The Mechanism Of Action Of The Anti-thrombosis Effect Of Total Flavones Of Hippophae Rhamnoides

Aim:To observe the impact of oral administration of total flavones of Hippophae Rhamnoides (TFH) on photochemical reaction model of thrombosis, investigate the mechanism of action of the anti-thrombosis effect of TFH and initially analyze the active anti-thrombosis ingredients of TFH.Methods:The impact of oral administration of total flavones of Hippophae hamnoides (TFH) on thrombosis was studied in a photochemical reaction model. The fully off-time of blood flow and the different blood flow velocities under the irradiation of 490nm green light of target blood vessels were calculated. The platelet aggregation activity of mice was studied by whole blood hemacytometry and the serum content of von Wilebrand Factor v (vWF) was measured by ELISA. Hydrogen dioxide (H2O2) was used to prepare the vascular endothelial cell injury model in ECV304 human umbilical vein endothelial cells (HUVEC). The relative vigor of endothelial cell was measured by MTT. The necrosis rate of endothelial cell and changes of the mean fluorecence intensity was measured by flow cytometry (FCM). The contents of t-PA, PAI-1, vWF, and TM of cell culture solution were measure by ELISA and the content of LDH was measured by pectrofluorometry. Intracellular free calcium concentration was measured by confocal laser-scan microscopy. The expression level of PAI-1 mRNA was detected by RT-PCR and the activity of caspase-3 in endothelial cell was detected by Western Blot.Results: The fully off-time of blood flow was prolonged to 41.0±2.1, 50.5±1.6 and 56.0±2.1min under the effect of 1.0, 3.0 and 10.0g/kg TFH respectively which showing a dose-dependent tendency (P<0.01); TFH significantly accelerated the velocity of blood flow in mice and the action time prolonged with the increase of dosage and significantly increased the PLT count in mice in all dosage group; Middle and high dose of TFH significantly decreased serum concentration of vWF in mice (P<0.05 or P<0.01) while low dose of TFH showed no obvious influence on it; H2O2 inhibited the vigor of ECV304 cell with a concentration-dependent tendency. 66% ECV304 cell could be inhibited by 200μM H2O2; 400μg/ml and 200μg/ml TFH could enhance the effect of H2O2 on ECV304 cell (P<0.05 and P<0.01 respectively); 11.4μg/ml and 5.7μg/ml isorhamnetin(ISO) could enhance the vigor of endothelial after H2O2 injury, while 22.8μg/ml ISO showed no this effect; The degree of endothelial cell inhibition of 400μg/ml TFH group was obviously lower than the corresponding ISO group; Different concentration of TFH, ISO and quercetin (Que) all cut down the mortality of endothelial cell after H2O2 injury(P<0.05) in which the effects of middle and low concentration of ISO were obviously stronger than that of the high concentration group(P<0.05 or P<0.01); The protective effect of high concentration of Que was obviously stronger than that of the high concentration of TFH while the protective effect of high concentration of ISO was poorer than that of high concentration of TFH. TFH and ISO could both inhibit the secretion of PAI-1 by ECV304 cell which showing a dose-dependent tendency. Compared with the control group, the significant difference was shown in both high concentration and middle concentration group(P<0.01) while not found in low concentration group. The same result was found in PAI-1 mRNA expression; No influence was found about TFH and ISO on the secretion of t-PA and vWF by ECV304 cell; The increase of protein TM in endothelial cell culture solution caused by H2O2 was inhibited by all sorts of concentration of TFH, Que and ISO except 22.8μg/ml ISO. No difference was found in TFH and corresponding concentration of ISO and Que; The leakage of LDH caused by H2O2 was inhibited by TFH, Que and ISO obviously(P<0.01) in which the effect of Que was augmented with the increase of concentration while the effect of high concentration of ISO was obviously poorer than that of the middle and low concentration group(P<0.05); The elevation of intracellular plasma calcium concentration caused by H2O2 was antagonized by TFH and Que , but no this effect was detected in ISO. To the contrary, high concentration of ISO could aggravate calcium overload; The damage of endothelial cell caspase-3 activity caused by H2O2 was inhibited by TFH, Que and ISO(P<0.01) in which the effect of high concentration of ISO was obviously poorer than that of the TFH (P<0.01) and the effect of high concentration of Que was obviously stronger than that of the TFH (P<0.05).Conclusions:(1) Oral administration of TFH has anti-thrombosis effect. (2) The inhibition of PLT activity is one of the mechanisms of its anti-thrombosis effect. (3) The protective effect of TFH on the injury of vascular endothelia caused by H2O2 is one of the mechanisms of its anti-thrombosis effect. (4) TFH can inhibit calcium overload and cut down the expression of Caspase-3, which is one of the mechanisms of its anti-thrombosis effect. (5)ISO and Que are active anti-thrombosis ingredients of TFH. (6) High concentration of ISO may injury endothelial cells. (7) The anti-thrombosis effect of TFH are multi-targetted .