Effects Of Scorpion Purified Liquid On The Release Of Cultured Vascular Endothelia Cells Induced The Release Of Vascular Active Substances By Thrombin

Objective:The human umbilical vein vascular endothelia cells (HUVECs) cultured in vitro was selected for the experiment method,to observe the effects of scorpion purified liquid(PLIS) on the release of HUVECs induced the release of vascular active substances by thrombin,and to investigate the mechanism of antithrombotic.Methods:HUVECs was cultured in vitro, then the well grown2-3generation HUVECs were divided into control group(1640culture liquid),thrombin group(thrombin10U/mL),high concentration PLIS group (PLIS24μg/mL+thrombin10U/mL),medium concentration PLIS group(PLIS12μg/mL+thrombin10U/mL),and low concentration PLIS group(PLIS6μg/mL+thrombin10U/mL). After HUVECs were cultured for12hours, the cell morphology was observed,Thrombin and drug concentrationt was screened by MTT,and the supernatant and cell lysates were collected,for determining the content of nitric oxide (NO)、 endothelin-1(ET-1) and the activity of lactate dehydrogenase (LDH) in the cells supernatant,and the same time, the content of total nitric oxide synthase (TNOS)、 inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were deteminded in the freeze-thaw fluid.Results:l.The contraction and smaller volume of HUVECs of thrombin group was obseved,the majority of HUVECs was integrated,the series of morphology was obseved,for example, the significantly widened intercellular, shrinkage cell,but clear cells and nuclear boundary,regular shape, reduced transparency, blur edge,it showed that HUVECs was injured by thrombin.The high,medium and low scorpion were respectively suppled into HUVECs,the cells growned well, the shape of cells was normal and similar to the blank control group,it showed that the scorpion could protective HUVECs which injuryed by thrombin.2.The OD value of the thrombin group detemined by MTT method was obviously lower than the blank group(P<0.01).The OD value of each PLIS groups determined by MTT method was significant difference compared with thrombin group (P<0.01),it showed that the HUVECs metabolic activity was lower after administed thrombin,but the inhibitory effects of PILS was obvious.3.Compared with the blank group,the LDH activity of thrombin group increased significantly (P<0.01).Compared with the thrombin group,the LDH of activity of each PILS groups inhibited significantly(P<0.01),it showed that the metabolism of HUVECs could inhibited by thrombin, the LDH leakage of HUVECs could promoted by thrombin,and thrombin damaged HUVECs.4.HUVECs released quantitative NO under basic condition, compared with the control group,the NO release of HUVECs promoted by thrombin was statistically significant (P<0.01);Compared with the control and thrombin group, the NO release of HUVECs promoted by PILS was marked (P<0.01).5.Thrombin stimulated the ET-1secrete of HUVECs,and which the ET-1secrete in the supernatant fluid was significantly higher than the control group (P<0.01);Compared with thrombin group,the ET-1secrete HUVECs induced by PILS was significantly lower (P<0.01).6.The release of TNOS、eNOS、iNOS of HUVECs suppled into PILS was not marked,compared with the thrombin and control group (P>0.05)Conclusion:PILS has significant anti-thrombosis effects,the mechanism is related to vascular endothelia cells expressing vascular active substances.