RESUMO
OBJECTIVE To investigate the expression differences of seven cytochrome P450 (CYP) isoforms between L02, LX-2 and HepG2 cells and identify the most suitable cell type for different CYP researches. METHODS L02, LX-2 and HepG2 cells were treated with omeprazole (Ome), dexamethasone(Dex), phenobarbital sodium (Phe), isoniazid (Iso) and rifampicin (Rif) at 5, 10, 20 and 40 μmol • L-1. Cell viability was analyzed by CCK-8 assay. The gene expressions of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 in LX-2 cells were assessed by real-time quantitative PCR (RT-qPCR). The protein expressions of these seven CYP isoforms in L02, LX-2 and HepG2 cells were assessed by Western blotting. Furthermore, CYP3A4 activity in the three types of cells treated with Rif 5, 10, 20 and 40 μmol • L-1 for 24 h was validated by Luciferin-PFBE. RESULTS Cell viabilities of all the three hepatocytes were over 80% when exposed to Ome, Dex, Phe, Iso and Rif (≤40 μmol • L-1) for 24 h. According to the RT-qPCR, Phe could significantly enhance the gene expressions of CYP2B6 (P<0.05) and CYP2D6 (P<0.01) in LX-2 cells. The results of Western blotting exhibited protein expression differences of seven CYP isoforms between L02, LX-2 and HepG2 cells treated with Ome, Dex, Phe, Iso and Rif (≤40 μmol-L-1) for 24 h. CYP2C9 [Integrated absorbance (IA)=1.58±0.07], CYP2C19 (IA= 0.95±0.03) and CYP3A4 (IA=1.29±0.05) had higher expression abundances in L02 cells, CYP2B6 (IA= 1.48±0.01) and CYP2D6 (IA=1.46±0.02) in LX-2 cells, and CYP1A2 (IA=1.62±0.19) and CYP2E1 (IA= 1.49±0.10) in HepG2 cells. Additionally, CYP3A4 activity in L02, LX-2 and HepG2 cells could not be up-regulated by Rif. CONCLUSION The most suitable cell type for different CYP researches is L02 for CYP2C9, CYP2C19 and CYP3A4, LX-2 for CYP2B6 and CYP2D6, HepG2 for CYP1A2 and CYP2E1, respectively.
RESUMO
This study is aimed to investigate the intervention effect and possible mechanism of ophiopogonin D( OPD) in protecting cardiomyocytes against ophiopogonin D'( OPD')-induced injury,and provide reference for further research on toxicity difference of saponins from ophiopogonins. CCK-8 assay was used to evaluate the effect of OPD and OPD' on cell viability. The effect of OPD on OPD'-induced cell apoptosis was measured by flow cytometry. Morphologies of endoplasmic reticulum were observed by endoplasmic reticulum fluorescent probe. PERK,ATF-4,Bip and CHOP mRNA levels were detected by Real-time quantitative polymerase chain reaction( PCR) analysis. ATF-4,phosphorylated PERK and e IF2α protein levels were detected by Western blot assay. RESULTS:: showed that treatment with OPD'( 6 μmol·L-1) significantly increased the rate of apoptosis; expressions of endoplasmic reticulum stress related genes were increased. The morphology of the endoplasmic reticulum was changed. In addition,different concentrations of OPD could partially reverse the myocardial cell injury caused by OPD'. The experimental results showed that OPD'-induced myocardial toxicity may be associated with the endoplasmic reticulum stress,and OPD may modulate the expression of CYP2 J3 to relieve the endoplasmic reticulum stress caused by OPD'.