The Measure Of Rhoa/Rho-kinase Signaling Pathway Activity Of Peripheral Circulating Blood Neutrophils In Patients With COPD Related Pulmonary Arterial Hypertension

Objective:To determinate p-MBS、t-MBS of the peripheral circulating blood neutrophils in healthy people、the healthy people who are smoking、COPD patients and patients with COPD related PAH. And then calculate the ratio of p-MBS/t-MBS, to find out weather RhoA/Rho-kinase signaling pathway is activitied and the role in the patients with COPD related PAH.Methods:1. ObjectsNine healthy people without smoking with normal pulmonary function (PF) were chosen as group A (male5,female4, average age61.1±8.32); Nine healthy people who are smoking(smoking index>100) with normal pulmonary function (PF) were chosen as group B(male6,female3; average age57.6±9.42); Nine chronic obstructive pulmonary disease(COPD) patients without pulmonary arterial hypertension(PAH), who were hospitalized in the third hospital of Changsha from December2010to April2011were chosen as group C(male, average age64.2±6.37);Nine COPD related PAH patients were chosen as group D (male7, female2, average age60.7±6.52);. All patients in group C and group D were diagnosed according to the COPD Guideline Published by Chinese Medical Committee in2007and the patients who meet the Guide-line of pulmonary hypertension made by ESC in2009were assigned to group D.2. Pulmonary function testAll objects were done the pulmonary function test(PFT), included FVC、FEV1and FEV1/FVC,the percentage of predict FEV1(FEV1%);The group C and group D are diagnosed by FEV1/FVC<70%after inhalation salbutamol(400ug).3. Pulmonary arterial systolic pressure testAll objects were done the PASP test by echocardiography.4. Western-Blot1) Sample collection:20milliliter blood was collected from each patients. The peripheral blood neutrophils were separated by percoll, and preserved for Western-Blot in a refrigerator with-80℃.2) Western-Blot:Extract the protein of the neutrophils, determine the concentration of the neutrophils with BCA; The membranes were immunoblotted with anti-phospho-MBS (Upstate) or anti-total-MBS (BD).And then do the as the instruction book, the next blots were visualized by the enhanced chemiluminescence system (ECL Western Blotting Detection Kit, GE Healthcare). Densitometric analysis was performed by Scion Image Software.Results:1.The PASP in group D was higher than group A、B and C(P<0.05); but the PASP of group C was no significant discrepancy with group A and B(P>0.05); The PASP of group B was higher than group A(P<0.05).2.The cell count of neutrophils:group D was significant higher than group A、B (P<0.01),and higher than group C(P<0.05); group C was significant higher than group A and group B(P<0.01); but there was no significant discrepancy between group B and group A(P>0.05).3.The ratio of p-MBS/t-MBS among four groups:group D was higher than group A、B and C (P<0.05); compare with group C with group A、B,there has discrepancy(P<0.05);but there was no significant discrepancy with group A and group B(P>0.05);4. The ratio of p-MBS/t-MBS has a positive relationship with PASP (r=0.535, P<0.01).5. The ratio of p-MBS/t-MBS has a positive relationship with the level of circulating blood neutrophils (r=0.836,P<0.01).Conclusions:1. The pathogenesis of COPD-related PAH may be related to systemic inflammatory2. RhoA/Rho-kinase signaling pathway plays a important role in the pathogenesis of COPD-related PAH.