A Research On The Expression Changes Of Akt Family In PMVECs Proliferation Induced By HPS Serum Of Rats

Implications and objectiveHepatopulmonary syndrome (HPS)--a pulmonary complication observed in patients who have chronic liver diseases and/or portal hypertension--is attributed to intrapulmonary vascular dilatation, gas exchange impairment and impaired arterial oxygenation, which incidence rate in hepatic cirrhosis is up to 15%, and is also the main reason of primary non-function and pulmonary infection during the perioperative period of liver transplantation surgery. Although extensive researches have been done, we still do not know much about its pathogenesis. Sindhu believe that intrapulmonary shunt is the main cause of HPS, and trichangiectasis is probably a morphological base of functional shunt. Transthoracic contrast-enhanced echocardiography, body scan with 99mTc-labeled macroaggregated albumin perfusion and pulmonary angiography also demonstrate the significance intrapulmonary vascular dilatation (IPVD). The pulmonary microvascular endothelial cells (PMVECs) are the major cells to structure the pulmonary capillaries. Therefore, significant pulmonary capillary dilation (expansion diameter up to 20-50μm) can only be completed by the abnormal proliferation of PMVECs. Thus, most scholars believe that the abnormal proliferation of PMVECs causes pulmonary capillary dilation, which leads to arterial oxygenation disturbance. Refractory hypoxemia and hypoxic pulmonary vascular remodeling induce the development and progression of the HPS. Its pathogenesis is not well defined, but the opinion that an association of liver-derived factors can precipitate proliferation of PMVECs by the blood circulation is more consistently agreed. Serine/ threonine kinase (Akt) is the important point of intersection of many signaling pathway, which can regulate cell viability signal such as cytokine and growth factor. Akt is ubiquitous in eukaryotic regulatory networks, with closely related to a variety of proliferation diseases. For these reasons, we speculate that what induces the development and progression of HPS is that an association of liver-derived factors in serum plays a role in proliferation of PMVECs via the Akt family on the basis of chronic liver disease and / or portal hypertension, which leads to significant pulmonary capillary dilation. Based on this, in order to elucidate the mechanism of development and progression of the HPS, methods and technologies such as MTT assay, 3H-thymidine incorporation assay, polymerase chain reaction (PCR) and western blot were performed in this study to investigate the effect of HPS serum of rats on PMVECs proliferation and the expression of Akt family (Akt1, Akt2, Akt3).MethodsTotal three parts:1. Rat model and serum preparationThe model was induced in rats by chronic bile duct ligation (CBDL). Serum of HPS rats was prepared after the model was examined by pathology and blood gas.2. Establishment of primary culture PMVECs and proliferation detecting Primary culture rat PMVECs was established and identified. PMVECs were randomly divided into two groups: C and HPS. Group C remained untreated as normal control, group HPS was treated by serum of HPS. After treatment for 24, 48, 72 hours (T₁³), the cell was determined by MTT assay and 3H-TdR assay.3. mRNA and protein expression levels detectingThe mRNA and protein expression levels of Akt1, Akt2 and Akt3 in each group were detected by RT-PCR and Western Blot, respectively.Results1. The Effect of HPS serum on PMVECs proliferationCompared with group C, PMVECs proliferation in group HPS increased (P<0.05). Proliferation increased with treating time in group HPS. Compared with HPS group T₁, PMVECs proliferation in HPS group T₂ increased (P<0.05). Compared with HPS group T₂, PMVECs proliferation in HPS group T3 increased (P<0.05).2. Result of Akt family mRNA expression levels detecting The result of RT-PCR showed that expression of Akt mRNA in group HPS increased (P<0.05) compared with group C. Expression of Akt mRNA increased with treating time in group HPS. Compared with HPS group T₁, expression of mRNA of Akt in HPS group T₂ increased (P<0.05). Compared with HPS group T₂, mRNA expression of Akt in HPS group T3 increased (P<0.05).3. Result of Akt family protein expression levels detectingThe result of Western Blot showed that expression of Akt protein in group HPS increased (P<0.05) compared with group C. Expression of Akt protein increased with treating time in group HPS. Compared with HPS group T1, protein expression of Akt in HPS group T2 increased (P<0.05). Compared with HPS group T2, expression of Akt protein in HPS group T3 increased (P<0.05).ConclusionsProliferation of PMVECs increased induced by the treatment of HPS serum. Expression of mRNA and protein of PMVECs Akt family increased. The results suggest that the change of PMVECs proliferation may be associated with the change of Akt family expression levels. Akt signal pathway may be one of the important way to regulate PMVECs proliferation, and play an important role in development and progression of the HPS.