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目的 探讨脂多糖(LPS)所致大鼠肺微血管内皮细胞(RPMVEC)炎性损伤中小窝蛋白-1(Cav-1)的作用机制.方法 将体外培养的RPMVEC,分别进行LPS刺激时效实验和蛋白激酶A(PKA)通路干预实验.①时效实验:以10 μg/mL LPS分别刺激0、1、3、6、12、24h后进行细胞单层通透性(伊文思蓝-白蛋白法)和Cav-1蛋白表达[蛋白质免疫印迹试验(Western blotting)]检测;并于LPS刺激0、10、30、60、90、120 min检测细胞磷酸化小窝蛋白-1(p-Cav-1)表达(Western blotting).②干预实验:以10μmol/L蛋白激酶A特异抑制剂(PKI)与RPMVEC预孵育30m in后再加入10 μg/mL LPS继续孵育,30 min后检测p-Cav-1表达,3h后检测细胞单层通透性及Cav-1蛋白表达;设未刺激组和LPS或PKI单独刺激组为对照.结果 ①LPS刺激RPMVEC后1 h Cav-1蛋白表达(积分A值)即开始上升(2.97±0.07),3h达峰值(3.77±0.37),之后逐渐下降,但至24 h时(1.45±0.18)仍明显高于LPS刺激0h时(1.12±0.08),组间比较差异有统计学意义(F=178.047,P=0.000);LPS可呈时间依赖性(0、1、3、6、12、24h)增加RPMVEC通透性(A值),变化趋势与Cav-1蛋白表达一致,分别为(99.67±4.32)％、(118.17±2.32)％、(159.00±2.61)％、(141.17±2.64)％、(120.17±2.79)％、(108.83±2.04)％,组间比较差异有统计学意义(F=345.869,P=0.000).LPS亦可呈时间依赖性增加Cav-1磷酸化:10 min时p-Cav-1蛋白表达(积分A值)即开始升高(2.41±0.11),30 min达高峰(2.83±0.10),然后逐渐下降,120 min时(1.04±0.04)恢复到基础水平(1.03±0.04),组间比较差异有统计学意义(F=519.417,P=0.000).②PKI与LPS预孵育可使Cav-1表达(积分A值:5.07±0.22比3.81±0.23,P＜0.01)及p-Cav-1表达(积分A值:3.93±0.23比2.77±0.10,P＜0.01)较LPS刺激组进一步增加,并破坏RPMVEC屏障功能,使RPMVEC通透性增加[(184.17±5.49)％比(151.50±3.08)％,P＜0.01].结论 Cav-1及其磷酸化表达增加参与LPS诱导RPMVEC屏障损伤,PKA信号通路通过调控Cav-1蛋白及其磷酸化表达参与LPS对RPMVEC的致损效应.

Objective To investigate the role of caveolin-1 (Car-1) in the modulation of rat pulmonary microvascular endothelial cells (RPMVEC) injury induced by popolysaccharide (LPS).Methods Cultured RPMVEC were randomly divided into time-dependent injury group induced by LPS and intervention group in which cells were pretreated by protein kinase A inhibitor (PKI).In the time-dependent injury group,monolayers of cells were constructed to determine permeability changes after 10 μg/mL LPS challenge for 0,1,3,6,12 and 24 hours with the method of Evans blue-labeled albumin flux across the monolayer (Pd).Western blotting was used to determine the Car-1 expression after LPS stimulation and the phosphorylation-Cav-1 (p-Cav-1) expression after LPS challenge for 0,10,30,60,90,120 minutes.In the intervention group,after pre-treatment with 10 μmol/L PKI for 30 minutes,RPMVECs were challenged with 10 μg/mL LPS,and the expression of p-Car-1 was determined 30 minutes after LPS challenge,the permeability and the Car-1 protein expression were assessed by Pd and Western blotting,respectively.Non-stimulation group and single PKI simulation group served as controls.Results Western blotting revealed that the expression of Cav-1 protein was elevated at 1 hour (2.97 ± 0.07),peaking at 3 hours (3.77 ± 0.37),then it lowered gradually,but it was still higher at 24 hours (1.45 ±0.18) when compared with 0 hour group (1.12 ±0.08) with significant differences (F=178.047,P=0.000).After RPMVEC monolayers were challenged by LPS for different periods (0,1,3,6,12 and 24 hours),there were significant increases in a time-dependent manner in Cav-1 expression in the permeability as measured by Pd [(99.67 ±4.32)％,(118.17 ±2.32)％,(159.00 ±2.61)％,(141.17 ±2.64)％,(120.17 ±2.79)％ and (108.83 ±2.04)％,F=345.869,P=0.000] which was similar to the changes in Cav-1 expression.LPS also increased Cav-1 phosphorylation in a time-dependent manner occurring at 10 minutes (2.41 ± 0.11),peaking at 30 minutes (2.83 ± 0.10),and then it decreased gradually,finally returned to basal levels (1.03 ± 0.04) at 120 minutes (1.04± 0.04) after LPS treatment with significant difference (F=519.417,P=0.000).When PKI was pre-treated with RPMVEC the expression of Cav-1 was significantly increased (5.07 ± 0.22 vs.3.81 ±0.23,P＜0.01),and p-Car-1 (3.93 ±0.23 vs.2.77 ±0.10,P＜0.01),and RPMVEC monolayers permeability was enhanced [(184.17 ± 5.49) ％ vs.(151.50 ± 3.08) ％,P＜ 0.01] after being challenged.Conclusion Up-regulated expression of Cav-1 and phosphorylation-Cav-1 that may be modulated by protein kinase A signal pathway plays an important role in RPMVEC permeability injury as induced by LPS.