내독소로 자극된 당뇨 쥐에서 단백분해효소와 그 억제제 발현

서기현 ( Ki Hyun Seo ),최재성 ( Jae Sung Choi ),나주옥 ( Joo Ok Na ),어수택 ( Soo Taek Uh ),김용훈 ( Yong Hoon Kim ),박춘식 ( Choon Sik Park ) 대한결핵 및 호흡기학회 2006 Tuberculosis and Respiratory Diseases Vol.61 No.3

연구배경: 급성폐손상에서 염증세포의 모집과 활성, 그 염증세포의 부산물인 활성산소, 사이토카인, 기질단백분해효소등과 기획성 세포사멸이 폐실질 파괴와 재구성에 중요한 역할을 하는 것으로 알려져 있다. 또한 당뇨환자에서 세균 감염 시 중성구의 기능 이상과 활성 산화기의 기능이상, 살균 능의 저하를 가져온다고 알려져 있으나 단백분해효소 대한 견해가 부족한 편이다. 본 연구에서는 동물 모델에서 내독소로 유발된 급성폐손상의 기전에 중요한 역할을 하는 단백분해효소와 그 억제제의 분비를 비교 관찰하였다. 방법: 생후 6주된 수컷 쥐 40마리를 정상군, 당뇨군, 내독소군, 당뇨-내독소군으로 나누어 당뇨군은 streptozotocin을 투여하였고 내독소군은 지질다당질을 투여하고서 희생시켰다. 각각 혈액, 기관지폐포세척액을 얻어 gelatin zymography를 이용하여 MMP-9의 활성을 측정하였고 Western blot으로 TIMP-1을 측정하였다. 결과: 1) 내독소군과 당뇨-내독소군은 정상과 당뇨군에 비해 폐 무게의 증가와 기관지폐포세척액과 폐 조직 소견상 염증세포, 특히 중성구의 증가를 보였고 기관지폐포세척액 내 단백질 양의 증가를 보였다 (p<0.05). 또한 당뇨-내독소군은 내독소군과 달리 기관지폐포세척액 내 총 염증세포 수가 유의하게 감소하였다 (p=0.001). 2) MMP-9의 활성은 혈청에서는 당뇨-내독소군이 다른 세 군보다 가장 높은 활성을 보였고 기관지폐포세척액에서는 내독소군과 당뇨-내독소군은 차이가 없었으나 정상과 당뇨군에 비해 높은 활성을 보였다 (p<0.05). TIMP-1 발현은 혈청에서는 네 군간의 차이가 없었고 기관지폐포세척액은 당뇨-내독소군이 가장 낮은 발현을 보였다 (p<0.05). MMP-9/TIMP-1 density ratio를 비교했을 때 기관지폐포세척액에서 당뇨-내독소군이 다른 세 군보다 높았고 내독소군도 정상과 당뇨군보다 높았다 (p<0.05). 결론: 급성폐손상이 있는 당뇨에서는 염증세포의 화학 주성 감소로 중증 감염에 민감해지고, 기질단백분해효소 활성 증가와 그 억제제의 감소로 폐손상이 가중될 것으로 예상된다. Background: An acute lung injury(ALI) is characterized by the recruitment, activation, and apoptosis of inflammatory cells, numerous products released by inflammatory cells such as reactive oxygen species, inflammatory mediators, and a variety of proteolytic enzymes. It was reported that bacterial infections in diabetics showed impaired PMN functions such as reduced PMN respiratory burst and decreased microbicidal activity in inflamed tissue. However, the effect of the proteinase-inhibitor (MMP-9 vs TIMP-1) in ALI in diabetics is unclear. This study evaluated the differences in the expression of MMP-9 and TIMP-1 after the stimulation of endotoxin in a rat model. Methods: Six-week-old male Sprague-Dawley rats were classified into normal, DM, LPS and DM+LPS groups. The peripheral blood, BAL fluids, and lung tissues were obtained from individual rats. The MMP-9 activity was measured by gelatin zymography and the TIMP-1 level was measured by Western blotting. Results: The total BAL cells of the DM-LPS groups were significantly lower than the LPS groups (p<0.01). The MMP-9 activities in the serum were higher in the DM+LPS groups than in the other groups. The MMP-9 activities in the BAL fluids were significantly higher in the DM+LPS group than in the normal and diabetic rats (p<0.05). TIMP-1 expressions in the BAL fluids were significantly lower in the DM+LPS group than other groups (p<0.05). The ratio between MMP-9 and TIMP-1 in the BAL fluids was significantly higher in the DM+LPS groups (p<0.05). Conclusion: In ALI in diabetics the higher MMP-9 activity and lower TIMP-1 level are believed to prolonged and intensify the course of inflammation. (Tuberc Respir Dis 2006; 61: 256-264)

