Deneysel sıçan diabetinde gelişen endotel disfonksiyonunda nitrik oksid sentaz ve nadph oksidaz ekspresyonundaki değişikliklerin rolü ve aposininle tedavinin etkisi
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Dosyalar
Tarih
2008
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Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Diyabetes mellitusta gelişen aterosklerozun başlangıcında gözlenen ilk bozukluk endotel
disfonksiyonudur ve bu bozukluğun temel nedenlerinden birisi hücrenin aşırı miktarlarda
reaktif oksijen türlerine (ROT) maruz kaldığı akut veya kronik oksidatif strestir. Memeli
hücrelerinde ROT’nin üretiminden sorumlu enzimlerden birisi olan NAD(P)H oksidazın
endotel hücrelerinde bazal koşullarda eksprese edildiği, vasküler düz kasta da bulunduğu ve
sitokinlerce aktive edildiği gösterilmiş; enzimi inhibe eden ve uyarılmış ROT üretimini
azaltan kimyasal ajanlar tanımlanmıştır. Bu projede, diabetik sıçanlarda bir NADPH oksidaz
inhibitörü olan aposininle tedavinin torasik aortada endotel disfonksiyonuna ve ayrıca nitrik
oksid sentazlar (NOS) ile NAD(P)H oksidazın ekspresyonuna etkisini araştırmak
amaçlanmıştır.
Streptozotosinle diyabet oluşturulan sıçanlarda 4 hafta sonra aposinin (16 mg/kg/gün)
tedavisi başlatıldı. 8 hafta sonra çıkarılan torasik aort halkalarında asetilkolinle endotel aracılı,
sodyum nitroprusidle endotel aracısız gevşeme yanıtı ve L-NAME kasılması ile bazal NOS3
aktivitesi değerlendirildi. Asetilkolin ve sodyum nitroprusid gevşeme yanıtları açısından
gruplararası fark bulunmadı. Diyabette L-NAME kasılma yanıtı azaldı, aposinin tedavisi
plazma glukoz düzeylerini değiştirmeden direkt etkiyle bu azalmayı önledi. Diyabette aort
dokusunda NOS3 protein ve mRNA ekspresyonu azaldı, NOS2 protein ekspresyonu arttı;
aposinin tedavisi NOS3 azalmasını ve NOS2 artışını önledi. NAD(P)H oksidazın p22phox alt
biriminin protein ve mRNA ekspresyonu diyabetik aortta arttı; aposinin tedavisi hem kontrol,
hem de diyabetik aortta p22phox ekspresyonunu azalttı. Enzimin diğer alt birimi olan gp91phox
ekspresyonu diyabette mRNA düzeyinde artmış bulundu, bu artış aposininle tedavi edilmiş
diyabetik aortta anlamlı olarak önlendi.
Sonuç olarak, sıçan diyabet modelinde diyabetin erken döneminde bazal NOS3 aktivitesi
ve ekspresyonu azalmakta ve bu azalmada diyabetik vasküler yapıda protein ve mRNA
düzeyinde ekspresyonu artan NAD(P)H oksidaz kaynaklı oksidatif stres rol oynamaktadır.
Bazal NOS3 aktivitesindeki azalmayı regüle etmek üzere ve oluşan oksidatif stresin etkisiyle
dokuda NOS2 ekspresyonunda artış tetiklenmekte ve bu tablo endotel disfonksiyonunun
devamlılığına katkıda bulunmaktadır. 16 mg/kg/gün dozda aposininin NAD(P)H oksidazı
etkili bir şekilde inhibe ederek bu değişiklikleri önlediği sonucuna varılmıştır.
