The Effect Of Bilirubin On Pulmonary Microvascular Endothelial Cells In Rat Hepatopulmoanry Syndrome

Background and purpose：Hepatopulmonary syndrome is a pulmonary complication mainly caused by chronicliver diseases or portal hypertension, manifests as pulmonary microvascular dilatation,impaired blood-gas exchange, abnormal arterial oxygenation. Presently, domestic andforeigen scholars believe that pulmonary microvascular dilatation and inropulmoanry shuntare the mainly cause of HPS development, which result in severe hypoxemia and thusmarkedly increases the mortality of patients. Liver transplantation is the only effectivetreatment. The current studies suggest that the abnormal proliferation of pulmonarymicrovascular endothelial cells (PMVECs) is the main cause of pulmonary microvasculardilatation. Meanwhile, our previous study found that pulmonary capillaries have thickenedwalls in5-week common bile duct ligation (CBDL) rats and PMVECs showed a tendencyof multiple growth (TGF-β1/Smad3and PI3K/Akt1abnormal increase) induced by serumof CBDL rats. In addition, bilirubin was the highest level in CBDL serum. However,whether bilirubin has effect in PMVECs proliferation is unclear. To explore thepathogenesis of HPS, in this study, we constructed HPS models by using CBDL, culturedPMVECs, evaluated the mRNA and protein levels of Akt1and Smad3in the lung of CBDLrats and PMVECs cultured by different concentration of bilirubin.Methods：1. The role of pulmonary Akt1and Smad3in the development of HPS2.1HPS models were constructed by CBDL, all animals were randomly divided intocontrol group (group C), sham operation group (group S), CBDL group, specimenscollection and blood gas analysis were performed in1W,3Wand5W following CBDL.2.2Pulmonary Akt1and Smad3levels in1W-,3W-and5W-CBDL animals weredetected by RT-PCR and western blotting.2. The effect of bilirubin on PMVECs. 2.3Isolation, culture and identification of rat pulmonary microvascular endothelialcells.2.4PMVECs were randomly divided into five groups: the control group, B1group (5μmol bilirubin),B2group (10μmol bilirubin), B3group (20μmol bilirubin), B4group(50μmol bilirubin). Akt1and Smad3levels in24h,48h and72h were detected by RT-PCRand western bloting, Cells proliferation were assessed by CCK-8and3H-TdRincorporation.Results：1. Compared with group C and S, the mRNA and protein levels of pulmonary Akt1and Smad3were significant up-regulated at3W-and5W-CBDL rats, microvessels hadenlarged lumens and the alveolar-arterial oxygen gradient was increased in5W-CBDLgroup (P<0.05).2. After48h culture of CBDL serum,the proliferation of PMVECs, the mRNA andprotein levels of Akt1and Smad3were declined in B4group (P<0.05). After72h cultureof CBDL serum, the proliferation of PMVECs, the mRNA and protein levels of Akt1andSmad3were decreased in B3and B4groups (P<0.05); compared withB3group, B4groupdecreased more significantly (P<0.05).Conclusions：1. In CBDL rats, pulmonary Akt1and Smad3upregulation may be a mechanism ofHPS development.2. Bilirubin inhibited PMVECs proliferation and Akt1and Smad3expression in adose and a time dependent manners.