肝素对急性肺损伤大鼠基质金属蛋白酶2和9活性的影响

Effects of unfractionated heparin on expression of metalloproteinase-2 and -9 in acute lung injury rats

目的探讨肝素对急性肺损伤（ALI）大鼠体内基质金属蛋白酶（MMP-2、MMP-9）蛋白及mRNA表达的影响。方法18只雄性Wistar大鼠，按随机数字表法分成3组，每组6只。经尾静脉注射脂多糖（LPS）6mg／kg复制ALI大鼠模型；肝素组在注射LPS前15min经尾静脉注射普通肝素100U／kg，对照组注射等量生理盐水。分别于制模后1、3、6h取股静脉血，采用酶联免疫吸附试验（ELISA）测定血清中MMP-2、MMP-9蛋白表达；制模后6h处死动物，取左肺，应用实时荧光定量聚合酶链反应（qRT—PCR）检测肺组织中MMP-2及MMP-9的mRNA表达。结果与对照组比较，ALI组血清MMP-2（μg／L）、MMP-9（μg／L）蛋白表达增高，6h达到高峰（MMP-2：2．86±0．40比1．21±0．24，MMP-9：2．54±0．29比1．15±0．34，均P〈0．01）；应用肝素后在6h时MMP-2、MMP-9蛋白表达明显降低（MMP-2：1．92±0．31比2．86±0．40，MMP-9：1．82±0．26比2．54±0．29，均P〈0．05）。制模后6h，ALI组肺组织MMP-2、MMP-9的mRNA表达明显高于对照组（MMP-2mRNA：1．88±0．09比1．00-±0．10，MMP-9mRNA：3．15±0．47比1．00±0．17，均P〈0．01）；肝素组MMP-2、MMP-9的mRNA表达较ALI组明显降低（MMP-2mRNA：1．26±0．14比1．88±0．09，P〈0．01；MMP-9mRNA：2．06±0．68比3．15±0．47，P〈0．05），但仍高于对照组（均P〈0．05）。结论ALI大鼠体内MMP-2、MMP-9表达增加，肝素能够减少血清及肺组织中MMP-2、MMP-9的表达，从而减轻肺损伤。

Objective To determine the activation of matrix metalloproteinase （ MMP-2, MMP-9 ） in rats with acute lung injury （ALI） and the effects of unfraetionated heparin （ UFH ） on the levels. Methods Eighteen male Wistar rats were divided into control group, ALI group and UFH group, with 6 rats in each group by means of random number table. ALI was induced by administering a bolus injection of lipopolysacchafide （LPS） via the caudal vein at a dose of 6 mg/kg. In UFH group the rats were treated intravenously with 100 U/kg of UFH 15 minutes before the injection of LPS. In control group, the rats were treated with the same volume of normal saline. Serum levels of MMP-2 and MMP-9 were measured by onzymelinked immunosorbent assay （ELISA） at l, 3, 6 hours via femoral vein. Six hours after the injection of reagents, the rats were sacrificed and lung tissue samples were collected for mRNA analysis of the MMP-2 and MMP-9 by real-time fluorescence quantitate reverse transcription-polymerase chain reaction （qRT-PCR）. Results Compared with control group, the content of MMP-2 （μg/L） and MMP-9 （μg/L） in serum of ALI group were increased, and reached the peak at 6 hours （MMP-2：2.86 ±0.40 vs. 1.21 ±0.24, MMP-9：2.54 ±0.29 vs. 1.15 ± 0.34, both P〈0.01 ）; they were down-regulated in UFH group at 6 hours （MMP-2：1.92 ± 0.31 vs. 2.86 ± 0.40, MMP-9 ： 1.82± 0.26 vs. 2.54 ± 0.29, both P〈0.05 ）. Compared with control group, the mRNA expressions of MMP-2 and MMP-9 in the lung tissue of ALI group were increased at 6 hours （MMP-2 mRNA： 1.88 ± 0.09 vs. 1.00± 0.10, MMP-9 mRNA： 3.15 ± 0.47 vs. 1.00 ± 0.17, both P〈0.01 ）; they were down-regulated in UFH group （MMP-2 mRNA： 1.26 ± 0.14 vs. 1.88 -＋ 0.09, P〈0.01 ; MMP-9 mRNA： 2.06 ± 0.68 vs. 3.15± 0.47, P〈0.05 ）, but still above the control group （ both P〈0.05 ）. Conclusions The present study demonstrated that the level of MMP-2 and MMP-9 increased in rats with ALL UFH could exert protective effects by inhibiting expression of MMP-2 and MMP-9 in serum and lung tissue, in both mRNA and protein expression.