血管生成素-1对小鼠早期急性肺损伤的保护作用

The protective effect of angiopoietin-1 on early acute lung injury induced by oleic acid in mice

目的观察血管生成素-1（Ang-1）对油酸致小鼠早期急性肺损伤的保护作用，探讨Ang-1对小鼠早期急性肺损伤血管内皮细胞生长因子（VEGF）表达的影响。方法96只BALB／c雌性小鼠按随机数字表法分为4组，每组24只。正常对照组：尾静脉注射生理盐水（0．9ml／kg）；急性肺损伤组：尾静脉注射油酸（0．9ml／kg）制备急性肺损伤模型；正常对照＋Ang-1组：注入生理盐水4h后腹腔内注射Ang-1（312．5μg／kg）；急性肺损伤＋Ang-1组：注入油酸4h后腹腔内注射Ang-1（312．5μg／kg）；8h后各组随机抽12只小鼠处死后行支气管肺泡灌洗（BAL），另外12只小鼠右肺称重后烘干，左肺留取病理标本；分别测定肺湿／干重比、支气管肺泡灌洗液（BALF）中细胞总数、中性粒细胞数及蛋白含量变化；采用酶联免疫吸附（ELISA）法测定血清和BALF中白细胞介素-6（IL-6）、VEGF含量；光镜观察并盲法评分比较肺组织病理学改变；采用免疫组化法检测肺组织中VEGF的表达。采用SPSS10．0软件。数据以x±s表示，多组间比较采用单因素方差分析，组间比较采用LSD-t检验，P〈0．05为差异有统计学意义。结果急性肺损伤＋Ang-1组肺湿／干重比、BALF中蛋白含量和细胞总数、中性粒细胞、血清和BALF中IL-6含量、血清VEGF含量分别为4．09±0．14、（176±13）μg／ml、（34．4±4．1）X10^4／L、（19．85±3．93）X10^3／L、（1318±62）pg／ml、（652±17）pg／ml、（48±5）pg／ml，与急性肺损伤组[5．32±0．51、（227±12）μg／ml、（42．2±5．2）X10^4／L、（26．22±2．22）X10^3／L、（1510±117）pg／ml、（744±13）pg／ml、（74±5）pg／m1]比较差异有统计学意义（t值分别为8．16、9．92、4．02、4．88、5．03、19．18、14．81，P均〈0．01）；而急性肺损伤＋Ang-1组BALF中VEGF含量[（179±15）pg／m1]与B组[（140±20）pg／m1]比较差异有统计学意义（t=5．31，P〈0．01）；急性肺损伤＋Ang-1组肺损伤评分为（1．84±0．12）分，急性肺损伤组为（3．44±0．21）分，两组比较差异有统计学意义（t=24．16，P〈0．01）。肺组织中VEGF表达吸光度（A）值急性肺损伤＋Ang-1组为549±72，与急性肺损伤组（342±85）比较差异有统计学意义（t=5．22，P〈0．01）；急性肺损伤＋Ang-1组与正常对照组（768±111）比较差异亦有统计学意义（t=5．35，P〈0．01）；正常对照＋Ang．1组和正常对照组肺组织中VEGF吸光度（A）值表达比较差异无统计学意义（t=0．69，P〉0．05）。结论Ang-1对油酸致小鼠早期急性肺损伤可减轻渗出、抑制炎性细胞浸润和炎症因子动员，同时Ang-1可引起小鼠早期急性肺损伤血清VEGF表达降低，而BALF和肺组织中VEGF表达增加。提示Ang-1对油酸致小鼠早期急性肺损伤有一定的保护作用。

Objective To observe the protective effects of angiopoietin-1 on acute lung injury （ALI） induced by oleic acid at early stage in mice and to investigate the expression changes of vascular endothelial growth factor （VEGF）. Methods Ninety-six female BALB/c mice were randomly divided into 4 groups（n =24）. The control group was given normal saline （0. 9 ml/kg） intravenously. The ALI group was treated with oleic acid （0. 9 ml/kg） intravenously to induce ALL In the control ＋ angiopoietin-1 group, the mice were injected intraperitoneally with angiopoietin-1 （312.5 μg/kg） 4 h after normal saline administration. In the ALI＋ angiopoietin-1 group, the mice were injected intraperitoneallv with angiopoietin-1 （312.5 μg/kg） 4 h after oleic acid treatment. Then 8 h later 12 mice were randomly taken from each group for bronchoalveolar lavage fluid （BALF） analysis including the protein level, cell count and differentials, and the level of IL-6 and VEGF . The wet/dry weight （W/D） of the right lung, the level of IL-6 and VEGF in serum, pathological changes of the left lung and VEGF expressions in lung tissues were examined for the rest of mice in each group. The level of IL-6 and VEGF in both serum and BALF were measured by enzyme-linked-immunosorbent assay （ELISA）. Pathological changes of the lung tissue were examined and scored with light microscrope. The expression of VEGF was immunohistochemically detected in lung tissues as integrated optic density （A） measured with Image Pro Plus 5.1 software. Results The W/D, the protein level, the total cell number and differential of polymorphonuclear leukocytes in BALF, the IL-6 level in both serum and BALF, the VEGF level in serum were significandy decreased in the ALl ＋ angiopoietin-1 group [ 4. 09 ± 0. 14, （ 176 ± 13 ） μg/ml, （ 34. 4 μ 4. 1 ） ~ 104/L, （ 19. 85 ± 3. 93 ） - 103/L, （ 1318 ± 62 ） pg/ml, （ 652 ± 17 ） pg/ml , （ 48 ± 5 ） pg/ml ] as compared to the ALI group [ 5.32 ± 0. 51, （227±12） pg/ml,（42. 2±5.2） x 104/L, （26.22±2.22） x 103/L, （1510 ± 117） pg/ml, （744 ± 13） pg/ml, （74 ± 5 ） pg/ml, t = 8. 16, 9. 92,4. 02,4. 88,5.03,19. 18,14. 81 respectively, all P 〈 0.01 ]. Pathological scores in the ALI＋ angiopoietin-1 group（ 1.84 ± 0. 12） improved as compared to those in the ALI group（ 3.44 ± 0.21, t = 24. 16, P 〈 0.01 ）, and the VEGF level in BALF of the ALI＋ angiopoietin-1 group （ 179 ± 15 ） pg/ml was higher than that of the ALI group （ 140 ± 20 ） pg/ml （ t = 5.31, P 〈 0. 01 ）. The expressions of VEGF in the lung tissues indicated by A （ 549 ± 72 ） in the ALI＋ angiopoietin-1 group were higher than those in the ALI group （342±85, t =5.22 ,P 〈0. 01 ）, but lower than those in the control group （768 ± 111 ） （ t = 5. 35, P 〈 0. O1 ）. The above measurements of the control ＋ angiopoietin-1 group showed no difference compared to the control group （ t = 0. 12,0.53,1.27,2. 28,1.18,0. 34,0. 13, 0. 25, 0. 58,0. 69 respectively, all P 〉 0. 05 ）. Statistical analysis was done using SPSS 10. 0 software. Data are expressed as mean ± s, with statistical significance deelaved for probility valaes 〈 0.05. LSD-t test was used for group comparison. One-factor ANOVA was used for mdlfi-group comparison. Conclusions Angiopoietin-1 is effective in relieving the severity of acute lung injury induced by oleie acid in mice at early stage and up-regulating VEGF expression in BALF and lung tissues but down-regulating VEGF expression in serum. The results suggest that angiopoietin-1 may exert beneficial effects on ALL