百草枯所致血管内皮损伤与p120连环蛋白的关系及芒果苷的保护作用

Relationship between endothelial damage and p120-catenin in paraquat intoxication and the protective effect of mangiferin

目的 探讨百草枯中毒导致的内皮屏障功能障碍与p120连环蛋白（p120-ctn）的关系,以及芒果苷对p120-ctn的调节作用.方法 采用二室弥散模型培养人脐静脉内皮细胞（HUVEC）,并分为对照组（给予含10％胎牛血清的DMEM培养液）、百草枯组（给予终浓度为0.05 μmol/L的百草枯培养液）和芒果苷干预组（百草枯培养基孵育30 min后加入终浓度为20 μmol/L的芒果苷继续孵育）.各组分别培养6、12、24、48、72 h后测定细胞通透性;采用逆转录-聚合酶链反应（RT-PCR）及蛋白质免疫印迹试验（Western Blot）测定p120-ctn异构体p120-ctn 1A、p120-ctn 3A的mRNA表达及p120-ctn蛋白表达;采用免疫荧光分析并镜下观察p120-ctn的分布.结果 与对照组比较,百草枯组和芒果苷组细胞通透性均随时间延长呈增加趋势,于72 h达峰值[（29.86±3.98）％、（24.39±2.79）％比（11.71±1.67）％,均P＜ 0.05];而芒果苷组各时间点细胞通透性均显著低于百草枯组（均P＜0.05）.百草枯处理6 h p120-ctn 1A、p120-ctn 3A的mRNA表达（灰度值）及p120-ctn蛋白表达（灰度值）即较对照组明显降低（p120-ctn 1A mRNA：0.150±0.024比0.433±0.024,p120-ctn 3A mRNA：0.316±0.043比0.701±0.020,p120-ctn蛋白：0.485±0.031比0.763±0.038,均P＜0.01）;而芒果苷组p120-ctn 1A、p120-ctn 3A的mRNA表达及p120-ctn蛋白表达于干预6h即较百草枯组明显增加（p120-ctn 1AmRNA：0.281 ±0.021比0.150±0.024,p120-ctn 3A mRNA：0.602±0.042比0.316±0.043,p120-ctn蛋白：0.675±0.031比0.485±0.031,均P＜0.01）,并均随时间延长呈增加趋势,于72 h达峰值（p120-ctn 1A mRNA：1.376±0.128比0.150±0.024,p120-ctn 3A mRNA：1.251±0.059比0.316±0.043,p120-ctn蛋白：0.844±0.050比0.485±0.031,均P＜0.01）.荧光显微镜下观察,对照组p120-ctn主要分布在胞膜上,胞质内略有表达,胞核无表达;百草枯组随时间延长,细胞膜上p120-ctn表达逐渐减少,胞质和核内表达增多,细胞膜边缘模糊,细胞间隙增宽;芒果苷组相应时间点细胞膜上p120-ctn表达有所改善,胞质和核内表达减少.结论 p120-ctn下调与百草枯中毒后内皮通透性增加的机制相关,芒果苷可通过保护p120-ctn而减轻内皮损伤.

Objective To investigate the relationship between endothelial damage and p120-catenin （p120-ctn） in a model of paraquat intoxication,and the modulatory effect of mangiferin on p120-ctn.Methods Human umbilical vein endothelial cells （HUVECs） were cultured in two compartment spreading apparatus in vitro.The endothelial cells were divided into three groups：control group （cultured in DMEM with 10％ fetal bovine serum）,paraquat group （paraquat was added to the medium with final concentration of 0.05 μmol/L） and mangiferin group （cultured in medium with addition of paraquat for 30 minutes,then mangiferin was added in a final concentration of 20 μmol/L）.The cellular permeability at 6,12,24,48,72 hours after culture in the three groups was measured.The expressions of p120-ctn 1A,p120-ctn 3A mRNA and p120-ctn protein were measured by reverse transcription-polymerase chain reaction （RT-PCR） and Western Blot analysis.The distribution of p120-ctn protein was observed by immunofluorescence.Results Compared with control group,cellular permeability in paraquat and mangiferin groups were increased with prolongation of time,and peaked at 72 hours [（29.86 ± 3.98）％,（24.39 ± 2.79）％ vs.（11.71 ± 1.67）％,both P＜0.05].The cellular permeability was significantly lower in mangiferin group than that in paraquat group at different time points （all P＜0.05）.At 6 hours after intoxication,the expressions of p120-ctn 1A,p 120-ctn 3A mRNA （gray value） and p 120-ctn protein （gray value） were significantly lower in paraquat group than those in control group （p120-ctn 1A mRNA：0.150 ± 0.024 vs.0.433 ± 0.024,p120-ctn 3A mRNA：0.316 ± 0.043 vs.0.701 ±0.020,p120-ctn protein：0.485 ±0.031 vs.0.763 ±0.038,all P＜0.01）.The expressions of p120-ctn 1A,p120-ctn 3A mRNA and p120-ctn protein were significantly higher in mangiferin group than those in paraquat group from 6 hours on （p120-ctn 1A mRNA：0.281 ± 0.021 vs.0.150 ± 0.024,p120-ctn 3A mRNA：0.602 ± 0.042 vs.0.316 ± 0.043,p120-ctn protein：0.675 ± 0.031 vs.0.485 ± 0.031,all P＜0.01）,and they were gradually increased with prolongation of time,and peaked at 72 hours （p120-ctn 1A mRNA：1.376 ±0.128 vs.0.150 ± 0.024,p120-ctn 3A mRNA：1.251 ± 0.059 vs.0.316 ± 0.043,p120-ctn protein：0.844 ± 0.050 vs.0.485 ± 0.031,all P＜ 0.01）.Under upright fluorescence microscope,p120-ctn was mainly distributed in the cell membrane in control group,with a slight expression in cytoplasm,and no expression in the nuclei.With prolongation of time,p120-ctn expression in the cell membrane was gradually decreased in paraquat group,while it was increased in the cytoplasm and nuclei,with blurring of cell membrane and widening of cellular gap.p120-ctn expression was improved on the cell membrane in mangiferin group at corresponding time points,with decreased in expression in nuclei and cytoplasm.Conclusion The p120-ctn protein plays an important role in the enhancement of endothelial permeability in paraquat intoxication,and mangiferin may attenuate endothelial injury in paraquat intoxication possibly through modulation of p 120-ctn protein.