中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
Chinese Journal of Experimental Ophthalmology
2015年
11期
1009-1014
,共6页
氧化应激/生理%叶黄素/药理%Müller细胞%年龄相关性黄斑变性%核因子E2相关转录因子2/代谢%血红素加氧酶-1/代谢
氧化應激/生理%葉黃素/藥理%Müller細胞%年齡相關性黃斑變性%覈因子E2相關轉錄因子2/代謝%血紅素加氧酶-1/代謝
양화응격/생리%협황소/약리%Müller세포%년령상관성황반변성%핵인자E2상관전록인자2/대사%혈홍소가양매-1/대사
Oxidative stress/physiology%Lutein/pharmocology%Müller cells%Macular degeneration,age-related%Nuclear factor-E2-related factor 2/metabolism%Heme oxygenase-1/metabolism
背景 氧化应激是年龄相关性黄斑变性(AMD)的主要因素之一.研究表明叶黄素对AMD有预防作用,但是其抗氧化机制仍不明确.Müller细胞是视网膜氧化应激反应的靶细胞之一,研究叶黄素对Müller细胞的氧化应激是否具有保护作用及其作用机制对于视网膜疾病的治疗具有重要意义. 目的 研究叶黄素对视网膜Müller细胞核因子E2相关转录因子2/抗氧化反应原件(Nrf2/ARE)信号途径的影响. 方法 人Müller细胞株进行常规培养,将对数生长期的细胞接种于96孔培养板,在培养液中分别加入不同浓度(40、80、160、320、640 μmol/L) H2 O2,绘制Müller细胞的凋亡曲线,取H2O2的半数致死量,即160 μmol/L制备Müller细胞氧化应激模型.将模型细胞分为模型对照组和不同质量浓度(12.5、25.0、50.0 mg/L)叶黄素组,根据分组情况分别在培养液中加入相应质量浓度的叶黄素,用常规培养的细胞作为空白对照组.采用MTT比色法测定各组中Müller细胞增生值(吸光度,A值)和凋亡率;采用流式细胞仪检测各组细胞中活性氧簇(ROS)的荧光强度;采用实时荧光定量PCR检测细胞中Nrf2 mRNA和血红素加氧酶-l(HO-1) mRNA的相对表达量;采用Western blot法检测各组细胞中Nrf2和HO-1蛋白的表达水平(灰度值). 结果 MTT比色法检测显示,随着H2O2浓度的增加,细胞增生抑制率逐渐增加,各组间总体比较差异有统计学意义(F=43.890,P＜0.01).空白对照组、模型对照组及12.5、25.0、50.0 mg/L叶黄素组细胞凋亡率的总体比较差异有统计学意义(F=346.770,P=0.000),其中随着叶黄素质量浓度的增加,细胞凋亡率逐渐下降,组间比较差异均有统计学意义(均P＜0.05).空白对照组、模型对照组及12.5、25.0、50.0 mg/L叶黄素组细胞中ROS含量分别为1.92±0.18、64.89±2.86、52.70±2.80、32.61±4.20和5.68±1.35,随着叶黄素质量浓度的增加,ROS含量逐渐下降,组间总体比较差异有统计学意义(F=324.900,P=0.000).各质量浓度叶黄素组细胞中Nrf2和HO-1mRNA相对表达量及细胞中HO-1和细胞核中Nrf2蛋白表达量均明显高于模型对照组,各组间总体比较差异均有统计学意义(F=236.960、242.620、186.830、263.120,均P=0.000),随着叶黄素质量浓度的增加,Nrf2 mRNA和HO-1 mRNA及其蛋白的表达量逐渐增加,组间比较差异均有统计学意义(均P＜0.05),但各组间Müller细胞的细胞质中Nrf2蛋白表达量总体比较差异无统计学意义(F=1.790,P=0.210).结论 叶黄素通过上调Nrf2的表达和核转位诱导抗氧化酶的表达,从而抑制Müller细胞的氧化应激反应.
