乌司他丁对大鼠内毒素急性肺损伤的治疗及米非司酮的干预效应

Effects of ulinastatin on endotoxin induced acute lung injury in rats and potential mechanism of mifepristone therapy

目的研究在米非司酮（minestrone，RU486）干预的条件下乌司他丁（ulinastatin，UTI）对内毒素（lipopolysaccharide，LPS）致大鼠急性肺损伤（acute lung injury，Au）的治疗作用。方法将30只成年雄性Sprague-Dawley（SD）大鼠按照完全随机分组法分为5组（每组6只）：空白对照（Con）组，LPS组，LPS＋UTI（LU）组，LPS＋RU486（LR）组，LPS＋RU486＋UTI（LRU）组，各组大鼠经相应处理后8h时处死，测定大鼠肺湿／干重比（wet／dry weight ratio，W／D）；光镜下观察肺组织苏木精伊红（hematoxvlin-eosin，HE）染色病理学变化并进行肺组织损伤评分；同时采用Western blot检测糖皮质激素受体（glucocorticoid receptor，GR）的表达，实时荧光定量聚合酶链式反应（polymerase chain reaction，PCR）检测GRmRNA的表达。结果①肺组织病理学检查：Con组大鼠肺大体形态正常，呈粉红色；肺泡结构正常，无出血、渗血等表现。LPS组大体形态见肺充血明显，并可见斑片状坏死；肺泡结构明显破坏，肺泡萎陷，肺弥漫性出血，肺间质大量炎性细胞浸润，肺泡间隔增宽、出血、水肿、渗出；肺损伤评分较Con组升高（P〈0.05）。LR组较LPS组损伤更加严重，肺组织损伤评分上升（P〈0．05）；LU组和LRU组肺组织病理损害较LPS组明显减轻，且肺组织损伤评分降低（P〈0．05），但两组之间比较差异无统计学意义（P〉0.05）。②肺组织W／D：LPS组（5．548±0．099）和LR组（6．202±0．128）较Con组（4．272±0．177）升高（P〈0．05），且两者比较差异有统计学意义（P〈0．05）；LU组（5．190±0．133）和LBU组（5．150±0．098）较LPS组、LR组均下降（P〈0．05），但两者比较差异无统计学意义（P〉0.05）。③GR蛋白的表达：LPS组（1．927±0．108）和LR组（0．966±0．003）大鼠肺组织GR蛋白的表达量较Con组（7．737±0．290）明显降低（P〈0．05），两者比较差异有统计学意义（P〈0．05）；LU组（5．026±0．556）和LRU组（5．026±0．556）表达量较LPS组、LR组升高（P〈0．05），但两者比较差异无统计学意义（P〉0．05）。④GRmRNA的表达：LPS组（0．700±0．020）和LR组（0．300±0．020）表达明显低于Con组（1．000±0．100），差异有统计学意义（P〈0．05）；LU组（0．700±0．030）表达比u）s组略高，差异无统计学意义（P〉0．05），但是仍低于Con组，差异有统计学意义（P〈0．05）；LRU组（0．853±0．031）表达高于LPS组、LU组、LR组，但是低于Con组，差异有统计学意义（P〈0．05）。结论UTI能上调GR和GRmRNA的表达水平，减轻LPS诱导的大鼠ALI程度，GR阻断剂RU486对其干预作用不明显,UTI效应的发挥可能与其他分子机制或信号通路有关。

Objective To study mechanism of mifepristone （RU486） intervention under the treatment of ulinastatin （UTI） for lipopolysaccharide （LPS）-induced acute lung injury （ALI） in rats. Methods Thirty adult male Sprague-Dawley（SD） rats were randomly divided into five groups（n=6）： control group（Con group）, LPS group,LPS plus UTI group（LU group）, LPS plus RU486 group （LR group）, LPS plus RU486 and UTI group （LRU group）. After the corresponding treatment rats were sacrificed at 8 h, the authors determined the rat lung wet/dry weight ratio（W/D）, observed the pathologics change of lung tissue by hematoxylin-eosin（HE ） staining and scored the lung injury mark, and the protein and mRNA expression levels of glucocorticoid receptor （GR） in the lungs were detected by Western blot and real time fluorescent quantitative polymerase chain reaction（PCR）. Results （1） Histopathologic examination of lungs： the rats in Con group of the lung were normal, Pathological morphology of alveolar structure was normal, and had no bleeding sites. In LPS group, alveolar structure was destroyed, alveolar atrophyed, pulmonary hemorrhage diffused, widened alveolar septum with bleeding and edema were found, the lung injury score was higher than that of the Con group （P〈0.05）.However, LR group is more severe, its lung injury score increased significantly （P〈0.05）, LU group and LRU group had less lung tissue damage compared to LPS group with lower scores （P〈0.05）, but there was no statistically significant difference between two groups （P〉0.05） . （2） The lung W/D ： compared to Con group （4.272±0.177 ）, LPS group （5.548±0.099） and LR group （ 6.202±0.128 ） lung W/D were increased（P〈0.05）, and both were statistically significant（P〈0.05）. LU group（5.190±-0.133） and LRU group （5.150±0.098） had greater decrease than those in LPS group, LR group （P〈0.05）, but they were no statistically significant （P〉0.05）. （3） The expression levels of GR： LPS group （1.927±0.108） and LR group（0.966±0.003 ） were lower than those of Con group （7.737±0.290） （P〈0.05）, both were statistically significant （P〈0.05）. LU group （5.026±0.556） and LRU group （5.026±0.556） had more increase than that of LPS group, LR group （P〈0.05）, but there had no statistically significant（P〉0.05）. （4） The expression levels of GR mRNA ： LPS group （0.700±0.020） and LR group （0.300±0.020） was lower than that in Con group （1.000±0.100） （ P〈0.05 ）, LU group （0.700±0.030） was higher than LPS group, but had no significant difference （P〉0.05）, but still lower than that in Con group, and the difference was statistically significant （P〈0.05）. LRU group （0.853±0.031） was higher than LPS group, LU group, LR group, but lower than Con group, the difference was statistically significant （P〈0.05）. Conclusions UTI can regulate the expression levels of GR and GR mRNA, and reduce the degree of LPS-induced ALl in rats, but the intervention of RU486 is not obvious, the effect of UTI may be associated with other molecular mechanisms or signaling pathways.