The Role Of Globular Adiponectin In The Damage Of Vascular Endothelial Cells Induced By Cigarette Smoke Extract

Research backgroundAt present,pulmonary vascular endothelial cell injury is considered to be one of the important pathogenesis of chronic obstructive pulmonary disease(COPD), Cigarette smoke,as the most important risk factors for COPD,can damage the lung vascular endothelial cells via direct action and/or the elevated levels of variety of inflammatory mediaters.Adiponectin is a kind of adipokines secreted by adipose tissue,which is considered to be have different kind of biological activities.The circulating adiponectin exists in different isoforms such as trimers, hexamers, a high-molecular weight form and a globular domain form. Nakanishi et al provided evidence that adiponectin-knockout mice showed progressive emphysematous phenotype age-dependently, and endothelial apoptosis has been proposed to be part of the pathogenesis. Studies about globular adiponectin in different cell lines demonstrated both proinflammatory and anti-inflammatory properties.This study examined the effect of different concertration of globular adiponectin on endothelial cells treated with cigarette smoke extract, we observed the cell apoptosis rate and the expression of inflammatory factors and oxidation materials to reflect the effct.ObjiectiveBy observing the the cell apoptosis rate and the expression of inflammatory factors and oxidation materials,we explore the effect of globular adiponectin pretreatment on the endothelial cells treated with cigarette smoke extract.MethodSelect the human umbilical vein endothelial cell lines (HUVEC) CRL-1730complete the following experiment.(1) Using the CCK-8to determine the toxic effect of cigarette smoke extract in HUVEC=.The HUVEC was cultured with high sugar DMEM medium containing10%fetal bovine serum, when the cells reaches70%confluence, digest it with0.25%trypsin-EDTA, centrifuge and then cells were resuspended with complete medium, adjust the density of the cells, seed the cells in the96-well plates at a concentration of4000cells each wells, the mediun volume was100μ1/well, using the wells containing same amount of cell medium and CCK-8as the blank control. When the cells reaches80%confluence, wash the cells with PBS and then added the midiun containing different concentration of cigarette smoke extract, the concentration of the CSE was0%,1%,2.5%,5%,7.5%,10%. Incubate another24hours, then add10μ1CCK-8to each well, put the cells into the incubator for1hours, messured the average optical density at450nm by Universal Microplate Spectrophotometer.(2) The effect of different concentration of gAd in cell apoptosis and the expression of inflammatory factors and oxidative materials induced by cigarette smoke extract.The HUVEC was cultured with high sugar DMEM medium containing10%fetal bovine serum, when the cells reaches80%confluence, digest it with0.25%trypsin-EDTA, centrifuge and then cells were resuspended with complete medium, seeded the cells in12-well plates at a density of1×105/well. When the cells reaches the logarithmic growth phase, replace the cell medium with medium containing different concentration of globular adiponectin (0.1μg/ml,0.5μg/ml,2.5μg/ml,5μg/ml,10μg/ml,20μg/ml), after4hours, added CSE solution to a final concentration of5%, put the12-well plate in the incubator for24hours, collect the cells, marked it with AnnexinV-FITC and then using flow cytometry to measure the apoptosis rate of the cells.Collect the supernatant of the cells, and then using the ELIS A method to detect the concentration of interleukin-6(IL-6)、tumor Necrosis Factor-a (TNF-a) and4-hydroxynonenal (4-HNE).Results1.The toxic effect of CSE in the cell viabilityAfter treated with different concentration of cigarette smoke extract, the cell viability decreased,the differences were statistically significant(F=180.620, P<0.05). The cell viability was significantly negatively correlated with the concentration of CSE(r value was-0.986, P<0.05),The cell viability decreased gradually along with the increase of the CSE concentration.The concentration of CSE make the cell viability decreased to half of the blank control was5%.2.The effect of5%CSE in cell apoptosis and the expression of IL-6^TNF-a and4-HNEWhen the endothelial cells was treated with5%CSE,the rate of apoptosis cells (43.673±0.900%) and the expression of IL-6(196.265±19.161pg/ml)、 TNF-a (297.410±12.232) and4-HNE (5.756±0.255μg/ml) protein increased compared with blank control (3.597±0.586%,23.994±4.509pg/ml,76.378±4.663pg/ml,0.865±0.110μg/ml), the differences were statistically significant (t values were-64.638,-15.158,-29.176,-30.482,P<0.05saparately) 3.The effect of different concentration gAd pretreatment in cell apoptosis and the expression of IL-6^TNF-a and4-HNE induced by5%CSEAfter pretreated with different concentration gAd for4hours, the rate of apoptosis cells and the expression of IL-6、TNF-a and4-HNE protein decreased compared with the5%CSE group, the differences were statistically significant (F value were74.295,50.630,64.012,61.822; P<0.05saparately).4.Correlation AnalysisIn the range of the concentration of gAd were<10μg/ml, the rate of apoptosis cells and the expression levels of IL-6, TNF-a and4-HNE were significantly negatively correlated with the gAd concentration,(r values were-0.794,-0.871,-0.859,-0.897, p<0.05saparately) With the concentration of the gAd increasing, the rate of apoptosis cells and the expression levels of IL-6, TNF-a and4-HNE decreased. At the point that the concentration of gAd was20μg/ml, the rate of apoptosis cells and the levels of the above factors increased compared with the group5%CSE+10μg/ml gAd, the differences were statistically significant(p<0.05saparately).Conclusion1. The cigarette smoke extract can damage the endothelial cells in a dose-dependent manner, with the concentration of the CSE increasing, the cell viability gradually decreased.2. When the concentration of the globular adiponectin was in the range of less than10μg/ml, gAd exert protective effects dose dependently on the HUVECs treated with CSE, but the protective effect decreased when the gAd concentration reaches20μg/ml. It indicate that gAd exert protective effect on the endothelial cells treated with CSE, and the protective effect is closely related with the concentration of the gAd.