缬沙坦抑制糖尿病大鼠视网膜内皮素－1表达的实验研究

doi:10.3877/cma.j.issn.2095-2007.2017.02.004

目的

观察缬沙坦对糖尿病大鼠视网膜内皮素－1(ET－1)表达的影响。

方法

将115只Wistar大鼠随机分为对照组、实验组及空白对照组。其中，对照组40只、实验组40只及空白对照组35只。建立糖尿病动物模型的方法为，按照65 mg/kg体重，给予对照组与实验组大鼠腹腔内单次注射浓度为1%的链脉佐菌素(STZ)溶液；给予空白对照组大鼠注射同等剂量的柠檬酸缓冲液。以模型大鼠空腹血糖>16.65 mmol/L者定为成功模型。成模后，给予实验组大鼠缬沙坦按24 mg/(kg·d)灌胃，给予对照组与空白对照组大鼠等体积蒸馏水灌胃。分别于干预后的2周﹑4周及6周采用5′－腺苷二磷酸二钠盐(ADP)酶视网膜铺片及免疫组织化学法观察视网膜血管的改变并检测视网膜ET－1的表达。ET－1平均积分光密度值以均数±标准差(±s)表示。采用两因素重复测量方差分析的方法，比较不同组间和不同时间点大鼠视网膜ET－1表达的差异有无统计学意义。当差异有统计学意义时，进一步采用Student－NewMan－Keuls法进行两两比较。

结果

视网膜显微形态学观察结果显示，空白对照组大鼠的视网膜组织结构层次分明，各层细胞排列整齐规则，细胞染色均匀且形态规整；对照组6周大鼠的视网膜神经节细胞排列紊乱、细胞数目减少及视网膜变薄；实验组6周大鼠的视网膜组织结构，并未见明显异常。对照组6周大鼠与空白对照组相比，视网膜周边血管走形迂曲。实验组6周大鼠视网膜血管走形未见明显异常。空白对照组、对照组及实验组三组之间ET－1表达的差异有统计学意义(F_组别＝95.15，P<0.05)。且糖尿病大鼠随病程进展ET－1表达逐渐增强，表现为对照组>实验组>空白对照组。经两两比较，结果差异有统计学意义(t＝－13.45，－9.47，8.57；P<0.05)。视网膜ET－1在不同时间点表达的比较，表现为6周>4周>2周，差异有统计学意义(F_时间＝72.12，P<0.05)。对照组大鼠视网膜ET－1的表达水平在不同时间点的两两比较，差异有统计学意义(t＝－7.63，－12.85，－7.32；P<0.05)。实验组大鼠视网膜ET－1的表达水平在不同时间点的两两比较，差异有统计学意义(t＝－4.03，－10.62，－7.68；P<0.05)。

结论

缬沙坦能够抑制糖尿病视网膜ET－1的表达，可能对糖尿病视网膜病变治疗起延缓作用。

关键词: 糖尿病, 视网膜, 缬沙坦, 内皮素－1

Objective

We aimed to observe the changes of the ET-1 protein expression in the retinea of diabetic rats which were treated by Valsartan, to reveal the therapeutical mechanism of suppressing the rennin-angitensin system in the diabetic retinopathy.

Methods

115 male Wistar rats were divided into normal, diabetes mellitus and treatment group randomly. The control group consisted of 40 rats, the experimental group consisted of 40 rats and the blank control group consisted of 35 rats. An animal model of diabetes was established. The control group and the experimental group were injected with the same dose of citric acid buffer solution in the same time by 65 mg/kg weight intraperitoneal injection of STZ solution with a concentration of 1%. Fasting blood glucose level of 16.65 mmol/L as a successful model. After the establishment of the model, the diabetes mellitus group was treated with Valsatan 24 mg/(kg·d), the other two groups are given distilled water. All the rats were normal bred. 2, 4 and 6 weeks after rat modal of diabetes, pluck eyeball after anesthesia. ADP enzymohisto-chemistry stain method of retinal stretched preparation was used to observe the change of retinal blood vessels. The immunohistochemistry method was used to measure the expression of ET-1 in the retina. The average integral optical density value of ET-1 was expressed as mean±standard deviation(±s). The two factor repeated measures analysis of variance was used to compare the differences between different groups and at different time points. The difference was statistically significant, and the Student-NewMan-Keuls method was used to compare the differences.

Results

The retinal optical microscopic observation showed that the level of blank control group rat retinal tissue structure is clear, the blank cells in each layer arranged, cell staining uniform and regular shape; the control group 6 weeks retinal tissue structure of rats, ganglion cells were arranged disorder, reducing the number of cells and the thinning of the retina; retinal tissue structure in experimental group 6 weeks in rats, and no obvious abnormalities. Normal rats have normal retinal blood vessels and Capillary tube was uniform. Diabetic rats had abnomal change in retinal vascular in the 6th week. 6 weeks of treatment group in rats had nonsignificant abnormal vascular. There was significant difference in ET-1 expression between the three groups of blank control group, control group and experimental group (F_group =95.15, P<0.05). The expression of ET-1 in diabetic rats increased gradually with the progression of the disease, the control group>the experimental group>the blank control group, the difference was statistically significant (t=-13.45, -9.47, 8.57; P<0.05). At different time points, 6 weeks>4 weeks>2 weeks, the difference was statistically significant (F_time=72.12, P<0.05). The expression levels of ET-1 in the retina of the control group were compared at different time points, the difference was statistically significant (t=-7.63, -12.85, -7.32; P<0.05). The expression levels of ET-1 in the retina of experimental group were compared with each other at different time points, the difference was statistically significant (t=-4.03, -10.62, -7.68; P<0.05).

Conclusion

Valsartan can reduce ET-1 in the retina of diabetic rat. Therefore, it is said to have treatment of diabetic retinopathy.

Key words: Diabetes mellitus, Retina, Valsartan, Endothelin-1

图1 实验大鼠视网膜光学显微镜下形态学观察图像　A图示空白对照组大鼠的视网膜组织结构层次分明，各层细胞排列整齐规则，细胞染色均匀且形态规整；B图示对照组6周大鼠的视网膜组织结构，神经节细胞排列紊乱，细胞数目减少且视网膜变薄；C图示实验组6周大鼠的视网膜组织结构，并未见明显异常(HE染色×400)

表1 实验不同时期三组实验大鼠视网膜ET－1表达平均积分光密度值的比较(±s)

组别	积分光密度值
组别	2周	4周	6周
空白对照组	16.10±11.01	15.35±8.38	15.22±4.71
对照组	21.20±11.84	60.19±12.95^ab	114.21±22.71^ab
实验组	18.14±9.67	32.37±8.10^a	64.03±12.22^a

表1 实验不同时期三组实验大鼠视网膜ET－1表达平均积分光密度值的比较(±s)

组别	积分光密度值
组别	2周	4周	6周
空白对照组	16.10±11.01	15.35±8.38	15.22±4.71
对照组	21.20±11.84	60.19±12.95^ab	114.21±22.71^ab
实验组	18.14±9.67	32.37±8.10^a	64.03±12.22^a

图2 实验大鼠视网膜的ET－1表达图像　A图示空白对照组大鼠视网膜ET－1表达；B图示对照组6周大鼠视网膜ET－1表达，随病程进展ET－1表达逐渐增强；C图示实验组6周大鼠视网膜ET－1表达，与同期的空白对照组相比，ET－1表达有所增高，但显著低于对照组(免疫组织化学染色×400)

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Effect of valsartan on the expression of Endothelin-1 in the retinea of diabetic rats

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Effect of valsartan on the expression of Endothelin-1 in the retinea of diabetic rats