Reversal effect of vitamin c on diabetes induced endothelial dysfunction in rats : roles of reactive oxygen species, tetrahydrobiopterin, and nitric oxide

Abstract

This study aimed to examine the effects of vitamin C on reversing endothelial dysfunction in diabetes mellitus. Male Spraque-Dawley rats weighing 200-250 g were divided into four groups: control (CON), diabetes (DM, using iv. injection of streptozotocin (STZ); 50 mg/kg BW), DM+Vit.C6wks, and DM+Vit.Cday2. For DM+Vit.C6wks, and DM+Vit.Cday2, they received vitamin C 1g/L on week 6th and on day 2 after STZ-injection, respectively. The diabetes-induced endothelial dysfunction was demonstrated by the impairment of mesenteric arteriolar acetylcholine (Ach)-induced vasodilation and the increase in leukocyte-endothelial cell interaction. These abnormalities were restored by delayed treatment of vitamin C (DM+Vit.C6wks) and prevented by early vitamin C supplementation (DM+Vit.Cday2). The importance of this present study was indicated that vitamin C supplementation could reverse diabetes-induced endothelial cell dysfunction in mesenteric microcirculation. Next, we aimed to examine the underlying mechanism(s) of reversal effect of vitamin C. Base on the first hypothesis, it is believed that vitamin C could restore endothelial function by its direct scavenging reactive oxygen species (ROS). In order to prove this hypothesis, therefore, we monitored hydrogen peroxide (H[subscript 2]O[subscript 2]), one stable form of ROS, by using the specific H[subscript 2]O[subscript 2] associated fluorescent dye called dihydrorhodamine 123 (DHR-123) with real time intravital fluorescence videomicroscopy. The results indicated that the H[subscript 2]O[subscript 2]-associated fluorescent intensity was greatly increased in 6wk-DM group compared to 6wk-CON. Interestingly, both vitamin C supplementation groups (DM+Vit.C6wks and DM+Vit.Cday2) have shown less H[subscript 2]O[subscript 2]-associated fluorescent intensity in the same manner. Together with this direct scavenging effect of vitamin C, therefore, we made a direct detection for nitric oxide (NO), since the idea was that NO should increase when ROS was scavenged. By using 4,5-diaminofluorescein-diacetate (DAF-2DA), the NO-associated fluorescent intensity was determined for each group; CON, DM, CON +2.6 mM Vit.C, and DM+2.6 mM Vit.C at 20-min vitamin C perfusion. The results showed that NO content was significantly decreased in 6wk-DM compared to 6wk-CON. However, vitamin C application could significantly increase NO contents in both CON and DM groups. Based on our finding, we indicated that vitamin C was able to scavenging ROS, as a consequence, it released NO. The second hypothesis for reversal effect of vitamin C was addressed by its possibly acting on preserving tetrahydrobiopterin (BH[subscript 4]) bioavailability. The experiments were conducted by dividing 6wk-CON and 6wk-DM into six groups: 1) Vehicle; 30-min Krebs-Ringer perfusion, 2) Veh+Vit.C; 1-min 2.6 mM vitamin C administration after 30-min Krebs-Ringer perfusion, 3) DAHP; 30-min BH[subscript 4] antagonist (20 mM, 2,4-diamino-6-hydroxypyrimidine (DAHP), 4) DAHP+Vit.C; 1-min 2.6 mM vitamin C administration after 30 min DAHP, 5) DAHP+BH[subscript 4]; 20-min BH[subscript 4] donor after 10-min DAHP, and 6) DAHP+BH[subscript 4]+Vit.C ; 10-min DAHP followed by 20-min BH[subscript 4] donor and 1-min vitamin C. The results indicated that the DAHP-mediated BH[subscript 4] deficiency significantly caused the reduction of Ach-induced vasodilatation in both CON and DM. Interestingly, this abnormality was improved by administration with vitamin C, or BH[subscript 4], or the combined vitamin C and BH[subscript 4]. Furthermore, the H[subscript 2]O[subscript 2]-associated fluorescent intensity was greatly increased when BH[subscript 4] synthesis was blocked by DAHP. Interestingly, this abnormality could be attenuated by the administration of vitamin C, or BH[subscript 4], or the combined vitamin C and BH[subscript 4]. Beside, it should be noted that the DAHP-induced the reduction of Ach-induced vasodilatation could be improved partially by vitamin C administration. Therefore, it may be concluded that vitamin C could regenerate BH¬[subscript 4] and consequently increase active form of coupled-eNOS. In conclusion, our results indicated that vitamin C could reverse diabetes-induced endothelial dysfunction at early stage of 6-wk diabetic induction. The possible mechanisms of vitamin C were worked through its potential on direct ROS scavenging and on enhancing BH[subscript 4] bioavailability. Therefore, it implied that vitamin C could increase NO bioavailability. And it may be used as a good therapeutic agent recommended for diabetic care.