| Sodium dehydroacetate(Na-DHA)is often used as a preservative in food,feed and cosmetics.But some studies reported that repeated oral administration of Na-DHA induces body weight and food intake of rats decreased,obvious bleeding of multiple organs observed,serum vitamin K(VK)level reduced,plasma prothrombin time(PT)and activated partial thromboplastin time(APTT)prolonged,and vitamin K epoxide reductase complex subunit 1(VKORC1)and VKORC1-like protein 1(VKORC1L1)expressions inhibited.VKORC1 is mainly distributed in the liver,while VKORC1L1 is mainly distributed in extrahepatic tissues.However,the effects of Na-DHA on VK and coagulation factors in rat hepatocytes and the regulatory roles of VKORC1 and VKORC1L1 in them are still unclear.In this study,we constructed buffalo rat liver BRL3A cells stably transfected cell lines overexpressing VKORC1 and VKORC1L1 by lentiviral vectors,respectively,to investigate the effects of Na-DHA on VK and coagulation factor ?(FIX)in rat hepatocytes and the regulation roles of VKORC1 and VKORC1L1 in them in vitro,providing a theoretical basis for the study of the mechanism of coagulation disorders induced by Na-DHAS and also providing a reference for exploring the corresponding drug prevention and treatment.In the selection of lentivirus infection conditions,BRL3A cells were treated with 1.0?5.0 ?g/mL polybrene for 24 h,polybrene above 2.0 ?g/mL had toxic effects on BRL3A cells,so 2.0 ?g/mL polybrene was selected for subsequent lentivirus infection.After BRL3A cells were infected with 0?40 multiplicity of infection(MOI)empty vector lentivirus(LV5-NC)for 96 h,the expression of labelled green fluorescence protein(GFP)was the highest at 30 MOI,so 30 MOI lentivirus was selected for subsequent construction of stably transfected cell lines.In the construction of stably transfected cell lines,BRL3A cells were treated with 0.5?2.5 ?g/mL puromycin for 3 d,respectively.When the concentration was higher than 1.5?g/mL,almost all BRL3A cells died,so 1.5 ?g/mL puromycin was selected for the screening of subsequent stably transfected cell lines.BRL3A cells were infected with lentivirus overexpressing rat Vkorcl and Vkorc1l1(LV5-Vkorcl and LV5-Vkorc1l1)respectively at 30 MOI and 2.0 ?g/mL polybrene for 96 h.After screening with 1.5 ?g/mL puromycin for 7 d,VKORC1 and VKORC1L1 overexpression BRL3A stably transfected cell lines(named BRL3A-VKORC1 and BRL3A-VKORC1L1 cells)were generated,respectively.The expressions of GFP,VKORC1 and VKORC1L1 were detected by fluorescence observation,reverse transcription-PCR(RT-PCR)and Western blot analysis,respectively.The results showed that BRL3A-VKORC1 and BRL3A-VKORC1L1 cells had high GFP expression,and the protein levels of VKORC1 and VKORC1L1 were increased by about 72.8%and 53.2%(P<0.01),respectively,indicating that VKORC1 and VKORC1L1 overexpression BRL3A stably transfected cell lines were successfully constructed.In the MTT and EdU assays to detect the effect of Na-DHA on the growth of BRL3A cells,2.0 and 5.0 mmol/L Na-DHA treatment for 24 h had no significant effect on cell viability and proliferation,but 10.0 mmol/L Na-DHA treatment for 24 h significantly reduced cell viability(P<0.001)and proliferation(P<0.05).Therefore,5.0 mmol/L Na-DHA was selected for subsequent experiments.After were treated with 5.0 mmol/L Na-DHA for 24 h,the mRNA levels of Vkorc1 and Vkorc1k1 in BRL3A cells and Vkorc1L1 in BRL3A-VKORC1L1 cells were not significantly affected,but the mRNA level of Vkorc1 in BRL3A-VKORC1 cells was remarkably reduced(P<0.01),the protein levels of VKORC1 or VKORC1L1 in BRL3A,BRL3A-VKORC1 or BRL3A-VKORC1L1 cells were obviously decreased(P<0.05),and the mRNA and protein levels of VKORC1 or VKORC1L1 in BRL3A-VKORC1 or BRL3A-VKORC1L1 cells were significantly higher than those in BRL3A cells(P<0.01).The VK and FIX contents in cells and medium were detected by enzyme linked immunosorbent assay(ELISA).When treated with PBS,the VK and FIX contents in cells of BRL3A-VKORC1 and BRL3A-VKORC1L1 cells were not significantly different from those of BRL3A cells,but the VK and FIX contents in medium of the two cells were remarkably higher than those of BRL3 A cells(P<0.01).After treatment with 5.0 mmol/L Na-DHA for 24 h,the VK and FIX contents in cells and medium of BRL3A,BRL3A-VKORC1 and BRL3A-VKORC1L1 cells were obviously reduced(P<0.05),the VK and FIX contents in cells of BRL3A-VKORC1 and BRL3A-VKORC1L1 cells were not significantly different from those of BRL3A cells,but the VK and FIX contents in medium of the two cells were remarkably higher than those of BRL3 A cells(P<0.05).In summary,VKORC1 and VKORC1L1 overexpression BRL3A stably transfected cell lines are successfully constructed,which can stably overexpress the mRNA and protein levels of VKORC1 and VKORC1L1,respectively.Na-DHA has no significant effect on the mRNA levels of Vkorcl and Vkorc1ll in BRL3A cells,but remarkably reduces the protein levels of VKORC1 and VKORC1L1,and obviously inhibits the production and secretion of VK and FIX in BRL3A cells.Overexpression of VKORC1 and VKORC1L1 increases the production and secretion of VK and FIX in BRL3A cells,and alleviates the inhibitory effects of Na-DHA on the expressions of VKORC1 and VKORC1L1 and the levels of VK and FIX in BRL3A cells.The results indicate that both VKORC1 and VKORC1L1 play regulatory roles in the effects of Na-DHA on VK and FIX of BRL3A cells,and the regulation of VKORC1 or VKORC1L1 activity is of great significance for alleviating coagulation disorders induced by Na-DHA. |
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