目的：

1、观察虾青素（Astaxanthin）对1型糖尿病大鼠晶状体组织的保护作用及相关机制。

2、观察虾青素（Astaxanthin，AST）对1型糖尿病大鼠视网膜病变的保护作用及相关机制。

方法：

1、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）方法制备大鼠糖尿病模型，连续3 d血糖值>16.7 mol/L者为造模成功，造模成功的24只大鼠按照随机方法分为糖尿病模型组、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20 mg/(kg·d)和100 mg/(kg·d)虾青素、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、脂质过氧化物/丙二醛（MDA）含量、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。

2、应用腹腔一次性注射1%链脲佐菌素（streptpzoxin，STZ）方法制备大鼠糖尿病模型（36只SPF雄性大鼠），随机分为糖尿病模型组（DM）、低剂量虾青素组(DM+AL)，高剂量虾青素组（DM+AH），DM+ AL组和DM+AH组分别给予20mg/kg/day ，100 mg/kg/day AST灌胃处理24周，DM 组给予等体积橄榄油，另有正常对照组（n＝12）。每两周测量大鼠的血糖和体重，24周后，处死大鼠，眼球称重标记， 经胰蛋白酶消化制作视网膜毛细血管铺片，每个样本随机取5个视野计算周细胞与无细胞性毛细血管的数量， 通过免疫组化，RT-PCR以及western测量白介素6（IL-6），肿瘤坏死因子（TNF-α），凋亡蛋白家族caspase-3在视网膜组织中的相对表达。

结果：  

1、12周后，DM+AL组及DM+AH组大鼠的体重高于DC组（p<0.05），而DM组、DM+AL组和DM+AH组之间的血糖的浓度水平无统计学差异（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含量分别为（7.23±0.5）μg/ml和（7.01±0.37） μg/ml，MDA含量分别为（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±0.132）μmol/(min·L)，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CAT含量明显低于高剂量虾青素组，差异有统计学意义（P<0.05）。

2、体重和血糖的测量结果表示，DM+AL组及DM+AH组大鼠的体重均高于DM组，血糖在糖尿病大鼠各组之间未呈现差异性表达；各组周细胞计数分别为466.4±23.2、207.3 ±31.7、298.1±27.1、312.2±19.5，组间总体比较差异具有统计学意义（F＝34.42，P＝0.047）。无细胞毛细血管计数显示各组分别为5.2±2.3、32.9±12.7、14.5±9.12、16.5±3.5，组间总体比较差异具有统计学意义（F＝47.34，P＝0.021 ）其中DM+AL、DM+AL的周细胞数量显著高于DM组，无细胞性毛细血管数量显著低于DM组（t1=3.9, P1=0.04; t2=4.2, P2=0.024) ,（t1=6.8, P1=0.041; t2=5.2, P2=0.034) 。 IL-6, TNF-α和 caspase-3的免疫组化染色的光密度值分析可见DM+AL,DM+AH组较DM组降低，差异均有统计学意义（P<0.05），RT-PCR提示，DM组 IL-6, TNF-α和caspase-3的mRNA相对表达量与正常对照组相比显著增加，DM+AL与DM+AH较 DM组均有所减少（t1-1=6.92, P1-1=0.03; t1-2=6.52, P1-2=0.03 ；t2-1=7.13, P2-1=0.03; t2-2=5.47, P2-2=0.04；t3-1=8.75, P3-1=0.03; t3-2=9.15, P3-2=0.02），差异具有统计学意义。Westernblot结果显示，DM组 IL-6, TNF-α和caspase-3的相对灰度值与正常对照组相比显著增加，组间总体比较差异具有统计学意义（F1＝61.44，P1＝0.017 F2＝33.18，P2＝0.027，F3＝91.11，P3＝0.021）， DM+AL、DM+AL的各个蛋白相对灰度表达显著高于DM组（t1-1=7.10, P1-1=0.03; t1-2=6.52, P1-2=0.02 ；t2-1=7.10, P2-1=0.03; t2-2=6.52, P2-2=0.02；t3-1=7.10, P3-1=0.03; t3-2=6.52, P3-2=0.02，差异具有统计学意义。

