Effect of vitamin C on endothelial nitric oxide synthase in streptozotocin-induced diabetic rats : quantitative comparison using image analysis

Abstract

To examine the effects of supplemented vitamin C on endothelial nitric oxide synthase (eNOS) in diabetes mellitus, the animal model of streptozotocin (STZ)-induced diabetic rats (a single intravenous injection of STZ ; 50 mg/kg BW) was used. Male Spraque-Dawley rats weighing 200-250 g were divided randomly into three groups of control (CON), diabetes (DM) and diabetic supplementation with vitamin C (DM+Vit.C). The supplementation of vitamin C was performed by allowing the animals freely assessed to drinking water added 1 g/L of ascorbic acid (Sigma, Chemical Co., USA). The experiment were performed at 12 and 24 weeks (wks) after injection of citrate buffer solution STZ. On the day of experiment, body weight (BW), plasma vitamin C, blood sugar (BS) were evaluated for all animals. Isolated heart, aorta and lung used for Western blot analysis were immediately collected from every rats. The results showed that both groups of 12 and 24 wks DM groups have the significantly increase in blood glucose (BS), but decrease in BW and plasma vitamin C levels as compared to their age-match control groups. However, the plasma vitamin C levels was significantly increased in DM+Vit.C group as compared to DM group (p<0.01). The eNOS protein bands obtained by Western blot analysis using monoclonal antibody against eNOS were quantified by Global Lab Image software analysis. The values of pixel numbers within each eNOS band from image analyzed were directly converted to amount of eNOS proteins of each sample by the standard equation, Y = 5.9 x 103X. After that all converted concentration of eNOS proteins were then normalized using the correlation by 100% equal to 5 mg total protein. It was found that the diabetic state caused the reduction of eNOS protein expression in the heart but not in the lung for both 12 and 24 weeks. Interestingly, it also found that lung have significantly higher eNOS protein levels in DM+Vit.C as compared to DM for both 12 and 24 wks (p<0.001). In conclusion, the present study has demonstrated that the application of digital image analysis can be used for converting qualitative method, such as Western blotting, to quantitative method. By using standard protein, the sensitivity of its application is well enough for differentiate the changes of protein content in microgram level. And the accuracy of this application is within +-15.76 %. From the present results indicated that the endothelial dysfunction induced by diabetic hyperglycemia has been resulted to the decrease in eNOS protein level in heart but not in lung of DM rats. Therefore, we hypothesized that it might due to the difference of high-and low-flow, that mediated eNOS protein synthesis, in heart and lung, respectively. However, this finding suggests that vitamin C supplementation could prevent the diabetic-induced endothelial impairment, especially to prevent eNOS protein damage in high flow systemic circulation. Therefore, it is suggested that vitamin C might be a great chemopreventive agent for diabetic cardiovascular complications.