【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。更多还原

【Abstract】 OBJECTIVE To explore the effect of apelin on the lipopolysaccharide（LPS）-induced apoptosis 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^-1treatment 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 nmol·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 AnnexinⅤ/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 mg·L^-1for 0,5,15,30,60,120 and 240 min or pretreated with apelin(1 and10 nmol·L^-1)for 2 h and then stimulated with LPS(10 mg·L^-1)for 30 min,and then the phosphorylation of p38 MAPK was detected by Western blotting. RESULTS The results of the laser confocal microscope showed that LPS significantly induced cytoskeleton rearrangement of rat PMVECs. Meanwhile,the formation of stress fiber and the morphological changes in cytoskeleton induced by LPS were obviously inhibited by apelin(1 and 10 nmol·L^-1)pretreatment. The results of AnnexinⅤ-FITC staining showed that apelin 1and 10 nmol ·L^-1inhibited 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 apoptosis rate decreased from（54.3±3.4）% to（29.5±4.6）% and（9.0±1.6）%,respectively（P<0.05）. Apelin 1and 10 nmol·L^-1also 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 min（P<0.05）and with the highest level observed at 30 min（P<0.01）,which was obviously inhibited by apelin(1 and 10 nmol·L^-1)pretreatment（P<0.01）. Meanwhile,SB203580 pretreatment significantly inhibited the LPS induced apoptosis of rat PMVECs,for the early and late apoptosis rate decreased from（36.7±3.8）% to（19.7±4.7）% and（38.3±7.5）% to（15.7±3.6）%,respectively（P<0.01）. CONCLUSION Apelin obviously inhibits the LPS induced cytoskeletal rearrangement and apoptosis injury,which is mediated by the inhibition of p38 MAPK signal pathway.更多还原