活体观察N-乙酰半胱氨酸防治白内障的实验研究

Preventive and therapeutical effects of N-acetylcysteine on selenite-induced cataract in vivo

背景亚硒酸钠诱导的白内障与年龄相关性白内障的形成机制具有一定的相似性，即氧化损伤，N-乙酰半胱氨酸（NAC）是一种有效的抗氧化剂，但其对白内障的预防和治疗作用研究尚少。目的观察NAC对亚硒酸钠诱导大鼠白内障的预防和治疗作用，为白内障的药物防治提供实验依据。方法实验分为预防部分和治疗部分。选取SD大鼠60只，随机数字表法分为正常对照组1、正常对照组2、硒性白内障组、NAC白内障预防组、硒性白内障生理盐水组及NAC白内障治疗组，每组10只大鼠。采用3．46mg／kg亚硒酸钠颈部皮下注射法制作硒性白内障模型，隔日1次，共3次。预防实验时在首次注射亚硒酸钠前30min大鼠腹腔内注射2mmol／L NAC，每日1次，共6次；治疗实验时，硒性白内障大鼠造模后1d腹腔内注射2mmol／L NAC，每日1次，共1个月；硒性白内障生理盐水组以同样方法注射生理盐水。每周各组大鼠在裂隙灯下观察晶状体混浊程度并参考LOCSⅢ标准进行分级。各实验组大鼠最后一次注药后制备晶状体组织切片，光学显微镜下观察晶状体上皮细胞（LECs）的组织病理学改变，扫描电子显微镜下观察晶状体上皮超微结构的改变。采用免疫组织化学法观察亚硒酸钠对晶状体中caspase-3的影响；对各组大鼠晶状体组织中超氧化物歧化酶（SOD）、丙二醛（MDA）含量的变化进行生化测定。结果实验后7d正常大鼠晶状体透明。硒性白内障组Ⅴ级晶状体混浊者有11只眼，NAC白内障预防组仅有Ⅱ级混浊8只眼和Ⅰ级混浊2只眼，差异有统计学意义（Х^2=40．000，P〈0．05）。实验后30d，硒性白内障生理盐水组和NAC白内障治疗组Ⅳ～Ⅴ级晶状体混浊均为20只眼，差异无统计学意义（Х^2=0．153，P〉0．05）。常规组织病理学检查表明，正常对照组LECs及晶状体纤维结构正常，硒性白内障组、硒性白内障生理盐水组和NAC白内障治疗组LECs与前囊部分分离，排列疏松紊乱，细胞膜破裂，细胞核呈椭圆形或长条形，晶状体纤维断裂，NAC白内障预防组晶状体结构破坏程度较轻。扫描电子显微镜下可见硒性白内障组、硒性白内障生理盐水组和NAC白内障治疗组晶状体前囊分层，外膜脱离，深层可见“变性球样小体”，纤维紊乱破碎，形成无结构的“水泥样”外观。硒性白内障组caspase-3和SOD的表达明显低于正常对照组，MDA的表达高于正常对照组，差异均有统计学意义（P〈0．05），而NAC白内障预防组caspase-3和SOD的表达明显高于硒性白内障组，差异均有统计学意义（P〈0．05）。硒性白内障生理盐水组与NAC白内障治疗组caspase-3、SOD和MDA表达均明显低于正常对照组2，差异均有统计学意义（P〈0．05），而硒性白内障生理盐水组与NAC白内障治疗组比较，caspase-3、SOD和MDA表达的差异均无统计学意义（P〉0．05）。结论NAC可以提高晶状体组织中SOD的活性，减少MDA生成，降低caspase-3的活性，从而减轻晶状体的氧化损伤，对早期白内障的发生、发展有一定的延缓和预防作用，但对于已经形成的白内障无明显治疗作用。

Background The mechanism of both selenite-induced cataract and age-related cataract is oxidative damage. N-acetylcysteine （NAC） is one of the effective antioxidants, but the literature is little about the preventive and treating effects of NAC on cataract. Objective This study attempted to investigate the preventive and therapeutical effects of NAC on the selenite-induced cataract, and to discuss the possible mechanism. Methods Sixty 10-day-old clean SD rats were randomly divided into normal control group-l, normal control group-2, selenite- induced cataract group,NAC preventive group,NAC＋normal saline group and NAC treatment group. Selenite cataract models were induced by subcutaneous injection of 3.46 mg/kg sodium selenite once daily for three days. The rats of NAC preventive group received the intraperitoneal injection of 2 mmol/L NAC 30 minutes before the injection of sodium selenite once daily for 6 days. In NAC treating group,2 mmol/L NAC was intraperitoneally injected 1 day after the injection of sodium selenite for 30 days, and the normal saline solution was injected at the same method in the NAC＋normal saline group. Lens opacification was graded according to LOCS HI criteria. Histopathological change of lens epithelium was examined under a light microscope after hemotoxylin and eosin staining, and the ultrastructure was observed under the scanning electron microscope. The expression of caspase-3 in lens was assayed using immunochemistry. The levels of superoxide dismutase （SOD）, malonaldehyde （MDA） in rat lens were detected respectively in corresponding time points. The use of the experimental animals complied with the Regulation for the Administration of Affairs Concerning Experimental Animals by State Science and Technology Committee. Results In 7 days after experiment,lenses were completely clear in the normal control group. Lens opacification of V grade was found in 11 eyes in selenite cataract model group, but no lens opacification of Ⅴ grade was seen in NAC preventive group, showing a significant difference （Х^2 = 40. 000,P〈0.05 ）. In 30 days after experiment, Ⅳ- Ⅴ grades of cataracts were found in 20 eyes both in NAC ＋ normal saline group and NAC treating group （Х^2 = 0. 153, P〉 0.05）. Histopathological examination showed that lens structure was normal, and the separation between LECs and anterior capsule,the rupture of cellular membrane, deformation of cellular nuclei and the feature of lens fiber were seen in selenite cataract group, but the damage of lens was mild in the NAC preventive group. Ultrastructure of lens was obviously abnormal in selenite cataract group, NAC ＋normal saline group and NAC treating group. Expressions of caspase-3 and SOD in lens were significantly lower,but that of MDA was significantly higher in the selenite cataract group than the normal control group （P〈0.05） ; while those of the NAC preventive group were significantly different from selenite cataract group（ P〈0.05 ）. No significant difference was found in the expressions of caspase-3 and the levels of SOD and MDA between NAC＋normal saline group and NAC treating group （P〉0.05）. Conclusions Selenite can induce the apoptosis of LECs. NAC can evidently postpone formation of selenite cataract by increasing the activity of SOD, decreasing the level of MDA and the expression of caspase-3. However, NAC could not reverse selenite-induced lens damage.