Background The pathogenesis of diabetic retinopathy (DR) might be related with oxidative stress and its mechanism has not been fully elucidated.Grape seed proanthocyanidin extracts (GSPE),a powerful antioxidant,plays roles in some systemic diseases.However,the effect and mechanism of GSPE in DR has not been illuminated clearly.Objective This study was to investigate whether GSPE has a protective effect on the retinas of diabetic subjects and explore its mechanism.Methods Thirty SPF adult Wistar rats were divided into the control group,diabetic group and diabetes + GSPE group according to the randomized number table.Diabetic models were induced by intraperitoneal injection of 100 mg/kg strebtozotocin (STZ) prepared with citrate buffer,and only the equal amount of citrate buffer was used in the same way in the control group.GSPE solution was intragastrally used 250 mg/kg daily in the rats of the diabetes+GSPE group from injective day through 8 weeks,and distilled water was used in the same way in the rats of the control and diabetic groups.The rats were sacrificed in the eighth week after injection and the retinas were isolated.The morphology of the retina were examined by hematoxylin and eosin stain.Retinal homogenatewas prepared for the assay of superoxide dismutase (SOD),glutathione peroxidase (GSH-Px) activity and malonaldehyde (MDA) content.The expressions and location of nuclear erythroid related factor 2 (Nrf2) was determined by immunochemistry,and the expressions of Nrf2 and heme oxygenase-1 (HO-1) were quantitatively analyzed by Western blot.The apoptosis of retinal cells was detected by TUNEL.Results In the eighth week after modeling,the blood glucose levels were significantly higher and the body weight was lower in the rats of the diabetic group and diabetes+GSPE group compared with the control group (all at P＜0.01).Retinal structure was normal in the rats of the control group.However,loose tissue,irregular arrangement of cells and thickness decrease of retinal fiber layer,retinal ganglion cell layer and inner plexiform layer were exhibited in the rats of the diabetic group,while the morphology abnormality was slight in the rats of the diabetes+GSPE group.The SOD and GSH-Px activities and MDA content were significantly different among the 3 groups (F =11.010,P =0.001 ; F =12.072,P =0.000 ; F =25.224,P=0.000),and activities of SOD and GSH-Px in the retinas were significantly lower,and the MDA level was higher in the rats of diabetic group than those of the diabetes+GSPE group and the control group (all at P＜0.01).The relative expressing levels of Nrf2 were (165.5±29.4) ％ and (134.8 ±7.8) ％ in the diabetes+ GSPE group and diabetic group,with a significant difference between them (t=2.450,P=0.044).Compared with the diabetic group,the expressing level of the HO-1 was sigficantly increased ([170.2±22.0)％ versus [125.3±9.2] ％,t =2.360,P =0.002).TUNEL showed that the retinal apoptotic cells of diabetic rats were mainly located in the retinal fiber layer,RGCs layer,inner and outer nuclear layer,and the number of apoptotic cells was less in the diabetes+GSPE group compared with the diabetic group under the fluorescence microscope.Conclusions GSPE can play a protective role on diabetic rats by activating Nrf2 pathway and therefore improving retinal oxidative stress and decreasing apoptosis.%背景 目前认为,糖尿病视网膜病变(DR)的发病和进展可能与氧化应激有关联.研究表明,葡萄多酚(GSPE)在多种系统性疾病中发挥强大的抗氧化作用,但其在DR中的作用及其机制尚不完全清楚.目的 探讨GSPE对糖尿病的视网膜是否具有保护作用,并分析其作用机制.方法 按照随机数字表法将30只SPF级成年Wistar大鼠随机分为对照组、糖尿病组和糖尿病+GSPE组,用100 mg/kg枸橼酸缓冲液制备链脲佐菌素(STZ)的一次性腹腔内注射法制备大鼠糖尿病模型,对照组大鼠以同样的方法注射等容量枸橼酸缓冲液.造模即日起,糖尿病+GSPE组大鼠用GSPE溶液灌胃,每天250 mg/kg,共56 d;糖尿病组和对照组大鼠用等量蒸馏水灌胃.动物饲养至第8周处死,分离视网膜,苏木精-伊红染色法,观察各组大鼠视网膜形态学改变;部分视网膜制备组织匀浆,分别检测大鼠视网膜中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性及丙二醛(MDA)含量;用免疫组织化学对各组大鼠视网膜中核红细胞相关因子2(Nrf2)的表达进行定性和定位分析,用Western blot法对大鼠视网膜中Nrf2和血红素加氧酶1(HO-1)蛋白的表达进行定量分析;TUNEL法测定各组大鼠视网膜细胞的凋亡情况并进行比较.结果 实验后8周,糖尿病组和糖尿病+GSPE组大鼠血糖水平明显高于对照组,而体质量明显低于对照组,差异均有统计学意义(P＜0.01).对照组大鼠视网膜结构正常,糖尿病组大鼠视网膜组织疏松,各层细胞排列不规则,视网膜神经纤维层、视神经节细胞(RGCs)层以及内丛状层(IPL)变薄,而糖尿病+GSPE组大鼠视网膜形态的异常轻于糖尿病组.3个组间SOD和GSH-Px活性及MDA含量均明显不同,差异均有统计学意义(F=11.010,P=0.001;F=12.072,P=0.000;F=25.224,P=0.000),其中糖尿病组大鼠SOD和GSH-Px活性均明显低于对照组和糖尿病+GSPE组,而MDA含量明显高于对照组和糖尿病+GSPE组,差异均有统计学意义(P＜0.01).糖尿病+GSPE组和糖尿病组大鼠视网膜中Nrf2表达量分别为(165.5±29.4)％和(134.8±7.8)％,差异有统计学意义(t=2.450,P=0.044);与糖尿病组比较,糖尿病+GSPE组大鼠视网膜中HO-1蛋白表达量明显升高[(170.2±22.0)％和(125.3±9.2)％],差异有统计学意义(t=2.360,P=0.002).TUNEL法检测表明,糖尿病组大鼠视网膜细胞凋亡主要分布在视网膜神经纤维层、RGCs层和内核层、外核层,荧光显微镜下糖尿病+GSPE组大鼠视网膜细胞凋亡数明显少于糖尿病组大鼠.结论 GSPE通过激活Nrf2通路而改善糖尿病大鼠视网膜氧化应激状态,减少视网膜细胞凋亡,对糖尿病大鼠的视网膜发挥保护作用.
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