CAJ | 학술논문

目的葡萄籽原花青素(grape seed proanthocyanidin extract,GSPE)能保护人眼晶状体上皮细胞免受氧化应激.该研究探讨葡萄籽原花青素对防止氧化应激相关的线粒体锰超氧化物歧化酶(mitochondrial-associated manganese superoxide dismutase,MnSOD)的表达(活力)的影响.方法人晶状体上皮细胞(human lens epithelial cells,HLE-B3)与GSPE共同预孵育相应时间段后收集mRNA和蛋白质的裂解物.Real-time PCR检测晶状体上皮细胞MnSOD基因的表达;Western印迹分析研究对其蛋白表达的影响;同时测定MnSOD活性.结果 HLE-B3细胞与GSPE共同作用6 h MnSOD活性无统计学意义的变化(67.5 ±6.5 u/mL,t=1.56,P＞0.05);12h后,MnSOD活性呈现一个显著的快速增长(110±10.3 u/mL,t=4.03,P＜0.05),24 h后下降到对照组水平(49.3±6.9 u/mL,t=1.69,P＞0.05).实验组的MnSOD基因和蛋白的表达检测没有显著变化.结论该研究表明,GSPE能增加MnSOD的活性,但是并不影响其mRNA和蛋白的表达.MnSOD通过减少ROS的生成从而在线粒体氧化应激中起到重要作用,最终促进细胞的存活.
목적 포도자원화청소(grape seed proanthocyanidin extract,GSPE)능보호인안정상체상피세포면수양화응격.해연구탐토포도자원화청소대방지양화응격상관적선립체맹초양화물기화매(mitochondrial-associated manganese superoxide dismutase,MnSOD)적표체(활력)적영향.방법 인정상체상피세포(human lens epithelial cells,HLE-B3)여GSPE공동예부육상응시간단후수집mRNA화단백질적렬해물.Real-time PCR검측정상체상피세포MnSOD기인적표체;Western인적분석연구대기단백표체적영향;동시측정MnSOD활성.결과 HLE-B3세포여GSPE공동작용6 h MnSOD활성무통계학의의적변화(67.5 ±6.5 u/mL,t=1.56,P＞0.05);12h후,MnSOD활성정현일개현저적쾌속증장(110±10.3 u/mL,t=4.03,P＜0.05),24 h후하강도대조조수평(49.3±6.9 u/mL,t=1.69,P＞0.05).실험조적MnSOD기인화단백적표체검측몰유현저변화.결론 해연구표명,GSPE능증가MnSOD적활성,단시병불영향기mRNA화단백적표체.MnSOD통과감소ROS적생성종이재선립체양화응격중기도중요작용,최종촉진세포적존활.
Objective Grape seed proanthocyanidin extract (GSPE) protects human lens epithelial cells against oxidative stress.The study described herein examined whether GSPE also elicits genomic protection by influencing the expression (and activity) of mitochondrial-associated manganese superoxide dismutase (MnSOD) as a possible mechanism by which GSPE protects against oxidative stress.Methods human lens epithelial cells (HLE-B3) were pre-incubated with GSPE,and mRNA or protein lysates were collected over a time course ranging from 90 min to 24 h.Positive expression of lens epithelial cell MnSOD mRNA was determined by real-time PCR up to 24 h after GSPE.Western blot analysis was used to examine the pattern of protein expression as influenced by GSPE treatment.MnSOD activity as influenced by GSPE was determined by measuring enzymatic activity.Results There was no significant change in the activity of MnSOD of 6 h (67.5 ±6.5 u/mL,t =1.56,P ＞0.05).A significant rapid increase in the activity of MnSOD(110 ± 10.3 u/mL,t =4.03,P ＜ 0.05) was observed with HLE-B3 cells treated with 12h addition of GSPE,which returned to control level (49.3 ± 6.9 u/mL,t =1.69,P ＞ 0.05) by 24h.Neither an increase in MnSOD mRNA nor in protein expression was detected up through 24 h.Conclusion These data demonstrate that GSPE increases the activity of MnSOD but influences neither its mRNA expression nor its protein expression.The results suggest that MnSOD plays an important role against mitochondrial oxidative stress by diminishing reactive oxygen species,thus promoting cell survival.