화관운동장애의 발생원인 및 치료에 계기를 마련할 수 있을 것으로 사료된다. Interstitial cells of Cajal (ICC) are the pacemakers in gastrointestinal slow wave, and also transduce signal inputs from the enteric nervous system to smooth muscle. The abnormal motility corresponded to a lack or decreasing of ICC and a disruption of electrical slow waves. So we developed partial obstruction model in murine small intestine and investigated changes in the ICC networks and electrical activity in the obstructed bowel using c-kit immunohistochemistry and intracelluar electrophysiological techniques. Two weeks following the onset of a partial obstruction, the small intestine increased in diameter and muscular hypertrophy was developed oral to the obstruction site. ICC were absent or only weak at 1~25 mm oral to the occlusion site, and this disruption was accompanied by the loss of electrical slow wave. ICC networks and slow waves were normal appearance aboral to the clip. In conclusion, The present results showed that partial intestinal obstruction induced the loss of ICC networks and slow waves. These result will provide a valuable aid for understanding pathogenesis of intestinal motility disorder, and this model may be an important tool for evaluating genetic or molecular factor for the therapeutic opportunities of motility disorder in human.

안구적출에 따른 위둔덕의 Calbindin D-28k과 c-fos의 변화에 관한 연구

김명,김기훈,김주영,하현철,안명수,김장민,조향훈,정명섭,장인엽 朝鮮大學校 附設 醫學硏究所 2005 The Medical Journal of Chosun University Vol.30 No.2

Changes of Calbindin D-28k- and c-fos-immunoreactivities in the superior collicuclus after Eye Enucleation. Objective and methods: Calcium-binding proteins (CBPs) play an important role in the protection, differentiation and reorganization of the central nervous system. The effects of neonatal retinal deafferentation on a CBPs, calbindin D-28k were examined immunohistochemically in the superficial layer of the rat superior colliculus. Also early gene familly c-fos was examined to evaluate the neuronal characteristics in the superior colliculus after monocular enucleation. Results: On the experimental side of superior colliculus, the number of calbindin D-28k-immunoreactive (IR) cells was reduced (77.4% compared to control), but not fibers. Appearance of c-fos-like immunoreactivity was represented much more in the ipsilateral superior colliculus than contralateral side within 24h after eye enucleation. Conclusion: These results suggest that the changes of retinotectal projection may alter the expressional pattern of calbindin D-28k and c-fos expression.

생쥐 위장관의 Interstitial cells of Cajal(ICC)에 대한 면역조직화학적 연구

김영철,차경훈,신무경,임건한,김주영,안병수,김장만,양경철,박도영,오재욱,장인엽 조선대학교 2003 The Medical Journal of Chosun University Vol.28 No.1

Background and Objectives : Interstitial cells of Cajal(ICC) are the pacemakers in gastrointestinal tract that modulates gastrointestinal motiliey and these cells also transmit neural input from enteric nerves to smooth muscles. Recent work on tissues from patients with motility disorders that suggest that loss or defect in ICC could be related to pathophysiology in human and animal models. Immunolabelling of ICC in intestinal wall is recently developed by using specific marker, anti-c-kit antibody. Immunohistochemistry was done for ICC network in attempt to provide a morphological basis for the mechanism regulating gastrointestinal motility Methods : Cryosection was done, and whole-mount preparations of mouse stomach, gastrointestinal tract were immunolabelled using the anti-c-Kit. Immunolabelled ICC networks were observed under a confocal laser scanning microscopy. Results : According to three dimensional reconstruction study, we found that the c-Kit-positive celluar networks were widely distributed in the gastrointestinal muscle (1) circular muscle layer(IC-IM), (2) myenteric plexus(IC-MY), (3) deep muscular plexus(IC-DMP) in ileum, (4) submucosal plexus(IC-SMP) and longitudinal muscle layer(IC-LM) in colon. Conclusion : The characteristic profiles of ICC celluar networks provide a morphological basis upon the mechanism regulating gastrointestinal motility. Additional studies for the enteric nerves-ICC interaction are need to evaluate the detailed roles of Icc in gastrointestinal tract.

개 대뇌겉질에서 Platelet-Derived Growth Factor α-Receptor의 출생 후 발달에 관한 면역조직화학적 연구

윤영,안병수,김인정,양경철,박선홍,김기훈,박도영,김장만,문정석,장인엽,조하영 조선대학교 부설 의학연구소 2002 The Medical Journal of Chosun University Vol.27 No.1

Background and Objectives : The localization of platelet-derived growth factor-α receptor (PDGF-α R) was commonly restricted to oligodendrocyte progenitors during late embryonic and postnatal development. However, several studies recently demonstrated that mature neurons could also synthesize PDGF-α, Materials and Methods : In the present study, to analyze the distributional pattern of PDGF-αR during postnatal development of the canine cerebral cortex, we used immunohistochemistry on sections of canine brain tissue. Results : We found that neurons of various regions of cerebral cortex exhibited the immunoreactivity to PDGF-αR as early as postnatal day 0, and slightly decreased after postnatal day 14. Whereas neuronal PDGF-αR were maintained at all ages, the oligodendroglia-like expression of PDGF-αR could not be confirmed. Conclusion : The localization of PDGF-αR in immature and mature neurons supports the several roles of PDGF during development, protection and survival of neurons.