Effect Of Saikosaponin-d On CYP450 In HepaRG Cell

Objective:To investigate the effect of Saikosaponin d(SSd)on some subtypes of CYP450 enzymes in Hepa RG cells,CYP3A4,CYP1A2 and CYP2D6,providing experimental evidence and theoretical reference for the study of Bupleurum and formulations containing SSd or Bupleurum,mechanism research of drug metabolism and related drug interactions and the rational combined use of related drugs in clinical practice.Methods:(1)MTT was used to investigate the inhibitory effect of SSd on the proliferation of Hepa RG cells;(2)Hepa RG cells were cultured with SSd at concentrations of 0.5,1,5 and 10μM for 72 hours,and the m RNA and protein expression of CYP3A4,CYP1A2 and CYP2D6 were analyzed with real-time quantitative reverse transcription polymerase chain reaction(RT-q PCR)and Western blot(WB)analysis,and relative enzyme activities were analyzed with HPLC based on consumption of the specific probe substrate;(3)The binding between SSd and CYP3A4,CYP1A2 and CYP2D6 proteins was analysed by molecular docking;(4)The surface morphology and internal structure of self-assembling peptide RADA16-I hydrogel three-dimensional(3D)scaffolds were investigated by inverted microscope and scanning electron microscope(SEM),respectively,and the formation of hydrogel and the network structure in self-assembling peptide RADA16-I hydrogel were also observed;(5)The growth of Hepa RG cells in 3D scaffolds was investigated by inverted microscope,calcein AM/PI staining and SEM method,and the two-dimensional(2D)cell culture was used as the control group;(6)The effects of SSd on m RNA and protein expressions of CYP3A4,CYP1A2 and CYP2D6 and the enzyme activities in Hepa RG cells cultured in 3D and 2D environments were compared via RT-q PCR,WB and HPLC method,respectively.Results:(1)The results of in vitro cell viability assay indicated that the Hepa RG cell viabilities at doses of 0.1,1,5 and 10 μmol/L in all SSd treatment groups were not significantly different from the control,suggesting that the concentration of 0.1,1,5and 10 μmol/L can be used in the subsequent drug interaction studies;(2)RT-q PCR results demonstrated that SSd can induce the m RNA expression of CYP2D6 and CYP1A2 in Hepa RG cells,while exerting a certain inhibitory effect on CYP3A4.The results of WB showed similar trend with that in RT-q PCR experiment that SSd can induce protein expression of CYP1A2 and CYP2D6 in Hepa RG cells in a concentration-dependent manner,while showing a slight inhibition effects on CYP3A4.The results of the probe drug method showed that SSd induced relative activity of CYP2D6 in Hepa RG cells at concertrations of 5 and 10μmol/L,while inhibiting that of CYP3A4.In addition,SSd can significantly upregulate the enzyme activity of CYP1A2 at concertrations of 1,5 and 10 μmol/L;(3)Molecular docking studies illustrated that hydrogen and hydrophobic bonds play a dominant role in the binding of SSd to amino acid residues of several key active sites of CYP1A2,CYP2D6 and CYP3A4 proteins,and their binding energies were-11.3 kcal/mol,-8.4 kcal/mol and-8.5 kcal/mol,respectively;(4)SEM showed that the self-assembling peptide RADA16-I hydrogel can form a three-dimensional network structure,which can be used for Hepa RG cells adhesion and growth;(5)The inverted microscope and SEM showed that the cells can adhere to the wall and grow in a flat shape in 2D culture.While,it was observed that the Hepa RG cells adhered to the peptide hydrogel scaffold and grow in stereo in 3D culture being constructed with self-assembling peptide RADA16-I hydrogel.Calcein AM/PI staining revealed that Hepa RG cells cultured in 2D and 3D all showed good viabilities;(6)In the 3D culture system of Hepa RG cells,the m RNA and protein expression levels and relative enzyme activities of CYP1A2 in the control,omeprazole group and SSd group were all higher than those in 2D group.Conclusion:The results demonstrated for the first time that SSd can induce CYP1A2 and CYP2D6,and inhibit CYP3A4 in Hepa RG cells.SSd may bind to CYP3A4,CYP1A2 and CYP2D6 proteins with hydrogen bonds and hydrophobic interactions.It is suggested that more special attention should be paid to avoid the drug-drug interactions or to take advantage of the drug interactions to seek beneficial effects and avoid harmful adverse drug reactions when formulations containing SSd or Bupleurum were co-administrated with drugs metabolized by CYP3A4,CYP1A2 and CYP2D6 in clinical practice.The in vitro 3D culture model for Hepa RG cells was successfully constructed by taking hydrogels of the self-assembling peptide RADA16-I as the cell culture scaffold.This novel model can be expected to provide a new research tool for in vitro drug metabolism and drug interaction.