Tissue Inhibitor of Metalloproteinase-1 Pro-motes NIH3T3 Fibroblast Proliferation by Activating p-Akt and Cell Cycle Progression

Yang Lu,Shuxin Liu,Shujia Zhang,Guangyan Cai,Hongwei Jiang,Huabin Su,Xiaofan Li,Quan Hong,Xueguang Zhang,Xiangmei Chen 한국분자세포생물학회 2011 Molecules and cells Vol.31 No.3

Tissue inhibitor of metalloproteinase-1 (TIMP-1) plays various roles in cell growth in different cell types. However, few studies have focused on TIMP-1’s effect on fibroblast cells. In this study, we investigated the effects of TIMP-1 overexpression on NIH3T3 fibroblast proliferation and potential transduction signaling pathways involved. Overexpression of TIMP-1, by transfection of the pLenti6/ V5-DESTTIMP-1 plasmid, significantly promoted NIH3T3 proliferation as determined by the BrdU array. Neither 5 nor 15 nM GM6001 (matrix metalloproteinase system inhibitor) affected NIH3T3 proliferation, but 45 nM GM6001 inhibited proliferation. TIMP-1 overexpression activated the p-Akt pathway, but not the p-ERK or p-p38 pathway. In TIMP-1-transfected cells, cyclinD1 was upregulated and p21CIP1 and p27^(KIP1) were downregulated, which promoted cell entry into the S and G2/M phases. The PI3-K inhibitor LY294002 abolished the TIMP-1-induced effects. Overexpression of intracellular TIMP-1 stimulated NIH3T3 fibroblast proliferation in a matrix metalloproteinase (MMP)-independent manner by activating the p-Akt pathway and related cell cycle progression.

