apelin对脂多糖诱导的大鼠肺微血管内皮细胞凋亡和骨架改变的影响及机制  

Effect of apelin on lipopolysaccharide-induced apoptosis and cytoskeleton rearrangement in rat pulmonary microvascular endothelial cells and the mechanisms

作　　者：刘焕龙[1] 朱忠宁[2] 鹿梦溪 苏素文[2] 裴庭梅[2] 陈雪彦[2] 

机构地区：[1]河北医科大学第二医院药学部,河北石家庄050000 [2]河北医科大学药理学教研室,河北石家庄050017 [3]华北理工大学基础医学院,河北唐山063009

出　　处：《中国药理学与毒理学杂志》2015年第6期905-911,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金资助项目(81273600);河北省自然科学基金资助项目(H2013206147)~~

摘　　要：目的探讨apelin对脂多糖(LPS)诱导的大鼠肺微血管内皮细胞(PMVEC)凋亡、骨架改变的影响及作用机制。方法采用体外组织贴块法培养大鼠PMVEC,激光共聚焦显微镜观察LPS 10 mg·L^(-1)分别处理0,3,6,12,24和48 h PMVEC骨架结构的改变;另取PMVEC加入apelin 1和10 nmol·L^(-1)或p38抑制剂SB203580 10μmol·L^(-1)预处理,2 h后加入LPS 10 mg·L^(-1)作用24 h后,AnnexinⅤ/PI染色法检测大鼠PMVEC凋亡,Western蛋白印迹法检测凋亡相关蛋白BAX和BCL-2的水平;同时还采用Western蛋白印迹法检测LPS 10 mg·L^(-1)作用0,5,15,30,60,120和240 min,或apelin 1和10 nmol·L^(-1)预处理2 h后加入LPS 10 mg·L^(-1)作用30 min后p38丝裂原激活蛋白激酶(MAPK)磷酸化水平的变化。结果激光共聚焦显微镜观察结果显示,LPS明显诱导了大鼠PMVEC骨架重排,apelin 1和10 nmol·L^(-1)预处理明显干预了LPS诱导的PMVEC应力纤维的形成和细胞骨架形态的改变。AnnexinⅤ/PI染色结果表明,apelin 1和10 nmol·L^(-1)预处理明显抑制了LPS诱导的大鼠PMVEC凋亡,早期凋亡率由(43.8±4.6)%分别降至(33.7±6.9)%和(11.2±3.0)%(P<0.05),晚期凋亡率由(54.3±3.4)%分别降至(29.5±4.6)%和(9.0±1.6)%(P<0.05),并不同程度地逆转了BAX和BCL-2的表达失衡情况(P<0.05)。Western蛋白印迹结果显示,LPS作用5 min即明显诱导大鼠PMVEC中p38 MAPK磷酸化水平增高(P<0.05),且在30 min时磷酸化水平达到最高(P<0.01),apelin预处理明显抑制了LPS作用30 min诱导的p38 MAPK磷酸化水平的增高(P<0.01)。p38抑制剂SB203580预处理明显抑制了LPS诱导的大鼠PMVEC凋亡,早期(36.7±3.8%)和晚期凋亡率(38.3±7.5%)分别降至(19.7±4.7)%和(15.7±3.6)%(P<0.01)。结论 apelin明显干预了LPS诱导的大鼠PMVEC骨架蛋白重排以及凋亡损伤,这一保护作用与其抑制p38 MAPK信号通路有关。OBJECTIVE To explore the effect of apelin on the lipopolysaccharide （LPS）-induced apop- tosis and cytoskeleton rearrangement in rat pulmonary microvascular endothelial cells （PMVECs） and the underlying mechanisms. METHODS PMVECs were cultured with the explant technique. The cytoskeletal rearrangement after LPS 10 mg. L-1 treatment for 0, 3, 6, 12, 24 and 48 h was detected by the laser confocal microscope. Quiescent PMVECs were pretreated with apelin （ 1 and 10 μmol. L-1） or p38 inhibitor SB203580 （10 μmol. L-1） for 2 h and stimulated with LPS （10 mg. L-1） for 24 h before the apoptosis of PMVECs was evaluated by Annexin V/PI staining assay, while the levels of apoptosis-related proteins BAX and BCL-2 were evaluated by Western blotting analysis. Meanwhile, quiescent PMVECs were treated with LPS 10 rag. L-1 for 0, 5, 15, 30, 60, 120 and 240 min or pretreated with apelin （1 and 10 nmol. L-1 ） for 2 h and then stimulated with LPS （ 10 rag. L-1 ） for 30 rain, and then the phosphorylation of p38 MAPK was detected b）/ Western blotting. RESULTS The results of the laser confocal microscope showed that LPS significantly induced cytoskeleton rearrangement of rat PMVECs. Meanwhile, the forma- tion of stress fiber and the morphological changes in cytoskeleton induced by LPS were obviously inhibited by apelin （1 and 10 nmol. I.-1） pretreatment. The results of Annexin V-FITC staining showed that apelin 1 and 10 nmol- L-1 inhibited the LPS-induced apoptosis of PMVECs significantly. The early apoptosis rate decreased from （43.8±4.6）% to（33.7±6.9）% and （11.2±3.0）%, respectively（P〈0.05） and the late apop- tosis rate decreased from （54.3±3.4）% to （29.5±4.6）% and （9.0±1.6）%, respectively（P〈0.05）. Apelin 1 and 10 nmol- L-1 also reversed the imbalance of the protein expression of BAX and BCL-2 caused by LPS （P〈0.05）. Western blotting analysis suggested that the phosphorylation of p38 MAPK began to increase after LPS treatment for 5 rnin（P〈0.05）and

关 键 词：APELIN 脂多糖 肺微血管内皮细胞 细胞凋亡 细胞骨架 

分 类 号：R965[医药卫生—药理学]