Study On The Screening Of Active Anti-thrombotic Fractions And The Cytoprotection Of Thrombin-induced Injury From Salvia Deserta Schang

Objective:1.To screen the fractions with antithrombotic activity from the various extracts of Salvia deserta Schang.2.To study the protective effect of antithrombotic fractions from Salvia deserta Schang on the HUVECs induced by thrombin.Methods:1.All extracts of Salvia deserta Schang were respectively extracted there times?3h,3h,1h?,with water,methanol,trichloromethane from the aerial parts of Salvia desterta Schang and with vaccum drying,to get SDS-W,SDS-M,SDS-T;other extracts were extracted with ethyl acetate and n-butyl alcohol from aqueous extracts and methanol extracts and with vaccum drying,to get SDS-WE,SDS-WN,SDS-WH,SDS-ME,SDS-MN,SDS-MH.120 Male SD rats were randomly assigned into 12 groups,there are:sham group,model group,positive drug group,SDS-W group,SDS-WE group,SDS-WN group,SDS-WH group,SDS-M group,SDS-ME group,SDS-MN group,SDS-MH group,SDS-T group.All rats were given by gavage once a day for 20 days with the extracts of the Salvia deserta Schang,and were fed with standard diet during the administration.All groups were narcotized in 1 h after the last administration,and were induced with FeCl₃by the left common carotid artery thrombosis model in rats,and then the vessels were clipped and done hematoxylin-easin staining to product paraffin sections,and to observe changes of thrombosis in all rats;blood was collected from the abdominal aorta,and the thromboxane B₂?TXB₂?,6-keto prostaglandin F₁??6-Keto-PGF₁??,ET-1 content in plasma were detected by radioimmuno assay.The v WF content were detected using double antibody sandwich ABC-ELISA assay in order to observe the effects of drugs for thrombosis.2.The HUVECs were cultured 3th to 5th generation,and seeded in 6-cell culture plates.The cells were divided into 8 groups:normal group,model group,SDS-W low dose group,SDS-W Middle dose group,SDS-W high dose group,SDS-MH low dose group,SDS-MH low dose group and SDS-MH high dose group.SDS-W group was administered by 0.7?g/mL,7?g/m L,70?g/m L with SDS-W for 24h after serum starvation with 12h,and administered by thrombin?final concentration of 60U/m L?for 24h;SDS-MH group was administered by 1.2?g/m L,12?g/m L and 120?g/m L with SDS-MH for 24h after serum starvation with 12h,and administered by thrombin?final concentration of 60U/mL?for 24h,collect all cells.The levels of PECAM-1,TLR4,TF and GP?b/?a were examined by QRT-PCR and WB.To observe the effect of active fractions from Salvia deserta Schang on the HUVECs.Results:1.Compared with the model group,the thrombus weight of rats were decreased in SDS-W group and SDS-MH group with statistical significance?P<0.01?,and which were decreased in SDS group,SDS-MN group with statistical significance?P<0.05?,the SDS-WN group and SDS-T group increased the thrombus weight in rats with statistical significance?P<0.01?;the levels of TXB₂ and the ratio of TXB₂/6-keto-PGF_1?decreased in SDS-W group and SDS-MH group,which increased the level of 6-keto-PGF_1?with statistical significance?P<0.01?,SDS-WE group,SDS-WN group,SDS-WH group,SDS-M group,SDS-ME group,SDS-MN group,SDS-T group reduces the levels of TXB₂ and TXB₂/6-keto-PGF_1?,they increased the level of6-keto-PGF_1?with statistical significance?P<0.05?;SDS-W group,SDS-MH group could decrease the level of ET-1 in plasma with statistical significance?P<0.01?,SDS-WE group,SDS-WN group,SDS-WH group,SDS-M