Effect of Cardiopulmonary Bypass on Annexin A1 Expression in Peripheral Blood Mononuclear Cells of Children with Congenital Heart Disease

Abstract

Summary: This study aimed to investigate the effect of cardiopulmonary bypass (CPB) on Annexin A1 expression in the peripheral blood mononuclear cells (PBMCs) of children with congenital heart disease (CHD). A total of 30 children receiving CPB for interventricular septal defect were included. Peripheral blood was collected before and after CPB. PBMCs were collected by density gradient centrifugation. Protein extra ction was performed by lysis and subjected to 2D-QUANT for protein quantitation. Isoelectric focusing electrophoresis (IEF) was carried out followed by gel image analysis. Protein spots with a difference in expression of >1.5 fold were collected as candidate proteins which were subjected to mass spectrometry for the identification of diffe rentially expressed proteins. Western blot assay was emplo yed to confirm the expressions of target proteins. Peripheral blood collected at two time points was subjected to two-dimensional electrophoresis, and a total of 12 differentially expressed proteins were identified. Of them, 5 proteins had decreased expression before CPB (T0) but their expressions increased after CPB (T1); the remaining 7 proteins had in cre ased expres sions before CPB but their expressions re du ced after CPB. One of these differentially expres sed proteins was Annexin A1. Western blot assay confirmed that Annexin A1 expression began to increase at 0.5 h after CPB, and the increase of Annexin A1 was more obvious after CPB. Our findings primarily indicate the potential mechanism underlying the role of PBMC in inflammatory response following CPB, and provide a target for the prevention and control of post-CPB systemic inflammatory response syndrome (SIRS).Keywords: annexin A1, cardiopulmonary bypass, peripheral blood mononuclear cells, congenital heart disease Kratak sadr`aj: Svrha studije bila je da se istra`i uticaj kardiopulmonarnog bajpasa (KPB) na ekspresiju aneksina A1 u mononuklearnim }elijama periferne krvi (M]PK) dece sa uro|enim sr~anim oboljenjima (SO). Istra`ivanjem je obuhva}eno ukupno 30 dece kod koje je zbog interventrikularnog septalnog defekta izveden kardiopulmonarni bajpas. Uzorci periferne krvi uzeti su pre i posle zahvata. M]PK su sakupljene centrifugiranjem u gradijentu gustine. Proteini su ekstra hovani lizom a kvantitacija proteina obavljena je metodom 2D-QUANT. Izvr{ena je elektroforeza izoelektri~nim fokusiranjem (EIF) a potom analiza gel image. Proteinske ta~ke sa razlikom u ekspresiji >1,5 puta uzete su kao proteini kandidati, koji su podvrgnuti masenoj spektrometriji radi identifikovanja razli~ite ekspresije proteina. Za potvr|ivanje ekspresija ciljnih proteina upotrebljen je test Western blot. Uzorci periferne krvi uzeti su dva puta u odre|enom vremenskom razmaku i podvrgnuti dvodimenzionalnoj elektroforezi te je identifikovano ukupno 12 diferencijalno izra`enih proteina. Od toga je 5 proteina imalo smanjenu ekspresiju pre KPB (T0), ali se njihova ekspresija posle KPB pove}ala (T1); preostalih 7 proteina imalo je pove}anu ekspresiju pre KPB, ali se njihova ekspr...