기계적 자극과 interleukin-1ß가 치주인대 섬유아세포의 collagenase와 TIMP-1의 발현에 미치는 영향

김명립(Myung-Lip Kim),배창(Chang Bae) 대한치과교정학회 1998 대한치과교정학회지 Vol.28 No.1

교정력이 치아에 가해지면 치주인대의 재생과 치조골의 개조가 일어난다. 치주인대 섬유아세포는 collagenase와 TIMP-1을 분비하여 치주조직의 교원질의 분해와 합성을 담당한다. 본 연구에서는 치주인대 섬유아세포에 기계적자극과 interleukin-1β를 가해 collagenase와 TIMP-1의 발현을 RT-PCR과 면역조직화학 염색을 사용하여 알아보았다. 4명의 10대 남자 교정환자에게서 아무런 병소가 없는 제 1소구치를 발치후 치주인대 섬유아세포를 배양하여 4-6세대의 세포를 사용하였다. 대조군 Petriperm dish^® 바닥의 표면적을 5% 증가시킨 기계적 자극을 가한 군, interleukin-1β를 1.0ng/ml를 가한 군과 기계적 자극과 interleukin-1β를 같이 가한 군으로 나누어 4명의 환자에서 얻은 세포군을 각 군별로 2, 4, 8시간 후 RT-PCR을 시행하여 그 산물을 반정량하여 대조군에 대한 각 실험구의 생대적인 증감을 나타내었고, 24시간 후 면역조직화학 염색을 시행하여 다음과 같은 결과를 얻었다. 1. 광학 현미경으로 세포의 형태를 관찰한 결과 대조군에서는 전형적인 별모양과 길쭉한 모양을 함께 보였으나 기계적 자극과 interleukin-1β 를 각각 혹은 동시에 준 군들에서는 별모양의 세포가 사라지고 모양이 더욱 길어졌다. 2. collagenase는 대조군에 비해 기계적 자극과 interleukin-1β를 각각 혹은 동시에 준 군들에서 증가하였고, 실험 8시간 후에서는 interleukin-1β를 준 군, 기계적 자극과 interleukin-1β를 동시에 준 군에서 뚜렷한 증가를 보였다. 3. TIMP-1은 세포 자극 2, 4시간 후에는 대조군에 비해 기계적 자극과 interleukin-1β를 각각 혹은 동시에 준 군들에서 감소하였지만. 실험 8시간 후에서는 증가를 보였다. 4. 면역조직화학 염색을 통해 collagenase와 TIMP-1이 대조군에 비해 기계적 자극과 interleukin-1β를 각각 혹은 동시에 준 군들에서 더욱 강한 염색상을 나타내었다. 본 실험의 결과 섬유아세포는 외부 자극이 가해지면 collagenase와 TIMP-1의 발현 조절을 통해 치주인대 재생과 치조골의 개조에 영향을 미쳐 항상성을 유지하려고 함을 알 수 있었다. The turnover of collagen is controlled by the balance between collagen synthesis and degradation. The production of collagenase (matrix rnetalloproteinase-1). and its inhibitor, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) are onet of the substances which rgulate this balance. The periodontal ligament fibrobIast plays an important role in collagen metabolism during orthodontic treatlment and is believed to be an origin of the osteoblast in the alveolar bone. The collagenase secreted by the periodontal ligament fibroblast and the osteoblast initiates the bone resorption by removing the osteoid layer in the alveloar bone. The interleukin-1β is secreted by the macrophage during orthodontic treatment. The present study was undertaken to assess the effect of mechanical stress and interleukin-1β on the expression of collagenase and TIMP-1 in the periodontal ligament fibroblasts sing reverse transcription polymerase chain reaction and immunohistochemical staining. The periodontal ligament frbroblasts were stretched by placing the Petriperm^® dish on the top of spheroidal convex watch glass(5%, surface increase) and treated with interleukin-1β (1.0ng/ml), or treated with both of them. Treatrnent with mechanical stress and/or interleukin-1β resulted in increased collagenase mRNA expression. The mechanical stress treated group {161, 1.62, 1.37 fold increase), the interleukin-1β treated group (1.68 1.60 3.78 fold increase), the mechanical stress and interleukin-1β treated group (1.89, 1.72, 5.48 fold increase) induced increases in collagenase mRNA compared with the control group after 2, 4, 8 hours, respectively. But, TIMP-1 mRNA expressions at experimental groups were decreased after 2, 4 hours and increased after 8 hours. The mechanical stress treated group (0.16, 0.49 fold decrease and 3.77 fold incease), the interleukin-1β treated group (0.15, 0.44 fold decrease and 4.46 fold increase), the mechanical stress and interleukin-1β treated group (0.15, 0.69 fold decrease and 4.81 fold increase) induced changes in TlMP-1, mRNA compated with the control group after 2, 4, 8 hours, respectively. Immunohistochemical stain showed that increased collagenase and TIMP-1 staining of the mechanical stress treated group, the interleukin-1β treated group, and the mechanical stress and interleukin-1β treated group compared with that of the control group after 8 hours. These findings suggest that mechanical stress and interleukin-1β regulate expressions of collagenase and TIMP-1.

