The Protective Effect OF Lyophyllum Ulamarium Sereptokinase In Vascular Endothelial Cells

Objective:To investigate the protective effect and anti-platelet effect of Lyophyllum ulmarium streptokinase on ethanol-induced vascular endothelial cell damage.Method:(1) In vitro:The survival rate of HUVEC was detected after Lyophyllum ulmarium streptokinase treatment by the MTT method in the oxidative damage model induced with 200μmol/L ethanol. LDH activity was assayed in HUVEC culture fluid, apoptotic cell was observed by AO/EB double staining, and mitochondrial membrane potential and active oxygen content were measured by flow cytometry, the protein expressions of Caspase-8, Caspase-9, cleaved Caspase-3, and Cytochrome C were observed by the western blot method. (2) In vivo:The rat model of endothelial cell injury was established by 40% ethanol through the gastric gavage, then the morphology changes of carotid artery were tested by HE staining, blood platelet CD62p was detected by flow cytometry, serum Ang-II, ET-1, Renin, NOS, iNOS, and eNOS were detected by ELISA, the levels of SOD, MDA, T-AOC, and NO were assayed by the colorimetric method.Results:(1) MTT result showed that L.u streptokinase played a protect role for ethanol-induced oxidative damage. The LDH activity of cell culture fluid was significantly decreased than that of model group. AO/EB staining result showed that endothelial cells shrunk into rotundity and some of endothelial cells changed to orange, some showed vacuole-shape and red dying appeared in the structure of model group. But after the treatment with L.u streptokinase, morphological changes were attenuated, the cells gradually returned to normal structure, and the number of apoptotic cells reduced. Flow cytometry data showed that mitochondrial transmembrane potential of L.u streptokinase group was significantly increased (p<0.01), while level of total active oxygen was decreased significantly (p<0.01). Western bolt results showed that L.u streptokinase could reduce the protein expressions of cleaved Caspase-3, cytochrome C, increased Caspase-8 and Caspase-9, respectively. (2) HE staining results showed that ethanol induced severe shrinkage of blood vessel, intermittent of vascular endothelial layer, and severe exfoliation of vascular endothelial cells. After the treatment of L.u streptokinase, the shrunk of blood vessel, intermittent of vascular endothelial layer, and the exfoliation symptom were improved, respectively, and the structure of high dose group could recovered even to that of the normal control. Platelet CD62p of L.u streptokinase group was significantly decreased than that of model group, it suggested L.u streptokinase might inhibit platelet activation. The levels of serum NOS, iNOS, eNOS, Renin, Ang-Ⅱ and ET-1 were significantly reduced, SOD and T-AOC levels were significantly increased, while the MDA and NO levels were reduced.Conclusions:(1) L.u streptokinase could play a protection role on ethanol-induced vascular endothelial cell damage. (2) L.u streptokinase could inhibit the platelet activation.