β-抑制蛋白-1在盐酸戊乙奎醚抑制内毒素诱导人肺微血管内皮细胞NF-κB激活中的作用  

Roles of β-arrestin-1 in inhibition of endotoxin-induced activation of NF-κB in human pulmonary microvascular endothelial cells by penehyclidine bydrochloride

作　　者：王轶芃[1] 詹佳[1] 张怀奇 张宗泽[1] 陈凯[1] 王焱林[1] 

机构地区：[1]武汉大学中南医院麻醉科,430071

出　　处：《中华麻醉学杂志》2015年第6期736-739,共4页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金青年基金（81101408）

摘　　要：目的 评价β-抑制蛋白-1在盐酸戊乙奎醚抑制内毒素诱导人肺微血管内皮细胞NF-κB激活中的作用.方法 将人肺微血管内皮细胞以1×105个/ml的密度接种于6孔板（2 ml/孔）或培养瓶（4 ml/瓶）中,采用随机数字表法,将其分为5组（n=20）：空质粒转染组（C组）、脂多糖（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＋含β-抑制蛋白-1短发卡RNA（shRNA）的质粒转染组（LPS＋shRNA组）和盐酸戊乙奎醚＋LPS＋含β-抑制蛋白-1shRNA的质粒转染组（P＋LPS＋shRNA组）.以空质粒1.5 μg或含15 nmol/L B-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0.1μg/ml的LPS,孵育1h,进行下述指标的测定.取上清液,测定乳酸脱氢酶（LDH）活性,收集细胞悬液,测定血管细胞粘附分子-1（VCAM-1）表达、NF-κB活性、NF-κB抑制蛋白（I-κB）及β-抑制蛋白-1的表达水平.结果 与C组比较,LPS组和LPS＋shRNA组上清液LDH活性升高,VCAM-1表达上调,NF-κB活性升高,I-κB和β-抑制蛋白-1的表达下调（P＜0.01）;与LPS组比较,P＋LPS组上清液LDH活性降低,VCAM-1表达下调,NF-κB活性降低,I-κB和β-抑制蛋白-1的表达上调（P＜0.05或0.01）,P＋LPS＋shRNA组上述指标差异无统计学意义（P＞0.05）;与P＋LPS组比较,P＋LPS＋shRNA组上清液LDH活性升高,VCAM-1表达上调,NF-κB活性升高,I-κB和β-抑制蛋白-1的表达下调（P＜0.05或0.01）.结论 盐酸戊乙奎醚完全通过上调β-抑制蛋白-1表达抑制内毒素诱导人肺微血管内皮细胞NF-κB激活.Objective To evaluate the role of β-arrestin-1 in inhibition of endotoxin-induced activation of nuclear factor kappa B （NF-κB） in human pulmonary microvascular endothelial cells （HPM-VECs） by penehyclidine hydrochloride （PHC）.Methods HPMVECs were seeded in 6-well plates （2 ml/hole） or in culture flasks （4 ml/flask） at the density of 1 × 105/ml,and were randomly divided into 5 groups （n =20 each） using a random number table：empty plasmid transfection group （group C）,lipopolysaccharide （LPS） ＋ empty plasmid transfection group （group LPS）,PHC ＋ LPS ＋ empty plasmid transfection group （group P＋LPS）,LPS ＋ β-arrestin-1 gene-shRNA transfection group （group LPS＋shRNA） and PHC ＋ LPS ＋ β-arrestin-1 gene-shRNA transfection group （group P＋LPS＋shRNA）.HPMVECs were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1gene-shRNA.At 24 h of incubation,PHC with the final concentration of 2 μg/ml was added,the cells were incubated for 1 h,LPS with the final concentration of 0.1 μg/ml was then added,and the cells were continuously incubated for another 1 h.The supernatant was collected to measure the activity of lactic dehydrogenase （LDH）.The cell suspension was collected for determination of vascular cell adhesion molecule-1 （VCAM-1） expression and NF-κB activities and NF-κB inhibitor I-κB and β-arrestin-1expression.Results Compared with group C,the activities of LDH in supernatant were increased,VCAM-1 expression was up-regulated,NF-κB activity was significantly increased,and I-κB and β-arrestin-1 expression was down-regulated in LPS and LPS＋shRNA groups.Compared with group LPS,the activities of LDH in supernatant were decreased,VCAM-1 expression was down-regulated,NF-κB activity was significantly decreased,and I-κB and β-arrestin-l expression was up-regulated in group P＋LPS,and no significant change was found in the parameters mentioned above in group P＋LPS＋shRNA.Compared with group P＋LPS,the

关 键 词：抑制蛋白类 胆碱能药 NF—κB 内皮细胞 毛细血管 肺 内毒素血症 

分 类 号：R614[医药卫生—麻醉学]