β-抑制蛋白-1在盐酸戊乙奎醚抑制LPS致人肺微血管内皮细胞通透性升高中的作用  

Role of β-arrestin-1 in penehyclidine hydrochloride-induced inhibition of LPS-caused increase in pulmonary microvascular permeability in human pulmonary microvascular endothelial cells

作　　者：袁清红 颜学滔[2] 郑菲[1] 王轶芃[1] 张宗泽[1] 陈凯[1] 王焱林[1] 詹佳[1] 

机构地区：[1]武汉大学中南医院麻醉科,430071 [2]广东省深圳市宝安区妇幼保健院麻醉科,518133

出　　处：《中华麻醉学杂志》2017年第7期869-873,共5页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金青年基金（81101408）;武汉市科技局晨光计划项目（2016070204010150）

摘　　要：目的 评价β-抑制蛋白-1在盐酸戊乙奎醚抑制LPS致人肺微血管内皮细胞通透性升高中的作用.方法 人肺微血管内皮细胞以1×10^5个/ml的密度接种于6孔板（2 ml/孔）或培养瓶（4ml/瓶）中,采用随机数字表法分为5组（n=15）：空质粒转染组（C组）、LPS＋空质粒转染组（LPS组）、盐酸戊乙奎醚＋LPS＋空质粒转染组（P＋LPS组）、LPS＋β-抑制蛋白-1 shRNA转染组（LPS＋ shRNA组）和盐酸戊乙奎醚＋LPS＋β-抑制蛋白-1 shRNA转染组（P＋LPS＋shRNA组）.LPS组和LPS＋shRNA组分别以空质粒1.5 μg或含15 nmol/L β-抑制蛋白-1 shRNA的质粒转染细胞,孵育24 h时加入终浓度为0.1 μg/ml的LPS孵育1 h;P＋LPS组和P＋LPS＋shRNA组分别以空质粒1.5 μg或含15 nmol/L β-抑制蛋白-1 shRNA的质粒转染细胞,孵育24 h时加入终浓度为2μg/ml的盐酸戊乙奎醚,孵育1h时加入终浓度为0.1 μg/ml的LPS孵育1h.采用Transwell法测定细胞通透性,采用免疫荧光化学法检测热休克蛋白27（HSP27）的表达水平,采用Western blot法检测β-抑制蛋白-1、丝裂原活化蛋白激酶p38（p38MAPK）、磷酸化p38MAPK （p-p38MAPK）的表达水平,并计算p-p38MAPK/p38MAPK比值.结果 与C组比较,LPS组、LPS＋shRNA组和P＋LPS＋shRNA组细胞通透性升高,HSP27表达上调,p-p38MAPK/p38MAPK比值升高,β-抑制蛋白-1表达下调（P〈0.05）,P＋LPS组上述指标差异无统计学意义（P〉0.05）;与LPS组比较,P＋LPS组细胞通透性降低,HSP27表达下调,p-p38MAPK/p38MAPK比值降低,β-抑制蛋白-1表达上调,P＋LPS＋shRNA组p-p38MAPK/p38MAPK比值升高（P〈0.05）,其余指标差异无统计学意义（P〉0.05）;与P＋LPS组比较,P＋LPS＋shRNA组细胞通透性升高,HSP27表达上调,p-p38MAPK/p38MAPK比值升高,β-抑制蛋白-1表达下调（P〈0.05）.结论 盐酸戊乙奎醚抑制LPS导致的人肺微血管内皮细胞通透性升高的机制完全与β-抑制蛋白-1有关.Objective To evaluate the role of β-arrestin-1 in penehyclidine hydrochloride （PHC）-induced inhibition of lipopolysaccharide （LPS）-caused increase in pulmonary microvascular permeability in human pulmonary microvascular endothelial cells （PMVECs）.Methods Human PMVECs were seeded in 6-well plates （2 ml/well） or in culture flasks （4 ml/flask） at the density of 1 × 10^5 cells/ml and divided into 5 groups （n=15 each） using a random number table：empty plasmid transfection group （group C）,LPS plus empty plasmid transfection group （LPS group）,PHC plus LPS plus empty plasmid transfection group （P＋LPS group）,LPS plus β-arrestin-1 short hairpin RNA （shRNA） transfection group （LPS＋shRNA group） and PHC plus LPS plus β-arrestin-1 shRNA transfection group （P＋LPS＋shRNA group）.In LPS and LPS＋shRNA groups,the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA,LPS with the final concentration of 0.1 μg/ml was added at 24 h of incubation,and the cells were then incubated for 1 h.In P＋LPS and P＋LPS＋shRNA groups,the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA,PHC with the final concentration of 2 μg/ml was added at 24 h of incubation,LPS with the final concentration of 0.1 μg/ml was added at 1 h of incubation,and the cells were then incubated for 1 h.The cell permeability was measured using Transwell chambers.The expression of heat shock protein （HSP27） was detected by immunofluorescence.The expression of β-arrestin-1,p38 mitogen-activated protein kinase （p38MAPK） and phosphorylated p38MAPK （p-p38MAPK） was detected by Western blot.The ratio of pp38MAPK/p38MAPK was calculated.Results Compared with group C,the cell permeability was significantly increased,the expression of HSP27 was up-regulated,p-p38MAPK/p38MAPK ratio was increased,and the expression of β-arrestin-1 was down-regulated in LPS,LPS ＋ shRNA and P ＋ LPS ＋ shRNA gro

关 键 词：抑制蛋白类 胆碱能拮抗剂 内毒素类 肺 毛细血管通透性 

分 类 号：R614[医药卫生—麻醉学]