早期糖尿病大鼠视网膜中神经型一氧化氮合酶的表达改变在蛋白质氧化损伤中的作用

Effect of neuronal nitric oxide synthase expression alteration on the protein oxidative damage in the retina of rats with early diabetes

目的:研究糖尿病是否会改变视网膜中神经型一氧化氮合酶(neuronalnitricoxidesynthase,nNOS)的表达,以及其表达的改变在蛋白质氧化损伤中的作用。方法:Sprague-Dawley(SD)大鼠,分成对照组及2组实验组,每组5只。实验组腹腔注射链佐星(streptozotocin,STZ)(65mg/kg)制备糖尿病大鼠动物模型。分别在造模成功后2及20周,研究大鼠视网膜内源性nNOS和蛋白质损伤产物3-硝基酪氨酸(3-nitrotyrosine,NT)的表达及其改变。NT用免疫组化法和计算机图像处理检测,nNOS的表达采用免疫组化和反转录聚合酶链反应(RT-PCR)方法检测。结果:对照组大鼠视网膜内无NT的免疫反应,糖尿病大鼠视网膜神经节细胞层、内核层和外核层均有NT的免疫反应,计算机图像处理显示,糖尿病20周大鼠的视网膜内NT阳性细胞数显著高于2周的糖尿病大鼠。RT-PCR结果显示,糖尿病大鼠视网膜的nNOS的mRNA表达低于对照大鼠,免疫组化结果也显示糖尿病大鼠视网膜的nNOS阳性细胞数少于对照组。结论:早期糖尿病大鼠视网膜内3-硝基酪氨酸的含量升高。早期糖尿病视网膜内,降低的nNOS的mRNA和酶的表达显示,nNOS产生的一氧化氮(nitrogenmonoxide,NO)可能是糖尿病视网膜内NT的限制性因素。这些结果同时提示。

AIM:To investigate whether diabetes can alter the expression of neuronal nitric oxide synthase (nNOS) in retina and its effect on the protein oxidative damage. METHODS:Sprague Dawley(SD) rats were divided into control group(n=5),study group 1(n=5) and study group 2(n=5). Diabetic rats were established by intraperitoneal injection of streptozotocin(STZ, 65 mg/kg). Endogenous nNOS and the expression and changes of 3 nitrotyrosine(NT) caused by protein damage in retina were studied at 2 and 20 weeks after diabetic models were established.NT was detected by using immunohistochemistry and computer image analysis, nNOS expression was measured by using immunocytochemistry and reverse transcription polymerase chain reaction(RT PCR). RESULTS:No immunologic reaction to NT was found in the control group, while were observed in the ganglion cell layer, inner cell layer and outer cell layer of retina in rats with diabetes. Image analysis showed that the number of NT positive cells in retina was significantly higher in rats with diabetes at 20 weeks than in those at 2 weeks. The expression of nNOS mRNA was significantly lower in diabetic rats than in control rats as determined by RT PCR. The result of immunohistochemistry also showed that nNOS positive cells were fewer in diabetic rats. CONCLUSION: The content of NT increases in retina of rats with early diabetes. NO production by the neuronal form of NOS may be the rate limiting step in NT formation, as supported by the reduced mRNA and enzyme expression of nNOS in the diabetic retina. These findings also suggest that a loss of nNOS activity might contribute to retinal neurovascular dysfunction in experimental rats with diabetes.