group,SDS-ME group,SDS-MN group,SDS-T group decreased the level of ET-1 with statistical significance?P<0.05?;SDS-W group,SDS-MH group can significantly decreased the vWF level of in plasma with statistical significance?P<0.01?,SDS-WE group,SDS-WN group,SDS-WH group,SDS-M group,SDS-ME group,SDS-MN group,SDS-T group could increase the level of vWF with statistical significance?P<0.01?.2.The best time condition of such conditions of drugs is 24h?serum starvation with 12h?.The concentration of SDS-W respectively are:0.7?g/mL,7?g/mL,70?g/m L;the concentration of SDS-MH respectively are:1.2?g/mL,12?g/mL,120?g/mL,The best time condition of such conditions of thrombin is24h?serum starvation with 12h?,the final concentration is 60U/m L.1)The result of PCR:the level of GP?b/?a in HUVECs were significantly down-regulated in the SDS-W groups?the concentrations of drugs:7?g/mL,70?g/mL?and the SDS-MH groups?the concentrations of drugs:12?g/mL,120?g/m L?with statistical significance?P<0.05?;the level of TF in HUVECs were significantly down-regulated in the SDS-W groups?the concentrations of drugs:0.7?g/mL,7?g/mL,70?g/mL?and the SDS-MH groups?the concentrations of drugs:1.2?g/mL,120?g/m L?with statistical significance?P<0.05?;the level of P-selectin in HUVECs were significantly down-regulated in the SDS-W groups?the concentrations of drugs:0.7?g/m L,7?g/mL,70?g/mL?and the SDS-MH groups?the concentrations of drugs:1.2?g/mL,12?g/mL,120?g/mL?with statistical significance?P<0.05?;the level of PECAM-1 in HUVECs were significantly down-regulated in SDS-W groups?the concentrations of drugs:0.7?g/mL,7?g/m L,70?g/mL?and the SDS-MH groups?the concentrations of drugs:1.2?g/mL,12?g/m L,120?g/mL?with statistical significance?P<0.05?;the level of TLR4 in HUVECs was significantly down-regulated in SDS-W groups?0.7?g/m L,7?g/mL,70?g/m L?and the SDS-MH groups?the concentrations of drugs:1.2?g/mL,12?g/m L,120?g/m L?with statistical significance?P<0.05?.2)The results of Western Bloting:the protein expression level of P-selectin in HUVECs were significantly down-regulated in the SDS-W groups?the concentrations of drugs:0.7?g/mL,7?g/m L?and the SDS-MH groups?the concentrations of drugs:12?g/m L,120?g/mL?with statistical significance?P<0.05?;the protein expression level of PECAM-1 in HUVECs were significantly down-regulated in SDS-W groups?the concentrations of drugs:0.7?g/m L,7?g/m L,70?g/mL?and the SDS-MH groups?the concentrations of drugs:1.2?g/mL,12?g/m L,120?g/m L?with statistical significance?P<0.05?;the protein expression level of TLR4 in HUVECs was significantly down-regulated in SDS-W groups?0.7?g/m L,7?g/mL,70?g/mL?and the SDS-MH groups?the concentrations of drugs:1.2?g/mL,12?g/m L,120?g/m L?with statistical significance?P<0.05?.the protein expression level of GP?b/?a in HUVECs was significantly down-regulated in SDS-W groups?0.7?g/m L,7?g/m L,70?g/mL?and the SDS-MH groups?the concentrations of drugs:12?g/mL,120?g/mL?with statistical significance?P<0.05?.the protein expression level of TF in HUVECs was significantly down-regulated in SDS-W groups?0.7?g/m L,7?g/mL,70?g/mL?and the SDS-MH groups?the concentrations of drugs:12?g/m L,120?g/mL?with statistical significance?P<0.05?.Conclusion:1.The SDS-W and SDS-MH of extracts from Salvia deserta Schang have an antithrombotic effect.2.The SDS-MH fraction and the SDS-MH fraction had protective effects on thrombin-induced human umbilical vein endothelial cells,it was related to the effect of preventing platelet aggregation.