The VKOR Mechanism Of Anticoagulation In Rats Induced By Sodium Dehydroacetate

Objective:Sodium dehydroacetate(DHA-S) is widely used a food preservative and feed additives, which have obtained an excellent preservative effect. DHA-S has similar structure of dicoumarin as warfarin which have effect on anticoagulation through the inhibition of VK. In our previous investigation on toxicology of DHA-S as a feed fungicide, the anticoagulation effects similar as reported on rats were found, which obvious internal hemorrhage in liver, lung, kidney and spleen. This paper will study the mechanism of anticoagulation in rats induced by DHA-S by detecting PT and APTT coagulated indicators, the VK level in serum, the expression of Vitamin ring oxidase(VKOR) and its mRNA level in liver, the level of prothrombin precursor protein PIVKA-Ⅱ, and the effect of VKOR enzymatic activity in vitro.Methods:Wistar rats were fed with DHA-S by gastric infusion at doses of 50mg/kg, 100mg/kg,150mg/kg and 200mg/kg respectively for 14 d. Negative group was fed with normal saline. Each group had 48 rats with equal male and female. The samples of serum and liver of rats were collected at 5 d,7 d,9 d,11 d,13 d after feeding DHA-S with two male and female each group. The VK intervened treatment were designed:vitamin Ki was intraperitoneal injected at 0.1 mL/100 g b.w. when DHA-S was given at 14 and 16 days. The serum and liver were also collected after 24 h and 48 h of VK treated. The ELISA methods were used to detect the level of vitamin K and PIVKA-Ⅱ. Western Blot and RT-PCR were used to detect protein and mRNA levels of VKORC1 in liver. Furthermore, rat hepatic microsomal enzyme was used to the test of enzymatic reaction which DHA-S was added at the concentration of 2 mmol/L,5 mmol/L,10 mmol/L,50 mmol/L respectively in vitro. The enzymatic activity ratio was used to evaluate the effect of DHA-S on VKOR enzymatic activity of liver microsome through detecting OD560nm.Result:The value of PT appeared significantly elevated at 2～3 times to the normal rats during 5 d to 13 d after the rats were fed DHA-S at the dosage of 150 mg/kg and 200 mg/kg, and there were no significant differences between the two groups.The value of APTT also elevated significantly at 1～3 times compared with the normal control group during 5 d to 13 d given 150 mg/kg and 200 mg/kg DHA-S. The APTT in 200 mg/kg group was significantly higher thanthat of 150 mg/kg group. The elevated PT and APTT induced by DHA-S can be decreased by injection of VK significantly.Results of VK ELISA analysis shown that the serum VK of rats fed 50～200 mg/kg DHA-S were decreased at different degree. The VK level of 150 mg/kg and 200 mg/kg groups were significantly lower than that of control during 5-15 d feed. The seurm VK level was significantly increased after injection of VK for 24 h.In the test of VKOR enzymatic activity, the 100 μmol/L VKO was selected as the suited oconcentration in the enzymatic reaction system. Result showed that the activity of VKOR treated with DHA-S in 5～50 mmol/mL decreased significantly, that of 50 mmol/mL DHA-S group was only half of of control. The fitting curve of DHA-S on the enzymatic activity of VKOR was y=0.00083x2+0.9923, the coefficient of correlation R2=0.98502, and the IC50 was 11.67mmol/L.The positive VKORC1 immunohistochemical expression was mainly located in the cytoplasm of liver. The purple blue positive cells of live treated by different doses of DHA-S were decreased clearly. The protein expressions of VKORC1 measured by Western Blot in rats treated with DHA-S were decreased significantly compared to normal control, that in female more lower than that of male. The protein expressions level of VKORC 1 in the treatment group were decreased gradually with the DHA-S feed time prolonged, and were significantly lower in 150 mg/kg and 200 mg/kg group (P< 0.01) than those in control group. The lower protein level of VKORC 1 induced by DHA-S were increased at the administion of VK injection.The mRNA level of VKORC 1 measured by RT-PCR in rats showed that mRNA expressions level of VKORC 1 were significantly decreased in both male and female treatment group than those in control group, and were lower in male group than in female group treated by DHA-S. The mRNA of VKORC 1 were decreased gradually in the treatment group with the time prolonged, and were significantly lower in 150 mg/kg and 200 mg/kg group (P< 0.01) than those in control group. The lower mRNA level of VKORC1 induced by DHA-S were increased at the administion of VK injection.Results of ELISA analysis for PIVKA-Ⅱ showed that the PIVKA-Ⅱ were increased in DHA-S treated groups. The content of PIVKA-Ⅱ at 13 d administration of DHA-S were significantly increased compared to control group. After treatment with VK for 24 h and 48 h, the increased PIVKA-Ⅱ level induced by DHA-S were decreased at the administion of VK injection.Conclusion:The rats treated with 150 mg/kg～200 mg/kg DHA-S appeared hemorrhagic tendency with PT and APTT lengthen. The mechanism of anticoagulant effect of DHA-S on rats was related to the inhibition on expression of VKOR and its enzymatic activity, therefore the VK cycle was inhibited the VK level decrease and PIVKA-Ⅱ increase.