Experimental Study Of Vitis Quinquangularis Rehd Extract On Antithrombotic Effects And Its Mechanisms

Objective: To investigate the antithrombotic effects and mechanisms of Vitis quinquangularis Rehd extract and provide an experimental evidence for the further evaluation and research of the anti-thrombosis of Vitis quinquangularis Rehd .Methods:(1) Arteriovenous shunt thrombosis model: The rats were randomly divided into five groups:①Control group: with distilled water intragastric infusion;②VRE large dose group (VRE-LD): with VRE intragastric infusion, equivalent to crude drug by 4g/kg dose;③V RE medium dose group (VRE-MD): with VRE intragastric infusion, equivalent to crude drug by 2g/kg dose;④V RE small dose group (VRE-SD): with VRE intragastric infusion, equivalent to crude drug by 1g/kg dose;⑤C DDP group: with CDDP solution intragastric infusion by 67.5mg/kg dose. After giving prophylactic intragastric infusion once a day for ten continuous days, rats were anaesthetized by chloral hydrate(1ml/100g) at 30 minutes after the last administration in each group. The left external jugular vein we separated was ligatured at the distal end and in which a narrow opening was cut. Then the previously prepared polyethylene pipe, which was filled with heparin sodium(50U/ml) and with a 6cm size 4 medical wire in it, was inserted toward the proximal in the narrow opening followed by ligation fixed. At the common carotid artery end, the right common carotid artery we separated was clamped with a arterial clip at the proximal. Then the other side of polyethylene pipe was inserted toward the proximal in the narrow opening which was previously cut in the right common carotid artery followed by ligation fixed. Timing when the right common carotid artery was unclamped and the blood flow keep unobstructed for 15minutes. The medical wire was taken out to be measured the thrombus weight (either wet or dry) , morphology of thrombus was observed by HE staining. At last, 3ml blood was sampled from the abdominal aorta and centrifuged 3000 rpm / min for 15 minutes at 4℃. Then the plasma left was used to determine the content of TXB2 and 6-keto-PGF1α(metabolite of TXA2 and PGI2) by radio immunoassay.(2) Rat model of arterial thrombosis induced by ferric chloride: The rats were randomly divided into six groups:①Sham group: with distilled water intragastric infusion;②C ontrol group: with distilled water intragastric infusion;③V RE large dose group (VRE-LD): with VRE intragastric infusion, equivalent to crude drug by 4g/kg dose;④VRE medium dose group (VRE-MD): with VRE intragastric infusion, equivalent to crude drug by 2g/kg dose;⑤V RE small dose group (VRE-SD): with VRE intragastric infusion, equivalent to crude drug by 1g/kg dose;⑥C DDP group: with CDDP solution intragastric infusion by 67.5mg/kg dose. After giving prophylactic intragastric infusion once a day for ten continuous days, rats were anaesthetized by chloral hydrate(1ml/100g) at 15 minutes after the last administration in each group. Ambilateral common carotid artery was separated and the right common carotid artery was used for sampling blood. A piece of plastic film (4cm×1.2cm) was put under the 1.5cm long part of the separated left common carotid artery. A piece of filter paper(1cm×0.6cm) with the solution 10μl FeCl3 was put below the left common carotid artery for 20min in group 2 to 5. After the piece of filter paper removed, blood was sampled from the right common carotid artery and centrifuged 3000 rpm / min for 15 minutes at 4℃. The plasma left was used to determine the content of TXB2,6-keto-PGF1αand ET-1 by radio immunoassay. Then the thrombotic segement of the vessel was taken and weighted, morphology of thrombus was observed by HE staining.(3)Acute blood stasis model: The rats were randomly divided into four groups:①N ormal group: with distilled water intragastric infusion;②Control group: with distilled water intragastric infusion;③VRE medium dose group (VRE-MD): with VRE intragastric infusion, equivalent to crude drug by 2g/kg dose;④C DDP group: with CDDP solution intragastric infusion by 67.5mg/kg dose. After giving prophylactic intragastric infusion once a day for ten continuous days, rats of all groups except the normal group were conducted subcutaneous injections with 0.08ml epinephrine (1mg/ml) per 0.1kg weight at 60 minutes after the last administration. Rats of group 2 to 4 were put into ice-cold water for 5 min after 2 hours behind subcutaneous injections, and were administered the same dose epinephrine after 2 hours interval. Rats were fasted without water deprivation and 5ml blood was sampled from the abdominal aorta the next day. The anticoagulant agent is ethylenediamine tetraacetic acid disodium salt(EDTA-Na2). Then the blood samples viscosity were detected by Multifunction Intelligent Blood Coagulation Analyzer TYXN-96.Results:(1) Effect on thrombosis in arteriovenous shunt model: It was shown that each VRE group had significantly inhibited thrombosis and reduced the thrombotic weight compared with the control group(P<0.05). The effect of VRE-MD group was especially markedly, better than that of CDDP group too. Meanwhile, it could down regulate the level of TXB2 and TXB2/6-keto-PGF1αand up regulate the level of 6-keto-PGF1αin plasma, and also showed the best performance in VRE-MD group.(2) Effect on thrombosis in rat model of arterial thrombosis induced by ferric chloride: Thrombosis in lumen of blood vessel of each VRE group was reduced apparently compared with the control group. Thrombus of CDDP group was smaller than that of control group, but bigger than that of VRE group. The effect of VRE-MD group was especially markedly. Meanwhile, it could down regulate the level of TXB2,ET-1 and up regulate the level of 6-keto-PGF1αin plasma, and also showed the best performance in VRE-MD group.(3) Effect on viscosity of acute blood stasis model: Compared with the control group, the VRE group could significantly down regulate the whole blood viscosity (P<0.05), effect of which was better than that of CDDP group.Conclusions:1. It is proved that VRE had an effect on anti-thrombosis in arteriovenous shunt model and rat model of arterial thrombosis induced by ferric chloride. The effect of VRE group was better than that of CDDP group.2. By detecting the variation of TXB2,6-keto-PGF1α,ET-1 and the blood viscosity, we found VRE had the function to down regulate the level of TXB2,ET-1 and the blood viscosity and up regulate the level of 6-keto-PGF1α. Its main anti-thrombosis mechanism may be associated with this variation.