| OBJECTIVE:Sodium dehydroacetate(DHA-S)is widely used in the fields of food and feed as a food additive and feed antimold agent.However,the significant prolongation of the formation of thrombin and prothrombin activator in rats were reported at high doses of DHA-S,and there is a tendency to hemorrhage.In the previous study of our group,the main organs of rats and rabbits were found obvious bleeding in different organs,and the level of Vitamin K1 in serum was significantly decreased at 150 mg/kg and 200 mg/kg doses of DHA-S.There is little information about the anticoagulation mechanism induced by sodium dehydroacetate up now.In this study,DHA-S was orally treated to rats with high doses of DHA-S,and the blood coagulation parameters of prothrombin time(PT)and activated partial thromboplastin time.(APTT).DHA-S content in liver,lung and testis,and the mRNA and protein levels of VKORC1 and VKORC1L1 in liver,lung,testis or ovary were detected to study the VKORC1/VKORC1L1 mechanism of anticoagulation induced by DHA-S in rats.METHODS:200 mg/kg DHA-S were given continuously to Wistar rats by lavage.At different administration time of 3,5,7,9,]],14,17 and 21 days,the blood samples were collected to measure PT and APTT,and tissue samples of liver,lung,testis,and ovarian were for concentration detection by HPLC method,or for the expression levels of VKORC1 and VKORCIL1 by immunohistochemistry,Western Blot and qRT-PCR method.The relativity analysis between the values of PT,APTT and the DHA-S content in tissues were mead.The expression difference of VKORC1 and VKORC1L1 in male or female,in different tissues were compared.RESULTS:With the increasing of treatment days,the PT and APTT values were significantly prolonged in the 200 mg/kg DHA-S treatment group(P<0.05),and the PT values of female rats were significantly higher than that of male rats.The PT values of male and female rats in DHA-S treatment were about 1.11～1.48 and 1.12～2.02 times of the control respectively,and the APTT value in male or female were about 1.14～1.37 and 1.20～1.70 times of the control·The DHA-S concentration in rat tissues were detected by HPLC.The DHA-S content in the liver and lung of female rats was significantly higher than that in male rats during DHA-S treatment for 3～9 days(P<0.05).The DHA-S content in the liver and lung of female rats was 1.2 to 3.0 times and 2.5 to 5.5 times that of male rats,respectively.After male rats were 14 or 21 days of continuous administration of DHA-S at 200 mg/kg,different withdraw time was designed.The levels of DHA-S in liver,lung and testis with 3 or 7 days withdraw time were significantly decreased(P<0.05)with the order of DHA-S content of lung>liver>testis.The mRNA expression levels of Vkorcl/Vkorc1l1 in liver,lung,testis and ovary of DHA-S treatment group was significantly decreased by qRT-PCR(P<0.05).After 9 days administration of DHA-S,the Vkorc1 and Vkorc11 mRNA in the liver,lung and testis were respectively 0.69,0.57,0.75 and 0.75,0.48,0.72 times in male rats,and the Vkorcl and Vkorcll mRNA levels in the liver,lung and ovary of female rats were respectively 0.33,0.27,0.55 and 0.62,0.46 and 0.68 times compared to the normal group.The Vkorc1 expression was more inhibited by DHA-S treatment than the vkorc1l1 in female rats.Immunohistochemistry and Western Blot assay showed that the expression of VKORC1 and VKORC1L1 in liver,lung,testis and ovarian tissues of DHA-S treatment group was significantly lower than that of normal control group(P<0.05).After 200 mg/kg DHA-S administration for 11 days,the positive expression of VKORC1 and VKORC1L1 protein in each tissue were significantly reduced in immunohistochemistry.The expression levels of VKORC1 and VKORC1L1 in tissues detected by Western Blot were significantly lower than that in normal controls(P<0.05).After treatment of DHA-S for 9 days,the protein expression levels of VKORC1/VKORC1L1 in the liver,lung and testis of male rats were 0.15/0.33,0.64/0.51,0.34/0.23 times of the control group respectively;the VKORC1/VKORC1L1 level in the liver,lung and ovary of female rats were 0.12/0.56,0.23/0.30,0.42/0.13 times of the control group,respectively.DHA-S inhibited the protein expression of VKORC1 more strongly than VKORC1L1 in the liver.Correlation analysis showed that the correlation coefficient between DHA-S content in the liver of female rats and its PT and APTT values were higher than 0.795,which appeared a good correlation.The correlation coefficient between the protein expression of VKORC1 and PT,APTT in the liver of male and female rats were at 0.74-0.89 range.Conclusion:The prolongation of blood coagulation indexes PT and APTT of rats were found at 200 mg/kg DHA-S administration,indicting there may be a tendency to hemorrhage.The concentration of DHA-S in the liver and lung of female rats was significantly higher than that of males.A good correlation with PT,APTT of female rats and its content of DHA-S in liver were found.The mechanism VKORC1 and VKORC1L1 inhibition was involved in the anticoagulant effect induced by DHA-S in rat,in which the VKORC1 mechanism may play a more important role. |
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