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
자외선-B격광조사대α정상체단백적영향급3-신타갑순대기분자반려활성적보호작용
Effects of ultraviolet-B laser irradiation on lens α-crystallin and protection of indole-3-carbinol to chaperone activity of α-crystallin
  • 万方数据
    中华实验眼科杂志 중화실험안과잡지
    CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
    2012年 6期 538-542 ,共5页

    张钦%曹晓光%裴雪婷%鲍永珍

    장흠%조효광%배설정%포영진

    α晶状体蛋白%3-吲哚甲醇%紫外线-B激光%分子伴侣活性 α晶狀體蛋白%3-吲哚甲醇%紫外線-B激光%分子伴侶活性
    α정상체단백%3-신타갑순%자외선-B격광%분자반려활성
    α-Crystallin%Indole-3-carbinol%Ultraviolet-B laser%Chaperone activity
    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、11 875.00和23 750.00mJ/cm2的紫外线-B激光照射在凸透镜后一固定位置的α晶状体蛋白,然后通过改变α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28 535.00、6730.00、3435.00、1910.00、1040.00 mJ/cm2.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、11 875.00mJ/cm2照射后的α晶状体蛋白溶液中分别加入50μmol/L和100 μmol/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50 μmol/L和100 μmol/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360 nm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1187.50 mJ/cm2时,α晶状体蛋白A280值降到10％左右,当照射能量达到23.75 J/cm2时,α晶状体蛋白A280值降到2％以下,二者呈负相关(R2=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(R2=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(R2=0.949).CAT热凝聚实验表明,加入50 μmol/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.
    배경 자외선조사시년령상관성백내장형성적유인지일.연구표명,3-신타갑순(I3C)가억제양화작용도치적세포손해급정분양섬유변성적형성,양화손상급정분양섬유변성적형성균여백내장유관,이I3C여α정상체단백활성적관계상유대증실. 목적 평고자외선-B격광조사대α정상체단백결구화분자반려공능적영향,탐토I3C대α정상체단백분자반려공능적보호작용.방법 취신선1세령우안구적정상체,채용응효층석법제순우적α정상체단백,병안조쾌속단백액상색보( FPLC)흡수보선수집α정상체단백.연후분별이23.75、118.75、475.00、1187.50、2375.00、4750.00、11 875.00화23 750.00mJ/cm2적자외선-B격광조사재철투경후일고정위치적α정상체단백,연후통과개변α정상체단백재철투경후적위치체도개변조사능량적목적,사각조조사능량분별위28 535.00、6730.00、3435.00、1910.00、1040.00 mJ/cm2.사용자외분광광도의측량자외선-B격광조사전후α정상체단백적자외흡수보선(색안산형광보).재조사능량위475.00、1187.50、2375.00、4750.00、11 875.00mJ/cm2조사후적α정상체단백용액중분별가입50μmol/L화100 μmol/L적I3C,병진행과양화경매(CAT)열응취실험,판단α정상체단백분자반려활성,미가입50 μmol/L화100 μmol/L I3C적α정상체단백용액진행상동적실험작위대조,채용분광광도의측량360 nm파장처각조α정상체단백억제CAT열응취적흡광도(A360)치,계산각간예조여대조조A치적백분수작위평개분자반려활성적지표. 결과 α정상체단백자외흡수보측정발현,자외선-B격광조사능량위1187.50 mJ/cm2시,α정상체단백A280치강도10％좌우,당조사능량체도23.75 J/cm2시,α정상체단백A280치강도2％이하,이자정부상관(R2=0.925).색안산형광광보측정표명,자외선-B격광조사강도여색안산형광강도정부상관(R2=0.996),이여색안산대사산물N-갑선견뇨안산(N-FK)적형광강도정정상관(R2=0.949).CAT열응취실험표명,가입50 μmol/L화100μmol/L적I3C후,각강도적격광조사조α정상체단백적상대A360치균명현고우대조조,차이유통계학의의(P=0.000)；α정상체단백분자반려공능하강폭도저우대조조,차이유통계학의의(P=0.000).분자반려활성수착격광조사능량적증가여불가입I3C적α정상체단백상비강저변만.결론 자외선-B격광조사가이조성α정상체단백분자결구적변화급분자반려활성적강저,I3C대우α정상체단백분자반려활성구유보호작용.
    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 nmultraviolet-B at different irradiation intensities ( 23.75,118.75,475.00,1187.50,2375.00,4750.00,11 875.00,23 750.00 mJ/cm2 ) and then to ultraviolet-B 2:308 nm with irradiation intensities of 28 535.00,6730.00,3435.00,1910.00,1040.00 mJ/cm2.Ultraviolet-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-crystallin solution to perform a catalase (CAT) thermal aggregation to confirm the chaperone activity of the α-crystallin,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 ultraviolet-B irradiation intensity of 1187.5 mJ/cm2 and that at the intensity of 23.75 J/cm2 lowed to 2％.A negative correlation was seen between the ultraviolet-B irradiation intensity and the A280 value of the α-crystallin (R2=0.925 ) and a positive correlation was found between ultraviolet-B with N-FK ( R2 =0.949 ).Ultraviolet-B irradiation intensity showed a negative correlation with Trp fluorescence intensity (R2 =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-crystallin was lower than that of the control group ( P =0.000 ). 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.
    원문보기 원문다운로드 사이트로이동
저자의 최근 논문