虾青素对1型糖尿病大鼠代谢性白内障的预防作用及其机制

Proventive effects of astaxanthin on metabolic cataract and its mechanism in type 1 diabetic rats

背景 糖尿病性白内障的发病机制尚未完全明了，研究认为多种代谢通路参与其发病，其中包括氧化应激机制。研究表明虾青素有强大的抗氧化作用，可有效抑制氧化应激损伤及脂质过氧化，但目前鲜见虾青素对糖尿病性白内障防治作用研究的相关报道。目的 观察虾青素对1型糖尿病大鼠代谢性白内障的预防作用及其机制。方法 将38只SPF级6周龄雄性SD大鼠纳入研究，其中30只大鼠用一次性腹腔内注射质量分数1%链脲佐菌素（STZ）方法制备糖尿病大鼠模型，连续3 d血糖值〉16.7 mol/L者为造模成功，应用随机数字表法将造模成功的24只大鼠随机分为糖尿病模型组、低剂量虾青素组和高剂量虾青素组，正常对照组8只大鼠同法注射等容量生理盐水。低剂量虾青素组和高剂量虾青素组大鼠分别给予50 mg/（kg·d）和100 mg/（kg·d）虾青素以及橄榄油和饲料混合物连续喂养3个月，糖尿病模型组大鼠以等容量橄榄油混合饲料喂养，正常对照组以正常饲料喂养。造模后用裂隙灯显微镜行眼前节照相并对晶状体混浊的严重程度分为1～5级；收集大鼠双侧眼球制备晶状体切片，采用苏木精－伊红染色法观察大鼠晶状体的组织病理学改变；采用免疫组织化学法观察晶状体中糖基化终末产物（AGEs）的阳性表达并进行定量分析；采用ELISA双抗体夹心法测定各组大鼠晶状体内AGEs质量浓度、丙二醛（MDA）浓度、过氧化氢酶（CAT）、超氧化物歧化酶（SOD）水平以及谷胱甘肽（GSH）质量浓度。结果 造模后2、4、6、8、10和12周糖尿病模型组、低剂量虾青素组和高剂量虾青素组大鼠血糖水平均明显高于正常对照组，差异均有统计学意义（均P〈0.05），而糖尿病模型组、低剂量虾青素组和高剂量虾青素组间大鼠血糖水平的差异均无统计学意义（均P〉0.05）。正常对照组大鼠晶状体透明，为1级，糖尿病模型组晶状体混浊均为5级，不同剂量虾青素组大鼠晶状体混浊度多为3～4级。低剂量虾青素组和高剂量虾青素组大鼠晶状体匀浆内的AGEs质量浓度分别为（7.23±0.50）μg/ml和（7.01±0.37）μg/ml，MDA浓度分别为（1.43±0.22）mmol/L和（1.35±0.16）mmol/L，均低于糖尿病模型组的（7.61±0.45）μg/ml和（1.62±0.42）mmol/L，差异均有统计学意义（均P〈0.05）。低剂量虾青素组和高剂量虾青素组大鼠晶状体中GSH质量浓度分别为（272.70±12.53）ng/L和（283.52±16.17）ng/L，SOD含量分别为（55.45±6.47）μmol/（min·L）和（56.73±5.12）μmol/（min·L），CAT含量分别为（2.91±0.41）μmol/（min·L）和（3.02±0.13）μmol/（min·L），均明显高于糖尿病模型组的（241.52±15.13）ng/L、（51.67±5.45）μmol/（min·L）和（2.72±0.27）μmol/（min·L），差异均有统计学意义（均P〈0.05），低剂量虾青素组大鼠晶状体中GSH质量浓度和SOD含量明显低于高剂量虾青素组，差异均有统计学意义（均P〈0.05）。结论 虾青素能够延缓1型糖尿病大鼠代谢性白内障的发生和发展，其作用机制与其抗氧化应激反应有关。

Background The pathogenesis mechanism of diabetic cataract has not been fully elucidated.Researches showed that multiple biological pathways participate in the pathogenesis of diabetic cataract, including oxidative stress.Astaxanthin can inhibit oxidative stress-mediated injury and lipid peroxidation.However, whether astaxanthin has the preventive effects on diabetic cataract is unclear. Objective This study was to investigate the preventive effects of astaxanthin on metabolic cataract in type 1 diabetic rats.Methods Thirty-eight 6-week-old SPF male SD rats were used in this study, and 1% streptozocin was intraperitoneally injected to establish type 1 diabetic models in 30 rats, and 24 successful models were assigned to diabetic model group, low-dose astaxanthin group and high-dose astaxanthin group.Equal volume of normal saline solution was injected in the same way in 8 rats as the normal control group.Mixture foods containing 50 mg/（kg·day） or 100 mg/（kg·day） astaxanthin with olive oil and fodder were used continuously for 3 months in the rats of low-dose astaxanthin group and high-dose astaxanthin group, respectively, and mixture food of olive oil with fodder was used in the diabetic model group.Only fodder was used in the same way in the rats of the normal control group.The opacification of lens was examined by slit lamp section radiography system and graded on a scale of 1-5.The specimen of lens were prepared for the hematoxylin ＆ eosin stain.The expression and lation of advanced glycosylation end products （AGEs） in the lens was examined using immunochemistry.The contents of oxidative stress-related indicators in the lens, such as AGEs, malonydialdehyde （MDA）, catalase （CAT）, superoxide dismutase （SOD） and mass fraction of glutathione （GSH）, were assayed by ELISA.The experimental process complied with the national standard （Laboratory Animal Requirements of Environment and Housing Facilities [GB14925-2001]）.Results The blood glucose levels of the rats were significantly higher in the diabetic model group, low-dose astaxanthin group and high-dose astaxanthin group than those in the normal control group at 2, 4, 6, 8, 10 and 12 weeks after modeling （all at P〈0.05）, while the blood glucose levels of rats were not evidently different between low-dose astaxanthin group and high-dose astaxanthin group at various time points（all at P〉0.05）. The rat lenses were transparent in the normal control group with scale of grade 1, and serious lens opacification was seen in the rats of the diabatic model group, with the scale of grade 5, while the rat lenses in the low-dose astaxanthin group and high-dose astaxanthin group were in grade 3-4.The contents of AGEs in the lenses were （7.23±0.50）μg/ml and （7.01±0.37）μg/ml, and MDA contents were （1.43 ±0.22）mmol/L and （1.35±0.16）mmol/L in the low-dose astaxanthin group and high-dose astaxanthin group respectively, which were significantly lower than （7.61± 0.45）μg/ml and （1.62±0.42）mmol/L in the normal control group （all at P〈0.05）. GSH contents in rat lenes were （272.70±12.53）ng/L and （283.52±16.17）ng/L, and SOD concents were （55.45±6.47）μmol/（min·L） and （56.73±5.12）μmol/（min·L）, and CAT concents were （2.91±0.41）μmol/（min·L） and （3.02±0.13）μmol/ （min·L） in the low-dose astaxanthin group and high-dose astaxanthin group respectively, which were significantly higher than （241.52±15.13）ng/L, （51.67±5.45）μmol/（min·L） and （2.72±0.27）μmol/（min·L） in the normal control group （all at P〈0.05）. The GSH concent and SOD concent in rat lens were lower in the low-dose astaxanthin group than that in the high-dose astaxanthin group （both at P〈0.05）.Conclusions Astaxanthin can postpone the pathogenesis and development of diabetic cataract in type 1 diabetic rats by antioxydative stress.