Atherosclerosis in diabetes mellitus is associated with endothelial dysfunction and acute or chronic oxidative stress caused by the abundant production of reactive oxygen species (ROS) contributes to the pathogenesis of endothelial dysfunction. NAD(P)H oxidases are predominant sources of ROS in mammalian cells and the enzyme is expressed under basal conditions in both endothelial and vascular smooth muscle cells and is activated in response to cytokines. Several chemical agents inhibiting the enzyme and thereby preventing the stimulation of ROS generation have been defined. This project aims to investigate the effect of apocynin, an NAD(P)H oxidase inhibitor, on the endothelial dysfunction in thoracic aorta of diabetic rats and to determine its effect on the expression of nitric oxide synthetases (NOS) and NAD(P)H oxidase as well. Rats received apocynin (16 mg/kg/day) from 4 to 8 weeks after inducing diabetes with streptozotocin. Endothelium-dependent and –independent relaxations to acetylcholine and sodium nitroprusside respectively and L-NAME contractility, as a measure of basal NOS3 activity, were determined in thoracic aort rings. Relaxant responses to acetylcholine and sodium nitroprusside did not show any differences between groups. L-NAME contractility significantly decreased in diabetes and apocynin treatment directly prevented this decrease without changing plasma glucose levels. NOS3 protein and mRNA expression in aortic tissue exhibited significant decrease in diabetes, while NOS2 protein expression was significantly increased; these alterations were markedly prevented by apocynin. Protein and mRNA expression of p22phox subunit of NAD(P)H oxidase markedly increased in diabetic aorta, which was significantly decreased after apocynin treatment in both control and diabetic rats. However, expression of gp91phox subunit increased only at mRNA level in untreated diabetic aorta; apocynin treatment significantly decreased gp91phox expression in diabetic rats. In conclusion; basal NOS3 activity, as well as the expression, is impaired in the early phase of rat model of diabetes which was accompanied by NAD(P)H oxidase expression, indicating the contribution of NAD(P)H oxidase-derived oxidative stress in this impairment. NOS2 expression is induced as a result of oxidative stress in aortic tissue to counteract the decreased basal NOS3 activity and expression, contributing to the maintenance of endothelial dysfunction. It has been concluded that apocynin 16 mg/kg/day effectively inhibits NAD(P)H oxidase and prevents these changes and the associated endothelial dysfunction.
Atherosclerosis in diabetes mellitus is associated with endothelial dysfunction and acute or chronic oxidative stress caused by the abundant production of reactive oxygen species (ROS) contributes to the pathogenesis of endothelial dysfunction. NAD(P)H oxidases are predominant sources of ROS in mammalian cells and the enzyme is expressed under basal conditions in both endothelial and vascular smooth muscle cells and is activated in response to cytokines. Several chemical agents inhibiting the enzyme and thereby preventing the stimulation of ROS generation have been defined. This project aims to investigate the effect of apocynin, an NAD(P)H oxidase inhibitor, on the endothelial dysfunction in thoracic aorta of diabetic rats and to determine its effect on the expression of nitric oxide synthetases (NOS) and NAD(P)H oxidase as well. Rats received apocynin (16 mg/kg/day) from 4 to 8 weeks after inducing diabetes with streptozotocin. Endothelium-dependent and –independent relaxations to acetylcholine and sodium nitroprusside respectively and L-NAME contractility, as a measure of basal NOS3 activity, were determined in thoracic aort rings. Relaxant responses to acetylcholine and sodium nitroprusside did not show any differences between groups. L-NAME contractility significantly decreased in diabetes and apocynin treatment directly prevented this decrease without changing plasma glucose levels. NOS3 protein and mRNA expression in aortic tissue exhibited significant decrease in diabetes, while NOS2 protein expression was significantly increased; these alterations were markedly prevented by apocynin. Protein and mRNA expression of p22phox subunit of NAD(P)H oxidase markedly increased in diabetic aorta, which was significantly decreased after apocynin treatment in both control and diabetic rats. However, expression of gp91phox subunit increased only at mRNA level in untreated diabetic aorta; apocynin treatment significantly decreased gp91phox expression in diabetic rats. In conclusion; basal NOS3 activity, as well as the expression, is impaired in the early phase of rat model of diabetes which was accompanied by NAD(P)H oxidase expression, indicating the contribution of NAD(P)H oxidase-derived oxidative stress in this impairment. NOS2 expression is induced as a result of oxidative stress in aortic tissue to counteract the decreased basal NOS3 activity and expression, contributing to the maintenance of endothelial dysfunction. It has been concluded that apocynin 16 mg/kg/day effectively inhibits NAD(P)H oxidase and prevents these changes and the associated endothelial dysfunction.
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