紫外线-B激光照射对α晶状体蛋白的影响及3-吲哚甲醇对其分子伴侣活性的保护作用

Effects of ultraviolet-B laser irradiation on lens α-crystallin and protection of indole-3-carbinol to chaperone activity of α-crystallin

背景紫外线照射是年龄相关性白内障形成的诱因之一。研究表明，3-吲哚甲醇（I3C）可抑制氧化作用导致的细胞损害及淀粉样纤维变性的形成，氧化损伤及淀粉样纤维变性的形成均与白内障有关，而I3C与α晶状体蛋白活性的关系尚有待证实。目的评估紫外线-B激光照射对α晶状体蛋白结构和分子伴侣功能的影响，探讨I3C对α晶状体蛋白分子伴侣功能的保护作用。方法取新鲜1岁龄牛眼球的晶状体，采用凝胶层析法提纯牛的α晶状体蛋白，并按照快速蛋白液相色谱（FPLC）吸收谱线收集α晶状体蛋白。然后分别以23．75、118．75、475．00、1187．50、2375．00、4750．00、11875．00和23750．00mJ／cm^2的紫外线-B激光照射在凸透镜后一固定位置的α晶状俸蛋白，然后通过改变仅晶状体蛋白在凸透镜后的位置达到改变照射能量的目的，使各组照射能量分别为28535．00、6730．00、3435．00、1910．00、1040．00mJ／cm^2。使用紫外分光光度仪测量紫外线-B激光照射前后α晶状体蛋白的紫外吸收谱线（色氨酸荧光谱）。在照射能量为475．00、1187．50、2375．00、4750．00、11875．00mJ／cm^2照射后的α晶状体蛋白溶液中分别加入50μmol／L和100μmol／L的I3C，并进行过氧化氢酶（CAT）热凝聚实验，判断α晶状体蛋白分子伴侣活性，未加入50μmol／L和100μmol／L I3C的α晶状体蛋白溶液进行相同的实验作为对照，采用分光光度仪测量360nm波长处各组α晶状体蛋白抑制CAT热凝聚的吸光度（A360）值，计算各干预组与对照组A值的百分数作为评价分子伴侣活性的指标。结果α晶状体蛋白紫外吸收谱测定发现，紫外线-B激光照射能量为1187．50mJ／cm^2时，α晶状体蛋白A280值降到10％左右，当照射能量达到23．75J／cm^2时，α晶状体蛋白A280值降到2％以下，二者呈负相关（R^2=0．925）。色氨酸荧光光谱测定表明，紫外线-B激光照射强度与色氨酸荧光强度呈负相关（R^2=0．996），而与色氨酸代谢产物N-甲酰犬尿氨酸（N—FK）的荧光强度呈正相关（R62=0．949）。CAT热凝聚实验表明，加入50μmol／L和100μmol／L的I3C后，各强度的激光照射组α晶状体蛋白的相对A360值均明显高于对照组，差异有统计学意义（P=0．000）；α晶状体蛋白分子伴侣功能下降幅度低于对照组，差异有统计学意义（P=0．000）。分子伴侣活性随着激光照射能量的增加与不加入I3C的α晶状体蛋白相比降低变慢。结论紫外线-B激光照射可以造成α晶状体蛋白分子结构的变化及分子伴侣活性的降低，I3C对于α晶状体蛋白分子伴侣活性具有保护作用。

Background Ultraviolet radiation is one of factors of the formation of age-related cataract. Indole-3-carbinol（I3C） is a plant chemical material with inhibitory effect on oxidative-induced cell damage and formation of amyloid fibrils, and the oxidative damage and amyloid fibrils are associated with cataract. However, the relationship between I3C and α-crystallin is in study. Objective This study was to evaluate the effects of ultraviolet-B laser irradiation on the secondary structure of α-crystallin and to explore the protection of I3C to chaperone activity of α-crystallin. Methods The fresh eyeballs were obtained from 1-year-old cattle to prepare the purified lens α-crystallin by gel chromatography, α-Crystallin was isolated from cattle lenses using gel chromatography. The purified α-crystallin was collected using fast protein liquid chromatography （FPLC） and exposed to 1 ： 308 nm ultraviolet-B at different irradiation intensities （ 23.75,118.75,475.00, 1187.50,2375.00,4750.00, 11 875.00, 23 750. 00 mJ/cm^2） and then to uhraviolet-B 2：308 nm with irradiation intensities of 28 535.00,6730.00,3435.00, 1910.00,1040. 00 mJ/cm^2. Uhraviolet-absorbance spectra, tryptophan fluorescence and N-formylkynurenine （N-FK） fluorescence spectra of both irradiated and non-irradiated α-crystallin were measured. I3C at the concentrations of 50 μmol/L and 100 μmol/L were added to the α-erystallin solution to perform a eatalase （CAT） thermal aggregation to confirm the chaperone activity of the α-erystallin, and the α-crystallin solution without any I3C was used as control. The ratios of A360 between various intervene groups with control group were calculated using spectrophotometry. Results The A280 values of the α-crystallin declined to 10% at the uhraviolet-B irradiation intensity of 1187. 5 mJ/cm^2 and that at the intensity of 23.75 J/cm^2 lowed to 2%. A negative correlation was seen between the uhraviolet-B irradiation intensity and the A280 value of the α-crystallin （R^2 = 0. 925） and a positive correlation was found between ultraviolet-B with N-FK （ R^2 = 0. 949 ）. Ultraviolet-B irradiation intensity showed a negative correlation with Trp fluorescence intensity （R^2 = 0. 996 ）. CAT hot condensed experiment revealed that after addition of different concentrations of indole-3-carbinol,the relative A360 values at various ultraviolet-B irradiation group were significantly higher than those of the control group （P=0. 000） ,and the decreasing degree of chaperone activity of α-erystallin was lower than that of the control group （ P = O. 000）. Conclusions The study suggests that I3C can protect the chaperone activity of α-crystallin from photooxidation, and the ultraviolet-B laser may be a good exposure source compared with ultraviolet lamp. The ultraviolet-B laser irradiation causes the alteration of structure and chaperone activity of α-crystallin.