Studies on the Nitric Oxide Synthetase Inhibitor Combined Effects of Nitric Oxide Inhibitors and Antioxidants on Streptozotocin Toxicity : 산화질소 저해제와 수산기 Radical Scavenger가 산화질소 합성효소 저해제와의 병용시에 Streptozotocin의 독성에 미치는 영향에 관한 연구

국문 초록 (Abstract)

췌장 β-cell 특이독성이 있어, 당뇨유발에 쓰이는 streptozotocin(STZ)은 nitric oxide(NO)와 hydroxyl radical을 liberation하는 것이 증명되었다. 자가면역성 당뇨병에서도 특히 cytokine의 하나인 interleukin-1β(IL-1β)가 nitric oxide를 분비하여 당뇨병을 유발하는 것과 STZ에 의한 당뇨병이 그 결과가 같아서, STZ 역시 IL-1β와 같은 기전으로 당뇨병을 유발한다는 견해가 지배적이다.
본 연구에서는 STZ이 발생시키는 nitric oxide와 hydroxyl radical에 대한 저해약물을 처리하여 그 병용효과를 알아보고자, rat에서 분리한 췌장소도세포에 Nitric oxide synthetase (NOS) inhibitor인 N-Nitro L-Arginine Methyl Ester(NAME)와 2,4-Diamino-6-hydroxypyrimidine(DAHP), Nitric Oxide inhibitor인 methylene blue(MB), hydroxyl radical scavenger인 1,3-dimethyl-2-thiourea(DMTU), melatonin을 처리한 후 STZ에 노출시켜 각 군의 insulin 분비능과 nilric oxide 발생량으로 약물의 STZ 독성 저해능을 평가하고, 각 약물에 강력한 NOS 저해약물인 NAME와 병용했을 경우와 비교하여 다음과 같은 결과를 얻었다.
Control group이 24시간동안 분비하는 insulin의 양을 1로 보았을 때, 실험에 사용된 모든 약물은 저해효과가 있었고, 특히 melatonin 1mM군이 0.79±0.104, MB와 NAME의 병용군이 0.74±0.045, DAHP 와 NAME의 병용군이 0.76±0.071로 control과 통계적 차이가 없었다. 이것은 STZ의 0.16±0.012보다 3배이상의 기능적 독성 저해 효과를 보인 것이다.
Nitrite assay에서는 STZ만을 투여한 경우(3.839±0. 1756μM)와 비교할 때 모든 agent가 nitric oxide의 발생을 줄이는 효과가 있었고 특히 DAHP는 NO toxicity의 저해약물로 쓰던 NAME 보다도 효과적이었다.
위의 실험으로 미루어볼 때, nitric oxide liberation만으로 STZ의 β-cell 특이독성이 일어나지 않는 것이 insulin 분비량의 측정에서 나타났고, classic hydroxyl radical scavenger인 DMTU는 NOS 저해제인 NAME와 병용한 경우 insulin과 nitrite 분비량에서 단독 적용시보다 효과적임이 나타났다.

다국어 초록 (Multilingual Abstract)

Streptozotocin(STZ) has been used to induce Diabetes Mellitus(DM) in laberatories for decades, even though it is not fully proved how to induce DM. It was reported that the diabetogenesis of interleukin-1β (IL-1β) was similar to that of STZ, which liberated nitric oxide(NO) and produced hydroxyl radicals.
In this study, that the diabetogeneic mechanism of STZ and the synergic effects of hydroxyl radical scavengers and N-nitro L-arginine methyl ester(NAME) on STZ toxicity has been investigated. NAME is the clinical STZ toxicity inhibitor, as a L-arginine analogue prevents biosynthesis of nitric oxide synthetase(NOS).
The level of insulin secretion and nitrite concentration were investigated. Before STZ exposure in isolated rat pancreatic islets were treated 30 minutes with 2,4-diamino-6-hydroxypyrimidine(DAHP), NAME, methylene blue(MB), 1,3-dimethyl-2-thiourea(DMTU) and melatonin.
Considering the average insulin secretion as one in control group for 24 hours, all agents used in this study were prevented STZ toxicity. Especially, a novel hydroxyl radical scavenger, melatonin (1mM) treatment (0.79±0.104), DAHP combined NAME treatment(0.76±0.071), and MB combined NAME treatment(0.74±0.045)groups did not have stastical difference comparing it with the control group(1.00±0.056). These inhibited more over three times comparing with the STZ treatment group(0.16±0.012).
In nitrite assay, comparing it with the STZ treatment group(3.389±0.1756μM), all agents used in this study decreased NO induced by STZ and DAHP treatment group (0.869±0.2102μM)decreased NO production most powerfully. Totally the classical hydroxyl radical scavenger, DMTU inhibited STZ toxicity more potent NAME combined treatment than single treatment. NO inhibitor, MB appeared in the same manner.
NAME increased insulin secretion more than DAHP or NAME combined DAHP treatment groups, however, nitrite to concentration was less than these. The melatonin treatment groups showed same patterns. Even though nitrite concentration was decreased, insulin secretion level did not increase in combination experiments(melatonin and DAHP).
Considering above mentioned results, hydroxyl radical scavenger combined NAME inhibited synergically STZ toxicity in insulin secretion and nitrite assay, and NO liberation was not the main mechanism of STZ diabetogenesis.

목차 (Table of Contents)

• 목차 = ⅴ
• ABSTRACT (ENGLISH) = ⅰ
• 국문초록 = ⅲ
• LIST OF FIGURES = ⅵ
• LIST OF TABLES = ⅶ
• INTRODUCTION = 1
• SHORT REVIEWS = 5
• 1) Streptozotocin = 5
• 2) Nitric Oxide = 8
• 3) N-Nitro L-Arginine Methyl Ester (L-NAME) = 11
• 4) 2,4-Diamino-6-hydroxypyrimidine (DAHP) = 11
• 5) Methylene Blue = 13
• 6) 1,3-Dimethy-2-thiourea (DMTU) = 14
• 7) Melatonin (N-Acetyl-5-methoxytrypamine) = 15
• MATERIALS = 17
• METHODS = 17
• 1) Islet Isolation = 17
• 2) Islet Culture = 21
• 3) Islet Exposure to Treating Agents = 21
• 4) Cytotoxicity Assay = 22
• 5) Insulin Quantification = 22
• 6) Measurement of the Concentration of Nitrite = 22
• 7) Stastical Analysis = 24
• RESULTS = 24
• 1) Streptozotocin Toxicity on Islets = 24
• 2) The observations of Insulin Secretion = 28
• 3) The Observations of Nitrite Concentration = 31
• DlSCUSSION = 35
• APPENDIX = 44
• REFERENCES = 47