ABSTRACT
Background Trans-arachidonic acids (TAAs) are specific lipid markers of nitrative stress and the major products of NO2·-mediated ismoerization of aracchidonic acid(AA),and they represent a possible new mechanism of NO2·-induced toxicity in ischemic retinopathy.Our previous study demonstrated that high glucose induce nitrative stress response. Objective This study aimed to evaluate the effect of nitrative stress on retinal capillary in diabetic rats by detecting the TAAs with gas chromatography and mass spectrometry(GC/MS).Methods One hundred 2-week-old clean male SD rats were randomly divided into control group and diabetic group.Diabetic rat models were induced by intraperitoneal injection of streptozoticin of 60 mg/kg,and the successful models were identified as the blood glucose level>16.7 mmol/L.The synthesis method was modified in this study andaraehidonic acid w8$utilized as starting material.14E-AA was obtained as a standard sample via expoxidation and deoxidation in a safe and practical pattern.Serum TAAs and AA in diabetic rats were detected by GC/MS in 2,4,8,12 and 16 weeks after injection of streptozoticin.The ratio of peak area of TAAs to AA with selected ion of 79 was estimated and compared with normal rats. Results Fifty rats had been in Diabetic models were established in 50 rats with the successful rate 100%,and the high blood glucose level in model rats retained throughout the experimental duration.GC/MS results showed that the 14E-AA was obtained with the purity of 97.3%,which provided a standard sample to nitrative stress-related research as a premise. No significant differences were found in serum TAAs/AA values at 2 weeks and 4 weeks between model group and control group (t =-0. 376, t =-0. 642,P>0. 05). However,serum TAAs/AA values in at 8,12 and 16 weeks after streptozoticin injection were considerably elevated in comparison with those of control group (0. 0832 vs 0. 1042,0. 0910 vs 0. 1568, 0. 1100 vs 0. 1895;t=-36.409,t=-166.714,t=-148.212,P<0. 05). Conclusion Elevation of serum TAAs/AA is associated with diabetic duration,implying that damage of nitrative stress to retinal capillary is a parallel procedure to diabetic course.Establishment of GC/MS detection system offers a new evaluating indicator in the research on microvascular ischemic disease and could be implemented in clinical testing.
ABSTRACT
<p><b>BACKGROUND</b>Trans-arachidonic acids (TAAs), newly discovered markers of nitrative stress and the major products of nitrogen dioxide (NO2(·))-mediated isomerization of arachidonic acid (AA), represent a new mechanism of NO2(·)-induced toxicity. It has been reported that TAAs were generated in oxygen-induced microvascular degeneration model and TAAs were also generated in a diabetic retinopathy (DR) model. In this study, we examined high glucose-induced nitrative stress damage and TAAs levels and explored the possible mechanisms for DR caused by reactive nitrogen species.</p><p><b>METHODS</b>Diabetic rats were induced by intraperitoneal injection of streptozotocin (STZ) at 60 mg/kg. Bovine retinal capillary endothelial cells (BRECs) were selectively cultured and incubated with normal or high glucose. The serum TAAs and AA in diabetic rats were measured by the gas chromatography and mass spectrometry (GC/MS) method. The ratio of peak area of TAAs to AA with selected ion of 79 was estimated by a group t-test. Thrombospondin-1 (TSP-1) in the rat retinas and BRECs extracts were examined by Western blotting. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) protein was examined by Western blotting in BRECs incubated with high glucose.</p><p><b>RESULTS</b>The TAAs to AA ratio (TAAs/AA) was significantly increased in the serum at 8, 12 and 16 weeks after STZ injection (P < 0.05), with no noticeable change found at 2 or 4 weeks (P > 0.05). Expression of TSP-1 in the retina of diabetic rats was progressively elevated according to the duration of diabetes. TSP-1 expression was increased in BRECs incubated with high glucose at 48 hours. Moreover, high glucose also increased ERK1/2 expression, which peaked at 30 minutes and then decreased in the following 48 hours.</p><p><b>CONCLUSION</b>An elevation of TAAs/AA is associated with high glucose-induced nitrative stress, which probably involves upregulation of TSP-1 through activating ERK1/2.</p>