Effects Of Three Compounds Of Capillaris On The MRNA And Protein Expression Of CYP450s In Vitro

Objective:To investigate the up-regulation of three simple coumarins,scoparone,scopoletin and isoscopoletin,towards CYP1A2,CYP2C9 and CYP3A4mRNA and protein levels in HepG2 and HL7702 cells and analyze the structure-induction relationship between compounds and CYPs.Methods:1 Cultured the human hepatoma c ell HepG2 and human normal liver cells HL7702.2 MTT assay was used to detect cells proliferation activity of scoparone,scopoletin and isoscopoletin at different concentrations after treatment for 24,36and 48h,and calculated IC₅₀0 for screening concentrations.3 The expression of CYP1A2,CYP2C9 and CYP3A4 mRNA in HepG2 and HL7702 was determined by real-time PCR method after 24,36 or 48h incubation at concentrations from 25 and 50?M,respectively.4 CYP1A2,CYP2C9 and CYP3A4 protein levels in HepG2 and HL7702 cells were measured by western blotting assay.Results:1 Compared with the control group,SA,SO and ISO can significantly inhibit the proliferation of HepG2 cells after 24,36 or 48h incubation at concentrations from25-1000?M,and the inhibition showed significative positive correlation with concentration and action time of drugs.The suppression of SO to HepG2 cells was the strongest.At the same conditions,there was no obviously inhibition on the viability of HL7702 by three coumarins at the con centration blew 1mM,and the suppression of SO to HL7702 cells was the weakest.2 Compared with the control group,as demonstrated by real-time PCR method,SA,SO and ISO obviously increased the expression of CYP1A2,CYP2C9 and CYP3A4 mRNA in HepG2 and HL7702 cells after 24,36 and 48h incubation at concentrations from 25 and 50?M,respectively.Three compounds had a more significant induction to HL7702 cells,and the induction towards CYP1A2 and CYP2C9 mRNA decreased with the prolongation of action time,w hereas CYP3A4mRNA levels increase.3 Compared with the control group,as investigated by western boltting assay,SA,SO and ISO obviously induced the expression s of CYP1A2 and CYP2C9 proteins in HepG2 and HL7702 cells after 24h incubations and incrased the protein levels of CYP3A4 in HepG2 and HL7702.The inducing of SA and SO on CYP1A2 protin in HL7702 was stronger than that of HepG2.There is no obviously difference in HepG2 and HL7702 on CYP2C9 protein expression of three drugs.After 48h,the up-inducing of SO on CYP3A4 protein in HepG2 is more obvious than that of SA and ISO.Conclusion:1 SA,SO and ISO have obvious effect on the proliferation of HepG2 cells and has no cytotoxicity to normal liver cells.The strongest inhibitory effect on the growth of HepG2 cells was SA at 24h,and SO at 48h.At the same concentrations,the most toxic to HepG2 was SO.2 The mRNA levels of CYP1A2 and CYP2C9 in HepG2 and HL7702 were induced significantly by SA,SO and ISO at 25?M after 24h,but CYP3A4 mRNA were induced after 48h.The enzyme induction ability of SO was stronger than ISO relatively.3 The protein expression of CYP1A2,CYP2C9 and CYP3A4 in HepG2 and HL7702 were induced by SA,SO and ISO at 25?M.The most induced on CYP3A4protein in HepG2 and HL7702 was SO at 48h.