葡萄糖调节蛋白78在复合致病因素诱导的大鼠肝肺综合征中的作用

摘要

AIM: To explore the role of 78 kD glucose - regulated protein ( GRP78 ) in the development of hepatopulmonary syndrome ( HPS ) in rats and the relation to intestinal endotoxemia ( IETM ).METHODS: The experimental animals were randomly divided into HPS groups of the 4th week, the 6th week and the 8th week.Normal control groups at the corresponding time points were also set up.The Wistar rat model of HPS produced in the process of liver cirrhosis was induced by employing multiple pathogenic factors to the animals.The rats in normal control group were designed by feeding with standard diet and tap water.Histopathological changes of the lung and liver were observed under microscope with the staining of hematoxylin and eosin ( HE ).The concentrations of alanine amino transferase ( ALT ), endotoxin and tumor necrosis factor α ( TNF - α ) in plasma, the contents of TNF - α and malondialdehyde ( MDA ) in the lung tissues were detected.The expression of GRP78 at mRNA and protein levels in the lungs was measured by the methods of RT -PCR and Western blotting, respectively.RESULTS: The level of endotoxin in plasma was gradually increased with the HPS development.The expression of GRP78 at mRNA and protein levels was also gradually increased with the HPS development and was significant at every time point.The endotoxin in plasma was positively correlated with the expression of GRP78 protein in the lung tissues of the rats with HPS.With the HPS development, the levels of ALT and TNF - α in plasma and the contents of TNF - α and MDA in the lung tissues were gradually increased.The content of endotoxin in plasma and the protein expression of GRP78 in the lung tissues were positively correlated with the contents of TNF - α in plasma and TNF - α and MDA in the lung tissues.The contents of TNF - α in plasma and GRP78 at mRNA and protein levels and TNF - α in the lung tissues were higher in the rats with HPS at every time point than those in normal control group.At the 6th week and the 8th week, the contents of endotoxin and ALT in plasma and MDA in the lung tissues of the rats with HPS were significantly higher than those in normal control group.CONCLUSION: IETM originated from the liver cirrhosis acts as a critical stressor of endoplasmic reticulum ( ER ) stress and activates ER stress in the lung by oxidative stress, resulting in increased expression of GRP78.Therefore, the increased expression of GRP78 induced by ER stress may play an important role in the development of HPS in rats.%目的:探讨葡萄糖调节蛋白78(GRP78)在大鼠肝肺综合征发病中的作用及其与肠源性内毒素血症的关系.方法:Wistar大鼠被随机分为4周组、6周组和8周组3个时点,采用复合致病因素法制备大鼠肝硬化合并肝肺综合征(HPS)模型,并设标准饮食的正常大鼠作为对照组.采用HE染色观察肺组织病理变化;测定血浆中丙氨酸氨基转移酶(ALT)、内毒素、TNF-α和肺组织匀浆中的TNF-α、丙二醛(MDA)的含量.Western blotting和RT-PCR法检测肺组织标本中GRP78蛋白和mRNA表达水平.结果:模型组动物血浆内毒素含量随病程进展逐渐增高;肺组织中GRP78蛋白和mRNA的表达随HPS进展逐步增高,且各时点间的表达有显著差异(P<0.05);血浆内毒素与升高的GRP78蛋白水平间呈高度正相关(P<0.01).血浆ALT和TNF-α含量以及肺组织匀浆中TNF-α和MDA含量随病程进展逐渐增高;血浆内毒素含量以及肺组织中GRP78蛋白分别与血浆TNF-α和肺组织中TNF-α、MDA的含量呈高度正相关(P<0.01).在各时点,模型组动物血浆TNF-α含量、肺组织匀浆TNF-α、GRP78蛋白及mRNA均显著高于正常对照组(P<0.05).在第6周和第8周,模型组动物血浆内毒素和ALT的含量以及肺组织匀浆中MDA的含量均显著高于正常对照组(P<0.05).结论:肝硬化时形成的肠源性内毒素血症作为内质网应激的重要应激原,通过氧化应激激活肺组织的内质网应激反应导致GRP78表达增高,很可能是HPS发病的重要机制.