Résumé
Objective To investigate the expression of endothelin-1 (ET-1), ET-2, ET-3, endothelin receptor A (ETRA), ETRB and endothelin coverting enzyme (ECE) in the retinas of 8-week-old diabetic SD rats. Methods The retinal gene expression profiles of healthy and 8-week-old diabetic rats were constructed with restriction fragment differential display polymerase chained reaction (RFDD-PCR), and the differential expression of ET-1, ET-2, ET-3, ETRA, ETRB and ECE was verified using semi-quantitative RT-PCR and Western blotting analysis. Results The results of RFDD-PCR showed that the expression of ET-1, ET-2, ET-3, ETRA, ETRB and ECE was up-regulated in diabetic retina. The results of semi-quantitative RT-PCR and Western blotting analysis showed that the expression levels of the six genes and proteins (relative D ratio) in diabetic group were significantly higher than those in the normal retinas (P<0. 05 and P<0. 01, respectively). Conclusion The expression of ET-1, ET-2, ET-3, ETRA, ETRB and ECE is up-regulated in diabetic retina, suggesting that the six genes may be involved in the pathgenesis of diabetic retina.
Résumé
<p><b>OBJECTIVE</b>To investigate the expression of nitric oxide synthase (NOS) in the retina of 8-week-old diabetic rats, and explore the potential molecular mechanisms for the role of NO in diabetic retinopathy (DR).</p><p><b>METHODS</b>Retinal gene expression profile of normal and 8-week-old diabetic rats was constructed with restriction fragment differential display polymerase chain reaction (RFDD-PCR). Bioinformatic analysis of the differentially expressed gene identified the genes coding for 3 subtypes of NOS, namely eNOS, nNOS and iNOS as the candidate genes related to DR, which was verified using semi-quantitative RT-PCR and immunohistochemistry.</p><p><b>RESULTS</b>The results of RFDD-PCR revealed down-regulated expression of eNOS and nNOS and up-regulated iNOS expression in diabetic rat retina. RT-PCR showed that the expression levels of eNOS and nNOS in diabetic rat retina were obviously lower than that in normal retina (0.23-/+0.03 vs 0.32-/+0.03 for eNOS, P<0.05; 0.25-/+0.02 vs 0.36-/+0.02 for nNOS, P<0.05), but the expression level of iNOS obviously higher (0.27-/+0.02 vs 0.20-/+0.03, P<0.05). Immunohistochemistry of healthy retina visualized eNOS-, nNOS- and iNOS-positive cells, all located in the inner nuclear layer (INL) and ganglion cell layer (GCL), and eNOS-positive cells were also found in vascular endothelium. In diabetic retina, the number of eNOS- and nNOS-positive cells was significantly lowered in comparison with normal rat retina (14.33-/+3.19 vs 22.13-/+3.60 for eNOS, P<0.05; 21.87-/+3.62 vs 34.40-/+7.09 for nNOS, P<0.05), but the number of iNOS-positive cells significantly increased (17.60-/+2.58 vs 11.73-/+2.70, P<0.05).</p><p><b>CONCLUSION</b>The alterations in eNOS, nNOS and iNOS expression are associated with the deuelopmant and progression of DR.</p>