| Objective:The main purpose of this thesis is to investigate the effects of the water extract of Polygonum multiflorum(WE)and its main components on mRNA expressions of cytochrome P450 enzyme(CYP450)such as CYP1A2,CYP2C9 and CYP2E1 in L02 cells,and declare the effects of CYP2E1 gene expression and CYP2E1 activity on L02 cell viability treated with WE.Meaning:Clear the effects of WE and its main components on mRNA expressions of CYP450s will provide reference data for the safety of its clinical application,and in addition,we build up a L02 cell model which mRNA expression of CYP2E1 is inhibited by RNA interference technology,provides a platform for the study on the correlation between the cytotoxicity of traditional Chinese medicine and the gene expression of CYP450s.There we discussed the correlation between the hepatotoxicity of Polygonum multiflorum and the down regulation of mRNA expression and enzyme activity of CYP2E1.Methods:First,MTT assay is used to determine the non-toxic dose of WE and its main components,than extract RNAs of each groups at the doses above,detect the mRNA expressions of CYP1A2,CYP2C9 and CYP2E1 by qRT-PCR(Real time quantitative reverse transcription PCR).On the other hand,interference the mRNA expression of CYP2E1 at the efficient sequence site,use transfection technology build up NC cells(transfected vector of nonsense)and SH cells(interferenced the mRNA expression of CYP2E1),qRT-PCR is used to determine the mRNA expression of CYP2E1 in the model cells,and MTT assay is used to detect the cell viability in NC,SH and normal L02 cells treated with WE and its main components.Moreover,detect the L02 cell viability treated with diethyl dithiocarbamate(DDTC,chemical inhibitors of CYP2E1 activity in vitro)and WE.Results:WE(crude)4 mg/mL,stilbene glycoside 400 μg/mL,emodin-8-O-βD-glucopyranoside 100 μg/mL,emodin and physcion 10 μg/mL show none toxicity to L02 cells.WE 4 mg/mL(crude)can reduce the mRNA expressions of CYP1A2,CYP2C9 and CYP2E1.Stilbene glycoside can reduced the mRNA expression CYP1A2,but it is the only ingredient to increase the mRNA expression of CYP2C9.Emodin-8O-β-D-glucopyranoside can reduce the mRNA expressions of CYP1A2 and CYP2C9.Emodin and physcion can inhibit the expression of these three genes,but physcion 10μg/mL can increase the mRNA expression of CYP2E1.The mRNA expression of CYP2E1 in SH cells,reduced about 70%(p<0.05)with respect to the NC cells,and we found that WE 2 mg/mL(crude)and emodin 10 μg/mL show lower toxicity to SH cells than the normal cells and NC cells,but stilbene glucoside,emodin-8-O-β-D-glucopyranoside and physcion show none significantly different in the three cells.However,ours another work shows that DDTC can enhance the toxicity to L02 cells treated with WE.Conclusion:WE can cause the reduction of mRNA expressions of CYP1A2,CYP2C9 and CYP2E1.Emodin-8-O-β-D-glucopyranoside,emodin and physcion are the main ingredients to reduce the mRNA expressions of CYP1A2 and CYP2C9,emodin and physcion can reduce the mRNA expression of CYP2E1,but high dose physcion can increase the expression.Stilbene glucoside can reduce the mRNA expression of CYP1A2 and increase the mRNA expression of CYP2C9,attentions should be paid to avoid the adverse reactions between Polygonum multiflorum and other drugs in terms of mRNA expressions of CYP450s.Our studies show that when interference the mRNA expression of CYP2E1,WE and emodin’s hepatotoxicity to L02 cells is decreased,stilbene glycosides,emodin-8O-β-D-glucopyranoside and physcion show no changed in this case,but the hepatotoxicity to L02 cells of WE will significantly enhanced,when used DDTC to inhibit the enzyme activity of CYP2E1.This shows that to prove the correlation between gene expressions or activity of CYP450s and drug’s toxicity requires integration of a wide range of experimental data. |
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