Studies On Mechanism Of Ethanol From Bistortae And Pariphyllin For Inhibiting Injury On Vascular Endothelial Cell Induced By H₂O₂

Objectives : To extract traditional Chinese medicine bistortae,investigate the protection of the injury on the umbiliar vein endothelial cell ECV 304 induced by hydrogen peroxide(H₂O₂) of ethanol from bistortae and pariphyllin, and to study the mechanism of anti一atherosclerosis (AS) of these medicines.Methods: Prepration ethanol from bistortae(EFB) ,then extracte pariphyllin(Pa) from this ; examine the protection of the two above medicine to the injury in ECV 304 induced by H₂O₂,decide the protection dose of the two medicine by MTT reduction assay. ECV 304 had been cultured in vitro, a model of endothelial cell oxidative damage induced by H₂O₂ was established.then cells were divided by eight experimental groups:1 normal;2oxidative damage group;3 high density ethanol from bistortae(500pg/ml); 4 medium density ethanol from bistortae(50pg/ml); 5 low density ethanol from bistortae(5pg/ml) ;6 high density pariphyllin(1ug /ml); 7 medium density pariphyllin(100pg/ml); 8 low density pariphyllin(10pg /ml).In second part experiment, Flow cytometry (FCM) methods Annexin V/PI staining and PI staining were used to analysis the effection of two medicine to the apoptosis and cell cycle in all groups, Laser confocal microscopy(LCM) was used to observe the appearance of apoptosis and quiet state and the dynamic change of the density of calcium ion (Ca²⁺) , Reverse transcription PCR (RT-PCR) and immunofluorescene(IFM) staining were used to detect the protein express in mRNA level of apoptosis associated protein caspase-3 in all experiment groups. In third part experiment, we used RT-PCR to detect the protein expression in mRNA level of intracelluar adhesion molecule (ICAM-1),vasculer cell adhesion molecule (VCAM-1),monocyte chemoattractant protein(MCP-1)in all experiment groups,FCM were used to quantitate the ICAM-1 and VCAM-1 and in all group cells,then, and IFM was used to decide the qualitation expression of MCP-1 in all groups Results:The model of oxidative damage was established, ethanol from bistortae and pariphyllin were got out successfully ;MTT assay discovered that cell optical density in oxidation damaged group was lower than nomal control (P<0.01);when pretreated by the two kind of medicine ,the OD value was increased,and was not significant difference in the high density ethanol from bistortae and the high density pariphyllin than normal control(P>0.05).FCM Annexin V/PI staining result showed that early period and advanced stage apoptosis rate was lower when predisposed than that of damaged group,and the effection was dose dependent in pariphyllin groups. FCM PI staining showed that cells of G0/G1 stage were more in cell cycle in oxidation damage group,but less in S stage and hypodiploid peak could observe ,The LCM showed that there appeared apoptosis body in damage group, Furthermore we studied H₂O₂ mediated Ca²⁺ responses in ECV304 and found that the increase of Fluo-3 fluorescence intensity in the presence of external Ca²⁺: H₂O₂ elicited an transient peak of [Ca²⁺]i and then fell to a subsequent sustained higher plateau.When pretreatment by the ethanol from bistortae(500pg/ml)and pariphyllin(1ug/ml)for 10 min and which significantly prevented the transient increase and sustained increase of [Ca²⁺]i,RT-PCR showed that the expression of caspase-3 weakened when predisposed by the two kind of medicine than without this disposal(P<0.01);IFM staining result showed that same effection;RT-PCR result showed that the expression of ICAM-1,VCAM-1 and MCP-1 mRNA was significantly descended(P<0.01) in medicine treatment groups than in H₂O₂ damage group, IFM staining showed that MCP-1 had the greatest fluorescence intensity in H₂O₂ damage group,but in all groups pretreatment with medicine it obviously weakened(P<0.01); FCM result discovered that the numbers of masculine cells which express ICAM-1 or VCAM-1 was obviously more than other groups(P<0.01),but pretreated groups the was lower.Conclusion: Ethanol from bistortae and Pariphyllin has the protective action on oxidative damage of ECV304 induced by H₂O₂, the mechanism of protective action may relate to decrease the cell signal transduction mediated of apoptosis,which may be activated by calcium ion mediated signal transduction ; and related the stabilize the cell wall, inhibit the inflammatory reaction induced by ICAM-1,VCAM-1 and MCP-1, and then protect endothelial cell,prevent AS come true.