Analysis of Protease and Antiprotease Concentrations in Retired Workers Exposed to Inorganic Dusts

Jae Hoon Shin,JooHwan Hwang,Kyung Myung Lee,Jong Seong Lee,Jeong Oh Lee,Byung-Soon Choi,In Sik Kim 대한의생명과학회 2009 Biomedical Science Letters Vol.15 No.4

Occupational exposure to inorganic dusts such as coal and silica has been identified as a chronic obstructive pulmonary disease (COPD) risk factor. This risk factor causes lung inflammation and protease-antiprotease imbalance. This abnormal inflammatory response of the lung induces parenchymal tissue destruction and leads to progressive airflow limitation that is characteristics of COPD. The aim of this study was to determine the relationship of proteases such as neutrophil elastase (NE) and matrix metalloproteinase (MMP)-9 and antiproteases such as alpha-1 antitrypsin (AAT) and tissue inhibitors of metalloproteinase (TIMP)-1 with lung function. The study population contained 223 retired workers exposed to inorganic dusts. We performed lung function test, including percent of forced expiratory volume in one second (%FEV₁) predicted and %FEV₁/forced vital capacity (FVC). We analyzed serum MMP-9, AAT, TIMP-1 and plasma NE concentrations by sandwich enzyme immunoassay. NE, AAT, and TIMP-1 concentrations in workers, who had %FEV₁<80% predicted, were higher than those of workers who had %FEV₁ ≥80% (P<0.05). Both AAT and TIMP-1 concentrations in workers with airflow limitation were higher than those of workers with normal airflow (P<0.05). %FEV₁ predicted showed significant negative correlation with AAT (r=-0.255, P<0.01), TIMP-1 (r=-0.232, P<0.01), and NE (r=-0.196, P<0.01). %FEV₁/FVC predicted showed significant negative correlation with NE (r=-0.172, P<0.05). From the results of stepwise multiple regression analysis about %FEV₁ and %FEV₁/FVC, significant independents were NE (r=-0.135, P=0.001) and AAT (r=-0.100, P=0.013) in %FEV₁, and NE (r=-0.160, P=0.014) in %FEV₁/FVC. In the present study, there were significant correlations between airflow limitation and protease concentration and between airflow limitation and antiprotease concentration. Serum protease and antiprotease concentrations, however, may be affected by the biological and inflammatory responses. It is necessary to evaluate specimens more reflected the effects of proteases and antiproteases in the lung such as lung tissue, bronchoalveolar lavage fluid, and exhaled breath condensate (EBC).

설편평세포암에서 CD44, MMP-2, MMP-9, TIMP-1의 발현

이상화,표성운,김영실,최목균 大韓顎顔面成形再建外科學會 2003 Maxillofacial Plastic Reconstructive Surgery Vol.25 No.4

Adhesion molecules and proteinases play an important role in the invasion and metastasis of a malignant tumor. However, there have not been many studies on tongue squamous cell carcinoma at Saint Mary's Hospital and Kangnam Saint Mary's Hospital, the Catholic University of Korea, between 1994 and 2000, and had not received chemo-or radiotherapy. The expression of the adhesion molecules CD44H, proteinases MMP-2 and MMP-9, and TIMP-1, which controls MMP activity, was analyzed by immunohistochemistry. In addition their association with the tumor size, lymph node metastasis, clinical stage, and cell differentiation was evaluated. Increased MMP-2 expression was significantly correlated with lymph node metastasis and clinical stage. However, it did not correlate with tumor size or cell differentiation. CD44H, MMP-9 and TIMP-1 expression were not significantly related to tumor size, lymph node metastasis, clincial stage and cell differentiation. The results suggest that MMP-2 is a possible immunohistochemical prognostic factor in tongue squamous cell carcinoma. Futher reasearches will be needed to investigate the additional tumor prognostic factors and their mechanisms.