揹景 氧化應激是年齡相關性黃斑變性(AMD)的主要因素之一.研究錶明葉黃素對AMD有預防作用,但是其抗氧化機製仍不明確.Müller細胞是視網膜氧化應激反應的靶細胞之一,研究葉黃素對Müller細胞的氧化應激是否具有保護作用及其作用機製對于視網膜疾病的治療具有重要意義. 目的 研究葉黃素對視網膜Müller細胞覈因子E2相關轉錄因子2/抗氧化反應原件(Nrf2/ARE)信號途徑的影響. 方法 人Müller細胞株進行常規培養,將對數生長期的細胞接種于96孔培養闆,在培養液中分彆加入不同濃度(40、80、160、320、640 μmol/L) H2 O2,繪製Müller細胞的凋亡麯線,取H2O2的半數緻死量,即160 μmol/L製備Müller細胞氧化應激模型.將模型細胞分為模型對照組和不同質量濃度(12.5、25.0、50.0 mg/L)葉黃素組,根據分組情況分彆在培養液中加入相應質量濃度的葉黃素,用常規培養的細胞作為空白對照組.採用MTT比色法測定各組中Müller細胞增生值(吸光度,A值)和凋亡率;採用流式細胞儀檢測各組細胞中活性氧簇(ROS)的熒光彊度;採用實時熒光定量PCR檢測細胞中Nrf2 mRNA和血紅素加氧酶-l(HO-1) mRNA的相對錶達量;採用Western blot法檢測各組細胞中Nrf2和HO-1蛋白的錶達水平(灰度值). 結果 MTT比色法檢測顯示,隨著H2O2濃度的增加,細胞增生抑製率逐漸增加,各組間總體比較差異有統計學意義(F=43.890,P＜0.01).空白對照組、模型對照組及12.5、25.0、50.0 mg/L葉黃素組細胞凋亡率的總體比較差異有統計學意義(F=346.770,P=0.000),其中隨著葉黃素質量濃度的增加,細胞凋亡率逐漸下降,組間比較差異均有統計學意義(均P＜0.05).空白對照組、模型對照組及12.5、25.0、50.0 mg/L葉黃素組細胞中ROS含量分彆為1.92±0.18、64.89±2.86、52.70±2.80、32.61±4.20和5.68±1.35,隨著葉黃素質量濃度的增加,ROS含量逐漸下降,組間總體比較差異有統計學意義(F=324.900,P=0.000).各質量濃度葉黃素組細胞中Nrf2和HO-1mRNA相對錶達量及細胞中HO-1和細胞覈中Nrf2蛋白錶達量均明顯高于模型對照組,各組間總體比較差異均有統計學意義(F=236.960、242.620、186.830、263.120,均P=0.000),隨著葉黃素質量濃度的增加,Nrf2 mRNA和HO-1 mRNA及其蛋白的錶達量逐漸增加,組間比較差異均有統計學意義(均P＜0.05),但各組間Müller細胞的細胞質中Nrf2蛋白錶達量總體比較差異無統計學意義(F=1.790,P=0.210).結論 葉黃素通過上調Nrf2的錶達和覈轉位誘導抗氧化酶的錶達,從而抑製Müller細胞的氧化應激反應.
