The Research On Effect Of Syk On Pathological Angiogenesis In HPS

Objective:Hepatopulmonary syndrome?HPS?is one of the pulmonary vascular complications in patients with advanced liver disease.It is characterized by hepatic dysfunction and/or portal hypertension,intrapulmonary vasodilation and increased alveolar-arterial oxygen pressure difference.Numerous researches have shown that the mechanism of HPS is associated with dysregulation of ventilation/perfusion ratio?V/Q?ratio,pulmonary arteriovenous shunt and oxygen diffusion disorder caused by pulmonary pathological angiogenesis.The researchers found that spleen tyrosine kinase?Syk?is involved in endothelial cell migration,proliferation,and angiogenesis.This research aims to investigate the expression of Syk in the lungs of HPS rats and its relationship with pathological angiogenesis of HPS,and to explore the mechanism of Syk regulation of pathological angiogenesis in HPS.Methods:1.Grouping and the establishment of CBDL rat modelThe rat CBDL?Common bile duct ligation?model was used to simulate the pathogenesis of human HPS.The rats in the sham operation group were normal rats.Then the lung tissue was taken for pathological section observation and subsequent experiments and arterial blood was taken for the detection of arterial blood gas.?1?Grouping of non-intervened rats:40 adult healthy SD rats were randomly divided into four groups?n=10?:sham operation group?Sham?,one week after common bile duct ligation?CBDL1W,1W?group,and three weeks after common bile duct ligation?CBDL3W,3W?group,five weeks after common bile duct ligation?CBDL5W,5W?group.The rats in the CBDL1W group were fed to the first week after the establishment of CBDL rat model and then harvested samples.The rats in the CBD3W group were fed to the third week after the establishment of CBDL rat model and then harvested samples.The rats in the CBDL5W group were fed to the fifth week after the establishment of CBDL rat model and then harvested samples.?2?Grouping of intervened experimental rats:30 adult healthy SD rats were randomly divided into three groups?n=10?:Sham group,CBDL5W group,and R788 intervening group?R788?.Rats in R788 group were intraperitoneally injected with R788?20mg/Kg/d?,fed to the fifth week after the establishment of CBDL rat model and then harvested samples.Rats in Sham group and rats in CBDL5W group were injected with the same dose of physiological saline solution every day,fed to the fifth week after the establishment of CBDL rat model and then harvested samples.2.To detect of the expression levels of Syk and downstream p-Erk1/2 and p-Akt in the lungs of CBDL ratsThe expression of mRNA of Syk,Erk1/2?Mapk3/Mapk1?and Akt in the lungs of CBDL rats was detected by qPCR and and the expression of Syk,p-Erk1/2 and p-Akt protein in the lungs of CBDL rats was detected by Western blot.3.To detect the expression and localization of Syk and downstream p-Erk1/2 and p-Akt protein in lungs of CBDL ratsImmunohistochemistry and immunofluorescence can be used to observe the characteristics of the expression and distribution of the target protein,thereby localizing the expression of Syk.Results:1.General appearance,blood gas analysis and pathological changes of lungs in CBDL ratsThe appearance of lungs in Sham group was normal,while the appearance of lungs in CBDL rats showed congestion spots and necrotic lesions.The arterial blood gas showed decreased PaO2 and increased A-aDO2 in CBDL rats compared with that in Sham rats?P<0.05?.HE staining showed that the structure of lungs in CBDL rats was disordered,with inflammatory cell infiltration,alveolar septum thickening,and the diameter of pulmonary microvessels in CBDL rats was wider than that in Sham rats?P<0.05?.2.The changes in expression of Syk in the lungs of CBDL rats?1?Compared with that in lungs of Sham rats,the expression of Syk,Erk1/2?Mapk3/Mapk1?and Akt mRNA in lungs of CBDL rats increased significantly,and the increasing trend was time-dependent?P<0.05?.?2?The expression levels of Syk,p-Erk1/2 and p-Akt in lungs of CBDL rats were significantly higher than those in lungs of Sham rats,and the increasing trend was more significant in 3W group and 5W group?P<0.05?.3.Expression and localization of Syk in lung tissue of CBDL rats?1?The results of immunohistochemistry showed that Syk was expressed in pulmonary microvascular endothelial cells.And the expression levels of Syk,p-Erk1/2 and p-Akt in lungs of CBDL rats were significantly higher than that in lungs of Sham rats?P<0.05?.?2?The CD34 positive rate was used to represent mean microvessel density?MVD?.And the mean vessel density in lungs of CBDL rats was significantly higher than that in lungs of Sham rats?P<0.05?.?3?We observed the localization of Syk in the pulmonary microvasculature by immunofluorescence microscopy.The number of microvessels in the lungs of the CBDL rats was higher than that of Sham rats?P<0.05?.4.R788 as Syk inhibitor inhibits the expression of Syk,down-regulates the expression of p-Erk1/2 and p-Akt and reduce new vessels.?1?Compared with that in CBDL5W rats,the appearance and pathological changes of lung in R788 rats were improved.PaO₂ was increased and A-aDO₂ was decreased of R788rats compared with that of Sham rats?P<0.05?.?2?Compared with that in CBDL 5W rats,the expression of Syk,p-Erk1/2 and p-Aktl protein in R788 rats was decreased?P<0.05?.And the mean vessel density?MVD?was also significantly decreased in R788 rats compared with that in CBDL 5W rats?P<0.05?.?3?The number of new vessels in the R788 rats was less than that in the CBDL5W group?P<0.05?.Conclusion:1.The results above show that in the process of HPS pathological angiogenesis,the expression of Syk is up-regulated and promotes and activates the active expression of p-Erk1/2 and p-Akt,leading to pulmonary microvascular endothelial cell proliferation,migration and forming of new vessels.2.Syk inhibitor R788 inhibits the expression of Syk in pulmonary microvessels,and also down-regulates the expression of p-Erk1/2 and p-Akt,thereby inhibiting intrapulmonary angiogenesis and improving HPS oxygenation.