Monosodium iodoacetate에 의해 유도된 골관절염 마우스 모델에서 별갑 물추출물의 효과

천진미,이지혜,남현화,문병철,박준홍 한약정보연구회 2022 한약정보연구회지 Vol.10 No.2

This study aimed to investigate the effects of aqueous extract of Pelodiscis Carapax (PCE) in monosodium iodoacetate (MIA)-induced osteoarthritis (OA) mice. PCE (200 mg/kg) was orally administered to C57BL/6 mice for 2 weeks. After MIA injection (75 mg/mL), we performed a behavior test (open field test, rotarod test) followed by micro-computed tomography, histopathological analysis, and real-time quantitative PCR analysis on knee joint tissues. PCE treatment improved general gait locomotor activity, joint morphological features, and histopathological features compared to the MIA-induced mice. In addition, it confirmed that the mRNA expression levels of tissue inhibitor of metalloproteinases (TIMP)-1 in the joint tissues were increased in the PCE-treated group. Our result revealed that PCE treatment phenotypically improved OA symptoms.

Evaluation of Effective MMP Inhibitors from Eight Different Brown Algae in Human Fibrosarcoma HT1080 Cells

Min Joo Bae,Fatih Karadeniz,Byul-Nim Ahn,Chang-Suk Kong 한국식품영양과학회 2015 Preventive Nutrition and Food Science Vol.20 No.3

Matrix metalloproteinases (MMPs) are crucial extracellular matrices degrading enzymes that have important roles in metastasis of cancer progression as well as other significant conditions such as oxidative stress and hepatic fibrosis. Marine plants are on the rise for their potential to provide natural products that exhibit remarkable health benefits. In this context, brown algae species have been of much interest in the pharmaceutical field with reported instances of isolation of bioactive compounds against tumor growth and MMP activity. In this study, eight different brown algae species were harvested, and their extracts were compared in regard to their anti-MMP effects. According to gelatin zymography results, Ecklonia cava, Ecklonia bicyclis, and Ishige okamurae showed higher inhibitory effects than the other samples on MMP-2 and -9 activity at the concentrations of 10, 50, and 100 ?g/mL. However, only I. okamurae was able to regulate the MMP activity through the expression of MMP and tissue inhibitor of MMP observed by mRNA levels. Overall, brown algae species showed to be good sources for anti-MMP agents, while I. okamurae needs to be further studied for its potential to yield pharmaceutical molecules that can regulate MMP-activity through cellular pathways as well as enzymatic inhibition.

Evaluation of Effective MMP Inhibitors from Eight Different Brown Algae in Human Fibrosarcoma HT1080 Cells

Bae, Min Joo,Karadeniz, Fatih,Ahn, Byul-Nim,Kong, Chang-Suk The Korean Society of Food Science and Nutrition 2015 Preventive Nutrition and Food Science Vol.20 No.3

Matrix metalloproteinases (MMPs) are crucial extracellular matrices degrading enzymes that have important roles in metastasis of cancer progression as well as other significant conditions such as oxidative stress and hepatic fibrosis. Marine plants are on the rise for their potential to provide natural products that exhibit remarkable health benefits. In this context, brown algae species have been of much interest in the pharmaceutical field with reported instances of isolation of bioactive compounds against tumor growth and MMP activity. In this study, eight different brown algae species were harvested, and their extracts were compared in regard to their anti-MMP effects. According to gelatin zymography results, Ecklonia cava, Ecklonia bicyclis, and Ishige okamurae showed higher inhibitory effects than the other samples on MMP-2 and -9 activity at the concentrations of 10, 50, and $100{\mu}g/mL$. However, only I. okamurae was able to regulate the MMP activity through the expression of MMP and tissue inhibitor of MMP observed by mRNA levels. Overall, brown algae species showed to be good sources for anti-MMP agents, while I. okamurae needs to be further studied for its potential to yield pharmaceutical molecules that can regulate MMP-activity through cellular pathways as well as enzymatic inhibition.

Hepatotoxicity aggravates cisplatin-induced acute kidney injury in Sprague-Dawley rats

Ji Su Kim(김지수),Kyeong Seok Kim(김경석),Yu Jung Park(박유정),Su Hyun Lee(이수현),Hyung Sik Kim(김형식) 환경독성보건학회 2019 한국독성학회 심포지움 및 학술발표회 Vol.2019 No.4