Study On Activated Protein C Inhibiting Endotheliocyte Apoptosis By Endoplasmic Reticulum Stress

Backgroud and Objective Endotoxin-induced acute lung injury is a hot topic in critical respiratory disease. Endotoxin could directly or indirectly induce PVEC injury and apoptosis. In recent years, activated protein C has been used in the treatment of severe sepsis because of its pleiotropic activities, including anticoagulant, anti-inflammatory, antiapoptotic activity, and protection of endothelial barrier function, and improvement of the prognosis of patients. The antiapoptotic effect of activated protein C is related to its participation in the mitochondria apoptosis pathway and death receptor pathway. But, wheather endoplasmic reticulum(ER) could contributed to the antiapoptotic activity is not reported. This study aimed to investigate whether APC could induce ER stress and its relation with apoptotic activity while treat the apoptotic modle of HUVECs, and wheather APC could inhibite endotheliocyte apoptosis mediated by GSK-3P afert endoplasmic reticulum stress, which would indicate a new mechanism of antiapoptotic activity of APC and provide more laboratory evidence for the APC treatment of endotoxin-induced acute lung injury.Methods (1) Flow cytometry and Caspase-3 activity assay would be used for detecting apoptosis of human umbilical vein endothelial cell (HUVEC) while in challenge of Lipopolysaccharide (LPS). LPS would be used for constructing apoptotic model of HUVECs. (2) Western blot would be used for detecting the expression of glucose-regulated protein 78(GRP78) while stimulated by 150nM APC. Flow cytometry would be used for detecting the influence of LPS-induced apoptosis from APC. (3) Flow cytometry and Caspase-3 activity assay would be applied for detecting influenced antiapoptotic activity of APC by 8-[N, N-diethyla-mino]-octyl-3, 4,5-trimethoxybenzoate (TMB-8), an ER calcium inhibitor. (4) The changes of p53 and GSK-3β expression induced by APC treatment would be investigated by Western blot in LPS-induced apoptosis of HUVECs. (5) RT-PCR and Western blot would be used in siRNAs testing. After GSK-3β silence, flow cytometry would be used for detecting the changes of antiapoptotic effect from APC in LPS-induced HUVECs apoptosis.Results (1) 0.1、1、10、20μg/ml LPS were used for stimulating of HUVECs respectively. The morphologic changes of cutured HUVECs was inconspicuous while in challenge of 0.1μg/ml LPS, but impressive while in challenge of 1、10、20μg/ml LPS. More death cells were observed under inverted microscope while in challenge of 10 or 20μg/ml LPS. (2) After the treatment of 150μM APC for HUVECs preconditioning 24 hours with LPS, the expression of GRP78 increased at 6hours, and lasted to 24hours. At every timepoint, there were significant differences in the expression of GRP78 between control group(P<0.05) and stimulating with APC alone.(3) With the application of 150nM APC and TMB-8 at the same time for HUVECs preconditioning 24hours with LPS, the percentage of apoptotic cells and Caspase-3 activity assay did not show significant changes (P>0.05).(4) After preconditioning with 1μg/ml LPS in HUVECs, expression of GSK-3β increased slightlly, and p53 increased signifienctlly. But, with 150nM APC treatment, expression of GSK-3β increased significentlly and p53 decreased significentlly. (5) With the application of GSK-3β-siRNA transfecting HUVECs, the results of flow cytometry and caspase-3 activity showed that there were significant differences between GSK-3β-HUVECs and GSK-3β+HUVECs (P<0.05).There was not significant difference in caspase-3 activity between GSK-3β-HUVECs treated with APC or without APC (P>0.05).Conclusions It is concluded that APC as an ER stressor (1) could induce UPR of HUVECs and show its cytoprotective effect in inhibiting LPS-induced apoptosis. (2) do not affect LPS-induced apoptosis although transient calcium release from ER could be induced. (3) induced ER stress and inhibit apoptosis of HUVECs mediated by GSK-3β.