烟酸对p38丝裂原活化蛋白激酶信号通路介导的内皮细胞功能障碍的早期干预研究  

Effects of niacin on cell adhesion and early atherogenesis：involvement of the p38 mitogen-activated protein kinases pathway

作　　者：牛娜[1] 韩波[1] 孙书珍[1] 于永慧[1] 汪翼[1] 王立俊[1] 

机构地区：[1]山东大学附属省立医院儿科,济南250021

出　　处：《中华儿科杂志》2013年第11期825-830,共6页Chinese Journal of Pediatrics

基　　金：山东省优秀中青年科学家科研奖励基金（BS2010YY056）

摘　　要：目的 探讨烟酸对p38MAPK通路介导的内皮细胞功能障碍的早期干预作用及可能机制.方法 人脐静脉内皮细胞株（HUVECs）,用Medium200培养基培养,实验分组：①阴性对照组：培养基；②溶血磷脂酰胆碱（LPC）不同作用时间组：培养基中加入终浓度为20 μmol/L的LPC,分别培养10 min、8h、24 h；③LPC＋ p38MAPK的抑制剂（SB203580）组：培养基中加入SB203580 10 μmol/L培养1h,再分别加入LPC培养10 min、8h、24 h；④LPC＋不同剂量烟酸组：培养基中分别加入终浓度为0.25、0.5、1 mmol/L烟酸培养18h,再加入LPC培养10 min、8h及24 h.应用Western blot定量分析检测内皮细胞磷酸化的p38 MAPK（pp38 MAPK）、细胞间黏附分子（ICAM-1）蛋白含量,实时定量PCR方法检测内皮细胞ICAM-1 mRNA表达,细胞免疫荧光方法检测LPC诱导的ICAM-1蛋白表达.结果 ICAM-1的蛋白表达LPC 24 h组为0.786±0.021,LPC＋烟酸（1 mmol/L）组培养24 h为0.487±0.015,LPC＋ SB203580组培养24 h为0.461±0.011,LPC＋烟酸组和LPC＋ SB203580组均低于LPC 24 h组,差异有统计学意义（F=6.3,P＜0.01）,但均未达到阴性对照组水平（0.134±0.012）.pp38MAPK蛋白在LPC 10 min组最高,为0.47±0.02,烟酸能降低pp38MAPK,LPC＋烟酸（1 mmol/L）组为0.07±0.02,LPC＋ SB203580为0.11±0.02,均低于LPC 10 min组（F=91.91,P＜0.01）.加入LPC培养8h后,ICAM-1 mRNA的表达（8.16±0.15）,高于阴性对照组（1.00±0.02）,差异有统计学意义（t=24.34,P＜0.01）；与LPC培养8h比较,烟酸降低LPC诱导的ICAM-1 mRNA的表达,LPC＋烟酸（1 mmol/L）组为3.85±0.14（F =8.06,P＜0.01）,而SB203580则不能有效的降低ICAM-1的mRNA的表达（8.09±0.11）.结论 在LPC诱导的HUVECs中,ICAM-1的蛋白与mRNA的表达均明显增强,pp38MAPK蛋白明显增强,烟酸干预可降低ICAM-1的蛋白与mRNA的表达,同时亦可降低pp38 MAPK的蛋白表达,p38MAPK的抑制剂SB203580仅能降低ICAM-1的蛋白的表达,而不能影响其mRNA表达,�Objective To examine the effects of niacin on lysophosphatidylcholine （LPC）-induced intercellular adhesion molecule-1 （ICAM-1）, and gained insight to the mechanisms. Method Human umbilical vein endothelial cell line was cultured using Medium 200 medium in incubator at 37 ℃ and 5% CO2 condition. Experimental groups： （1） the negative control group： medium; （2） LPC different time groups：the medium added with 20 μmol/L final concentration of LPC, were cultured for 10 min and 8 h, 24 h ; （ 3 ） LPC ± p38-mitogen-activated protein kinase （p38MAPK） inhibitor （ SB203580 ） group： the medium added with 10 txmol/L p38 MAPK inhibitor （SB203580） was cultured for 1 h, then human umbilical vein endothelial cells （HUVECs） added with the LPC were cultured for 10 rain, 8 h and 24 h. （4） LPC ± different niacin dose group ~ after separately adding with O. 25, O. 5, 1 mmol/L niacin, the cells were cultured for 18 h, then HUVECs added with the LPC were cultured for 10 min, 8 h and 24 h. Cell concentration in each group was 5× 105/ml, inoculated in 6-well plates, each well 1 ml. Detected by Western blot analysis of pp38MAPK, ICAM-1 protein content, real-time quantitative PCR to detect endothelial cell ICAM-! mRNA expression, cell immunofluorescence to detect LPC-induced ICAM-1 protein expression. Result In LPC 24 h group, the expression of ICAM-1 protein was significantly increased 0. 786 ± 0.02, the LPC ± niacin group, ICAM-1 protein levels （0. 487 ±0. 015） was significantly lower than the LPC 24 h group （P 〈 0. 01 ）, in LPC ± SB203580 intervention group, ICAM-1 protein levels （0. 461 ± 0.011 ） was significantly lower than that of the LPC 24 h group （ P 〈 0.01 ）, but did not reach the level of the control group. Adding LPC to culture for 10 min, phosphorylation of p38MAPK （pp38MAPK） reached its peak （0. 47 ± 0. 02）, niacin could reduce the pp38MAPK （0.07 -± 0.02）, SB203580 could also reduce its activity （0. 11 ± 0.02 ） ~ Adding LPC to cult

关 键 词：内皮细胞 P38丝裂原活化蛋白激酶类 烟酸类 

分 类 号：R363[医药卫生—病理学]