Ecdysterone treatment restores constitutive NO synthesis and alleviates oxidative damage in heart tissue and mitochondria of streptozotocin-induced diabetic rats

Abstract

Constitutive NO synthases (cNOS) are the primary targets of diabetes mellitus and the impairment of cNOS functioning in cardiovascular system is one of the hallmarks of this disease. The aim of this work was to study the effect of a plant sterol ecdysterone (20-β-hydroxyecdysterone) on the NO synthases functioning and RNS metabolism in heart mitochondria and the heart tissue in the rat model of streptozotocin-induced type I diabetes. Diabetes development resulted in cNOS dysfunction both in heart mitochondria and heart tissue. cNOS activity was dramatically suppressed, but 3-fold and 6-fold rise of iNOS activity was observed in mitochondria and heart tissue respectively. Also, in mitochondria there was ~2.5 time’s increase in urea content and the activity of arginase 2 (ARG2), which could compete with NOS for the common substrate L-arginine. Total RNS production was dramatically elevated in mitochondria of diabetic animals, which well agreed with iNOS activation. Unlike this, in heart tissue dramatic increase of iNOS activity increased the content of nitrosothiols (RSNO), while total RNS production remained close to control. Both in the heart tissue and mitochondria, there was dramatic augmentation of superoxide production that correlated with sharp elevation of iNOS activity and steep rise of diene conjugates (DC) content, which indicated strong lipid oxidation. Ecdysterone treatment resulted in the reduction of iNOS activity and twofold elevation of mtNOS activity as compared to control. However, in the whole heart tissue eNOS was restored only by half of control level, which indicated specific action of ecdysterone on mtNOS isoform. RNS production returned to control in mitochondria, and was by half reduced in the heart tissue, which indicated the abolition of nitrosative stress. Correlation dependence between iNOS activity and superoxide production was found in mitochondria, which could indicate iNOS uncoupling. The restoration of cNOS activity and the reduction of iNOS activity to control level after ecdysterone treatment well correlated with the reduction of superoxide production and indicated possible ‘iNOS re-coupling‘, which resulted in the reduction of DC formation to control level. So, STZ-induced type I diabetes dramatically up-regulated iNOS activity and suppressed cNOS activity. Ecdysterone treatment reduced iNOS activity and restored constitutive NO biosynthesis to control level, which abolished oxidative and nitrosative stress in cardiac mitochondria and heart tissue of STZ-induced diabetic animals. Possible pathways involved in ecdysterone action on constitutive NO biosynthesis were discussed.