结论：

1、虾青素可以减轻1型糖尿病大鼠糖尿病性白内障的严重程度，在晶状体内发挥抗氧化作用，对晶状体组织具有保护作用，这种作用可能与虾青素能通过抑制氧化应激反应、提高机体抗氧化能力（抑制AGEs生成，提高SOD,CAT,GSH水平）以及减少氧化损伤（降低MDA含量）有关

2、虾青素可以抑制糖尿病大鼠的周细胞凋亡，对维持视网膜毛细血管正常形态起到保护作用，并可抑制视网膜组织内炎性因子及凋亡蛋白的表达。

Objective：

1. To investigate the effect of astaxanthin of metabolic cataract of rats with type 1 diabetes mellitus.  

2. To investigate the protective effect of astaxanthin on the retinapathy in rats with type 1 diabetes and related mechanism .

Methods：

1. Prepare diabetic rats model (n = 30) through the application of abdominal cavity chain of one-off injection of 1% urea with streptpzoxin(STZ). The model is built successfully with three consecutive days of blood sugar measurement value > 16.7 mol/L (n = 24).  Randomly divide the whole model into three groups of diabetic group (DM), low-dose astaxanthin group (DM+AL), high-dose astaxanthin group (DM+AH) (n=8). AL group was given 20 mg/kg/day AST mixed feed, AH group was given 100 mg/kg/day AST mixed feed for three months, and DM group was given same volume of olive oil. Additionally, there is a normal control group (n = 8) with no processing. Observe the turbid degree of rats lens by slit lamp section radiography system. View histopathological changes of rats’ lens with HE staining.  Use the double antibody method to determine the content of advanced glycation end-products (AGEs), Malonydialdehyde (MDA), catalase (CAT), the level of superoxide dismutase (SOD), and the mass fraction of glutathione (GSH). Use immunohistochemical quantitative method to observe the lens’ AGEs distributed in paraffin section.

2. Rats (n=36) were randomly assigned to diabetes +olive oil (DM) , DM+AL and DM+AH group (n = 12). The last groups received astaxanthin 20 mg/kg/day and 100 mg/kg/day dissolved in olive oil by gavage. The DM group received an equal volume of olive oil. An control group (n = 12) was included. During the study, blood glucose and body weight was measured every two weeks. After 24 weeks, the retina was digested to make retinal capillary network preparation. The number of pericytes and acelluar strands were calculated in the view and compared between the different experimental groups. Immunohistochemistry, Real-time polymerase chain reaction (RT-PCR) and western was used to detect the relative expression of anti-inflammatory cytokines in the in retinal tissue.

Results:

1. 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 lenes 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±0.5) 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 activities were (55.45±6.47) μmol/(min·L) and (56.73±5.12) μmol/(min·L), and  CAT activities 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) mmmol/(min·L) and (2.72±0.27) μmol/(min·L) in the normal control group (all at P<0.05). The CAT content in rat len was lower in the low-dose astaxanthin group than that in the high-dose astaxanthin group (P<0.05). Conclusions ：Astaxanthin can play a role of antioxidant in lens, therefore protect the lens. It can postpone the progress of the diabetic cataract.

2. The body weight of the rats in the DM+AL and DM+AH group was higher than that in the DM group. AST had no significant effect on rat blood glucose levels. The number of pericytes and acelluar strands in each group were 466.4±23.2、207.3 ±31.7、298.1±27.1、312.2±19.5; (F＝34.42，P＝0.047) 5.2±2.3、32.9±12.7、14.5±9.12、16.5±3.5 ;（F＝47.34，P＝0.021 ） In the DM+AL and DM+AH group, the relative expression of IL-6, TNF-αand caspase-3 in retinal tissues were decreased when compared with the DM group in levels of mRNA and protein. All differences between the groups were statistically significant (p < 0.05).

Concusions:

1. Astaxanthin can postpone pathogebesis and development of diabetic cataract in type 1 diabetic rats by  antioxydative stress.

2. AST may play an important role in protecting pericytes from apoptosis and may be delaying development and progression of diabetic retinopathy in diabetic rats. Additionally, it can inhibit the release of anti-apoptosis and anti-inflammatory cytokines.