배경 양화응격시년령상관성황반변성(AMD)적주요인소지일.연구표명협황소대AMD유예방작용,단시기항양화궤제잉불명학.Müller세포시시망막양화응격반응적파세포지일,연구협황소대Müller세포적양화응격시부구유보호작용급기작용궤제대우시망막질병적치료구유중요의의. 목적 연구협황소대시망막Müller세포핵인자E2상관전록인자2/항양화반응원건(Nrf2/ARE)신호도경적영향. 방법 인Müller세포주진행상규배양,장대수생장기적세포접충우96공배양판,재배양액중분별가입불동농도(40、80、160、320、640 μmol/L) H2 O2,회제Müller세포적조망곡선,취H2O2적반수치사량,즉160 μmol/L제비Müller세포양화응격모형.장모형세포분위모형대조조화불동질량농도(12.5、25.0、50.0 mg/L)협황소조,근거분조정황분별재배양액중가입상응질량농도적협황소,용상규배양적세포작위공백대조조.채용MTT비색법측정각조중Müller세포증생치(흡광도,A치)화조망솔;채용류식세포의검측각조세포중활성양족(ROS)적형광강도;채용실시형광정량PCR검측세포중Nrf2 mRNA화혈홍소가양매-l(HO-1) mRNA적상대표체량;채용Western blot법검측각조세포중Nrf2화HO-1단백적표체수평(회도치). 결과 MTT비색법검측현시,수착H2O2농도적증가,세포증생억제솔축점증가,각조간총체비교차이유통계학의의(F=43.890,P＜0.01).공백대조조、모형대조조급12.5、25.0、50.0 mg/L협황소조세포조망솔적총체비교차이유통계학의의(F=346.770,P=0.000),기중수착협황소질량농도적증가,세포조망솔축점하강,조간비교차이균유통계학의의(균P＜0.05).공백대조조、모형대조조급12.5、25.0、50.0 mg/L협황소조세포중ROS함량분별위1.92±0.18、64.89±2.86、52.70±2.80、32.61±4.20화5.68±1.35,수착협황소질량농도적증가,ROS함량축점하강,조간총체비교차이유통계학의의(F=324.900,P=0.000).각질량농도협황소조세포중Nrf2화HO-1mRNA상대표체량급세포중HO-1화세포핵중Nrf2단백표체량균명현고우모형대조조,각조간총체비교차이균유통계학의의(F=236.960、242.620、186.830、263.120,균P=0.000),수착협황소질량농도적증가,Nrf2 mRNA화HO-1 mRNA급기단백적표체량축점증가,조간비교차이균유통계학의의(균P＜0.05),단각조간Müller세포적세포질중Nrf2단백표체량총체비교차이무통계학의의(F=1.790,P=0.210).결론 협황소통과상조Nrf2적표체화핵전위유도항양화매적표체,종이억제Müller세포적양화응격반응.
Background Oxidative stress is a main cause of age-related macular degeneration (AMD).Lutein has a preventive role for AMD, but its antioxidant mechanism remains unclear.Objective Present study was to investigate the effect of lutein on oxidative stress of Müller cells and its signaling pathway.Methods Human Müller cells (human Müller cell strain) were cultured, and the cells at logarithmimic growth phase were incubated in 96 well plate overnightly.Oxidative stress cell models were established by adding 160 μmol/L H2O2, a median lethal dose for Müller cells.The models were divided into the model control group and 12.5,25.0,50.0 mg/L lutein groups,and the different concentrations of lutein were used to culture the cells for 24 hours, respectively.The routine cultured cells served as the blank control group.Growth of the cells was assayed by MTT method (absorbancy);the reactive oxygen species (ROS) content in the cells was assayed by flow cytometry;the mRNA and protein levels of nuclear factor-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in the cells were detected by quantitative real-time PCR and Western blot, respectively.Results The inhibitory effects on the cells were gradually enhanced with the increase of H2O2 concentrations,showing a significant difference among the groups (F =43.890,P＜0.01).A significant difference was found in apoptotic rate of the cells among the blank control group,model control group and 12.5,25.0,50.0 mg/L lutein groups (F =346.770, P =0.000) , and the apoptosis rate was significant elevated with the increase of lutein dose (all at P＜0.05).The ROS contents in the cells were 1.92±0.18,64.89±2.86,52.70±2.80,32.61 ±4.20 and 5.68 ± 1.35 in the blank control group, model control group and 12.5,25.0,50.0 mg/L group, respectively, with significant difference among the groups (F =324.900, P =0.000), and the ROS content was gradually reduced as the increase of lutein dose (all at P＜0.05).The relative mRNA and protein expressions of Nrf2 and HO-1 were remarkedly higher in the 12.5,25.0,50.0 mg/L lutein groups than those in the model control group (F =236.960,242.620,186.830,263.120, all at P =0.000) , and no significant difference was seen in the relative expression level of nuclear Nrf2 protein among the groups (F =1.790, P =0.210).Conclusions Lutein can induce the expression of antioxidant enzymes by inducing the expression of nuclear translocation of Nrf2 and consequently inhibit the oxidative stress status.
