Study On The Mechanism Of Coagulation Induced By Sodium Dehydroacetate Via Regulating MTOR And ERK1/2 In Rats

Sodium dehydroacetate(Na-DHA)has good anti-bacterial and fungal effects,and is widely used in beverages,food feed and cosmetics.But studies showed that high-dose Na-DHA can lead to significant food intake decrease,weight loss,prothrombin time(PT)and activated partial prothrombin time(APTT)prolongation,and severe and extensive bleeding in the internal organs of the rats.Previously,we observed that rabbits and rats were intragastrically treated with 150 mg/kg and 200 mg/kg b.w.Na-DHA that caused obvious bleeding symptoms,and also significantly reduced Vitamin K(VK)content in the serum,vitamin K2,3-epoxide reductase complex subunit 1(VKORC1)and vitamin K2,3-epoxide reductase complex subunit 1-like protein 1(VKORC1L1)expression level in the liver of rats.Recent study had showed that ERK1/2 and mTOR regulated the expression of VKORC1 in human liver cancer cells.However,the effects of Na-DHA on the expression of ERK1/2 and mTOR,and the roles of mTOR and ERK1/2 in Na-DHA-induced coagulation dysfunction is still unclear.The present research investigated whether and how Na-DHA regulates ERK and mTOR signals in rats and BRL3 A rat liver cell line,in order to further explore the mechanism of Na-DHA-caused coagulation dysfunction.Wistar rats were intragastrically treated with 200 mg/kg b.w.Na-DHA for 3～9 days.Blood was collected to determine PT and APTT.The protein expression levels of VKORC1,VKORC1L1,ERK1/2,p-ERK1/2,mTOR and p-mTOR were detected by using Western blot in rat liver tissues.The results showed that after administrated intragastrically with 200 mg/kg Na-DHA for 9 days,PT and APTT were significantly prolonged(P<0.05),and the values in female rats were significantly higher than that in male rats;the proteins expression levels of VKORC1 and VKORC1L1 were decreased,and p-ERK1/2,mTOR and p-mTOR expression levels were increased significantly(P<0.05)in liver tissues.BRL3A rat liver cell line were treated with 0.1,0.2,0.5,1.0,2.0,5.0 and 10.0 mmol/L Na-DHA for 24 h and 48 h,cell viability was detected by MTT method;after treatment with 0.2,0.5,1.0,2.0,5.0 and 10.0 mmol/L Na-DHA for 10 hours,the content of VK and coagulation factor FIX in the cells and culture medium were detected by enzyme linked immunosorbent assay(ELISA);cells were treated with 0.2,0.5,1.0,2.0 and 5.0 mmol/L Na-DHA for 10 h and 24 h,PCR and Western blot methods were used to detect the mRNA and proteins levels of VKORC1 and VKORC1L1,ERK1/2,p-ERK1/2,mTOR and p-mTOR expression levels were detected by Western blot method.The data showed that cell viability was significantly inhibited after treatment with 5.0 mmol/L Na-DHA for 48 h or 10.0 mmol/L Na-DHA for 24 and 48 h(P<0.05).After treatment for 10 h,2.0,5.0,10.0 mmol/L Na-DHA significantly decreased the VK content in cells(P<0.05),while 10.0 mmol/L Na-DHA decreased the coagulation factor FIX content in cells and medium(P<0.05).After treatment with 0.2～5.0 mmol/L Na-DHA for 10 h and 24 h,the mRNA expression levels of VKORC1 and VKORC1L1 did not change significantly,but the proteins expression levels of VKORC1 and VKORC1L1 were significantly reduced(P<0.05).At the same time,the protein expression levels of p-ERK1/2,mTOR and p-mTOR were increased significantly(P<0.05).BRL3A rat liver cell line were treated with Na-DHA and/or rapamycin(RP)for 24 h,then cell viability was detected by using MTT method;Compared with the control group,the cell viability of 50 nmol/L RP treatment group and the 1.0 mmol/L Na-DHA+50 nmol/L RP co-treatment group were decreased,while the other groups did not change significantly.In addition,after cells were treated with 25 nmol/L RP,1.0 mmol/L Na-DHA,1.0 mmol/L Na-DHA+25 nmol/L RP for 10 h,the proteins expression levels of mTOR,p-mTOR,ERK1/2,p-ERK1/2,VKORC1 and VKORC1L1 were detected by Western blot.Compared with the control group,1.0 mmol/L Na-DHA increased the proteins expression levels of p-ERK1/2,mTOR and p-mTOR significantly(P<0.05),decreased the proteins expression levels of VKORC1 and VKORC1L1 significantly(P<0.05);the proteins expression levels of mTOR and p-mTOR in the 25 nmol/L RP group,and 1.0 mmol/L+25 nmol/L RP group were significantly decreased(P<0.05);The proteins expression levels of ERK1/2 and p-ERK1/2 in the 25 nmol/L RP group were significantly decreased(P<0.05).Compared with the 1.0 mmol/L Na-DHA treatment group,the proteins expression of mTOR,p-mTOR and p-ERK1/2 was significantly decreased,while VKORC1 and VKORC1L1 expression was significantly increased in the co-treatment group(P<0.05).This study shows that Na-DHA can significantly prolong PT and APTT in rats,and female rats are more sensitive than males.Na-DHA decreases the content of VK and FIX in BRL3A rat liver cell line.Na-DHA inhibits VKORC1 and VKORC1L1 protein expression,while increases p-ERK1/2,mTOR and p-mTOR protein levels in in vitro and in vivo.Co-treatment with mTOR inhibitor RP can attenuates the inhibitory effects of Na-DHA on the protein expression of VKORC1 and VKORC1L1 in BRL3A cell line.These data demonstrate that Na-DHA induces ERK1/2 and mTOR signals in rat liver cell line,and these effects paly a critical role in Na-DHA-caused coagulation dysfunction.