Research Of PCA Extract From Agkistrodon Acutus Venom In Wan-nan Region On The Effect Of Hypoxia Reoxygenation Of Vascular Endothelial Cells And Its Mechanism

Objective: To discuss PCA extract from Agkistrodon acutus venom in wan-nan region on the effect of hypoxia reoxygenation of vascular endothelial cells and its mechanism, and analyse the characteristics of plasma protein C activity change in myocardial ischemia-reperfusion injury in rats.Methods: 1、Cells animal experiment: Human umbilical vein endothelial cells(HUVEC) strains were divided into normal control(NC), anoxia reoxygenation injury(AR), anoxia reoxygenation + PCA experimental group. AR group were divided into anoxia 2 h group(R0), reoxygenation 2 h(R2), 4 h(R4), 6 h(R6) group. To use the PCA effect on R6 group, and depending on the concentration of PCA, the PAR groups were divided into group 1, 2 and 4ug/ml groups with 6 in each group according to the concentration of the PCA. To detect the activity of HUVEC proliferation by MTT method; to detect the p H of Cell culture medium by acidity meter; to detect intracellular calcium ion concentration by fluorescence spectrophotometer; to observe expression of t-PA and PAI-1changes by immunocytochemistry method to; to detect protein content of t-PA and PAI-1 by western blot. 2、Animal experiments: 50 Sprague Dawley healthy male rats randomly divided into control group(C) and ischemia reperfusion group(IR), blocking heart blood flow 30 min by ligated left anterior descending coronary artery then restoring blood flow with IR group rats. The rats of IR group were randomly divided into reperfusion 0(R0), 30(R30), 60(R60), 120(R120) min group with 10 in each group. Just threading the left anterior descending coronary artery without ligating it in C group rats. To evaluate the myocardial IR model by continuous monitoring electrocardiogram(ECG) of rats, myocardial pathologic histology observation and TTC staining; took the plasma to detect APTT, PT, TT, FIB; to test PC activity by chromomeric substrate method; to detect the content of tissue factor(TF), plasma free protein S(FPS), fibrinolytic enzyme activator(t-PA), fibrinolytic enzyme activator inhibitors(PAI-1) by enzyme linked immunosorbent assay(ELISA); to detect platelet aggregation by turbidimetry.Results: 1、Cells experiments: compared with NC group, cell proliferation rate decreased(P < 0.05) in AR group, then recovered(P < 0.05) in PAR group, and the proliferation rate increased as the concentration of PCA rising; the PH of cells supernatant fluid decreased(P < 0.05) in AR group, cells supernatant fluid had a lower PH in PAR group, there were no statistically significant difference; compared with AR group(P > 0.05); intracellular Ca2+ concentration increased(P < 0.01) in AR group, reduced in PAR groups, and reduced significantly with the increasing concentration of PCA; immunocytochemistry displayed t-PA expression decreasing, PAI-1 expression increasing in AR group, rising of t-PA express, declineing of PAI 1 expression in PAR group along with the increasing concentration of PCA change significantly; Western blot also showed t- PA content decreasing(P < 0.05) and PAI- 1 content increasing(P < 0.05) in AR group, t- PA content increasing(P < 0.05) and PAI 1 content decreasing(P < 0.05) in PAR group, increasing with the concentration of the PCA change obviously. 2、Animal experiments: ECG showed T wave, ST segment elevation of ischemia reperfusion rats, myocardial pathology examination showed myocardial cell change; TTC stain showed pale infarct area; compared with control group, the plasma PC activity in ischemia significantly reduced(P < 0.01), restored in the early reperfusion, decreased again significantly(P < 0.01); the content of plasma TF increased in ischemia(P < 0.05), higher(P < 0.01) after reperfusion;the content of plasma FPS increased in the early reperfusion(P < 0.05), decreased significantly(P < 0.05) in the late reperfusion; the content of plasma PAI-1 reduced in ischemia(P < 0.05), to further reduce in the during reperfusion(P < 0.01),and plasma t-PA began to increase(P < 0.05) in reperfusion, further elevated longer duration of reperfusion(P < 0.05); Platelet aggregation rate increased significantly(P < 0.01) in early ischemia and reperfusion group; APTT significantly shortened(P < 0.01) after reperfusion 60 min, PT shortened(P < 0.01) in R120 group; the content of FIB(mg/dl) increased significantly(P < 0.01) in R120 group, TT had no obvious change.Conclusion: PCA extract from Agkistrodon acutus venom in the institute of snake venom of wannan medical college can not only directly activate plasma protein C, but also influence the endothelial cells of the release of cytokines as t-PA, PAI-1,TF, reduce cell damage caused by ischemia-reperfusion and indirectly change blood coagulation process.