背景 研究发现,白内障患者的血液、房水、泪液及晶状体内一氧化氮(NO)和一氧化氮合酶(NOS)含量均升高.NO是一种极不稳定的生物自由基,其生成依赖于NOS.目前NOS抑制剂对白内障防治作用的研究鲜有报道. 目的 制作大鼠半乳糖性白内障模型,应用NOS抑制剂-硝基左旋精氨酸甲酯(L-NAME)滴眼液进行干预,探讨L-NAME对大鼠半乳糖性白内障的作用. 方法 清洁级Wistar大鼠60只用随机数字表法随机分为对照组、模型组、L-NAME组3个组,每组各20只.对照组20只大鼠皮下注射质量分数0.9％生理盐水30 ml/kg;模型组与L-NAME组各20只大鼠均给予皮下注射等体积质量分数50％ D-半乳糖溶液,L-NAME组大鼠同时用L-NAME滴眼液点眼,每日3次.注射后第10、20、30天,在裂隙灯显微镜下检查各组鼠眼情况并对晶状体混浊程度进行评分,于注射后第30天摘出晶状体,检测各组大鼠晶状体中NO及NOS的含量.应用流式细胞分析技术检测各组大鼠晶状体上皮细胞(LECs)中凋亡基因半胱天冬酶-3(caspase-3)的含量.采用重复测量两因素方差分析比较各组大鼠不同时间点晶状体混浊评分的差异,采用单因素分析法比较各组大鼠晶状体中NO、NOS及caspase-3含量,以平均荧光强度指数(MFI)表示蛋白表达量.结果 模型组和L-NAME组大鼠皮下注射50％ D-半乳糖溶液后晶状体出现混浊,模型组大鼠晶状体混浊评分均明显高于L-NAME组;模型组和L-NAME组大鼠的晶状体混浊评分随着实验时间的延长均增加,差异均有统计学意义(F时间 =435.251,P=0.000;F分组=395.120,P=0.000).对照组大鼠晶状体中NO含量为(0.45±0.15) μmol/g,模型组为(2.67±0.47) μmol/g; L-NAME组为(1.68±0.34) μmol/g,3个组间差异有统计学意义(F=58.872,P=0.000).对照组大鼠晶状体中NOS含量为(0.0160±0.0020) U/ml(商品单位),模型组为(0.0370±0.0040) U/ml(商品单位),L-NAME组为(0.0270±0.0010) U/ml,3个组间的差异有统计学意义(F=66.174,P=0.000).对照组、模型组和L-NAME组大鼠晶状体中caspase-3含量分别为339.4±37.9、697.7±46.5和650.7±53.1,各组间差异有统计学意义(F=100.005,P=0.000). 结论 大鼠晶状体中NO、NOS和caspase-3含量的多少与晶状体混浊程度有关,L-NAME的局部应用可以抑制半乳糖性白内障晶状体中NOS的表达,减少NO的生成,抑制LECs的凋亡.%Background Researches showed that the content of nitric oxide (NO) and nitric oxide synthetase (NOS) increases in blood,aqueous humor and tear of cataract patient.But the function of NO and NOS in cataract formation is still elusive.Objective The aim of this study was to explore the prevention and treatment effect of NOS inhibitor,1-nitro-arginine methyl ester (L-NAME),on galactose cataract.Methods Sixty clean three-week-old Wistar rats were equally and randomly divided into 3 groups.0.9％ Normal saline solution (30 ml/kg) was subcutaneously injected every day for 30 days in the rats of the control group,and 50％ of D-galactose solution (30 ml/kg) was used in the rats of the model and L-NAME group at the same way.L-NAME eye drops was simultaneously administered in the L-NAME group 3 times per day for 30 days.The eyes of the rats were examined under the slit lamp in 10,20 and 30 days,and the degree of lens opacification was scored.Lenses of the rats were obtained at the end of this experiment for the detect of NO,NOS contents.Flow cytometry was used to assay the caspase-3 level of rat lens.Repeated measurement two factor analysis of variance was used to analyze the difference of lens opacification scores in different groups and different time points,and one-way ANOVA was used to analyze the differences of NO,NOS and caspase-3 contents in lens among the groups.Results Lens opacification appeared in 10 days after injection of 50％ D-galactose solution in the rats of the model group and L-NAME group.Lens opacification score was higher among the different groups and different time points (Ftime =435.251,P =0.000 ;Fgroup =395.120,P=0.000).NO content in the lens was (0.45±0.15) μmol/g,(2.67 ± 0.47) μmol/g and (1.68±0.34) μmol/g in the control group,model group and L-NAME group,showing a significant difference (F=58.872,P=0.000).The NOS contents in the lens was (0.0160±0.0020) U/ml,(0.0370±0.0040) U/ml and (0.0270±0.0010) U/ml in the control group,model group and L-NAME group,showing a significant difference (F =66.174,P=0.000).Caspase-3 contents in the lens was (339.4 ± 37.9),(697.7 ± 46.5) and (650.7 ± 53.1),Showing a significant difference among them (F =100.005,P =0.000).Conclusions The increase of NO,NOS and caspase3 levels are associated with lens opacification.Topical administration of L-NAME eye drops can down-regulate NOS content in lens,reduce the NO formation and inhibit the apoptosis of lens epithelial cells.
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