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

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。

Accurate quantification of astaxanthin from Haematococcus crude extract spectrophotometrically

The influence of alkali on astaxanthin and the optimal working wave length for measurement of astaxanthin from Haematocoecus crude extract were investigated, and a spectrophotometric method for precise quantification of the astaxanthin based on the method of Boussiba et al. was established. According to Boussiba＇s method, alkali treatment destroys chlorophyll. However, we found that： 1） carotenoid content declined for about 25% in Haematococcus fresh cysts and up to 30% in dry powder of Haematococcus broken cysts after alkali treatment; and 2） dimethyl sulfoxide （DMSO）-extracted chlorophyll of green Haematococeus bares little absorption at 520-550 nm. Interestingly, a good linear relationship existed between absorbance at 530 nm and astaxanthin content, while an unknown interference at 540-550 nm was detected in our study. Therefore, with 530 nm as working wavelength, the alkali treatment to destroy chlorophyll was not necessary and the influence of chlorophyll, other carotenoids, and the unknown interference could be avoided. The astaxanthin contents of two samples were measured at 492 nm and 530 nm; the measured values at 530 nm were 2.617 g/100 g and 1.811 g/100 g. When compared with the measured values at 492 nm, the measured values at 530 nm decreased by 6.93% and 11.96%, respectively. The measured values at 530 nm are closer to the true astaxanthin contents in the samples. The data show that 530 nm is the most suitable wave length for spectrophotometric determination to the astaxanthin in Haematococcus crude extract.