Study On The Material Basis Of Liver Injury Induced By Radix Polygoni Multiflori And Its Effect On The Expression Of CYP1A1 And CYP3A4

Objective The traditional Chinese medicine Polygonum multiflorum has the functions of Tonifying the liver and kidney,tonifying the muscles and bones,and tonifying the hair of the black hair.But in recent years,the practice of liver injury caused by Polygonum multiflorum has attracted extensive attention in the medical field.In this experiment,two highly toxic parts of Shouwu are screened by in vitro cells,and the main components contained in it are found by HPLC analysis;The target components are evaluated in vivo and in vitro.On this basis,RT-PCR and protein immuno-imprinting techniques are used to explore its toxicity mechanism,in order to provide a basis for the research on the clinical application of Polygonum multiflorum.Method 1.Taking the production of Radix Polygoni multiflorum as the research object,extracting different polar extracts of Radix Polygoni multiflori by systematic solvent method,and using CCK-8 method to screen out the two highly toxic parts of Polygonum multiflorum,namely dichloromethane extract and ethyl acetate extract.The main components in these two parts are detected by HPLC to determine the components that may cause liver damage.2.Observing the effect of different concentrations of emodin,rhein,and gallic acid on HepG2 cell morphology through an inverted microscope;The apoptosis of Hep G2 cells induced by different concentrations of emodin,rhein and gallic acid is determined by CCK-8 method and Annexin V-FITC / PI double staining method;The expression of CYP1A1,CYP3A4 and Caspase-3 mRNA in the cells of each group is detected by RT-PCR technology;And the expression of CYP1A1,CYP3A4 and cleaved Caspase-3 protein is detected by WB to study the mechanism of the apoptosis of HepG2 cells induced by different components of Shouwu.3.Sixty KM mice are randomly divided into 6 groups: blank group(0g /(kg·d)),raw Shouwu group(8g /(kg·d)),Shouwu group(8g /(kg·d)),emodin group(37.8mg /(kg·d)),rhein group(312?g /(kg·d)),gallic acid group(4.58 g /(kg·d))(40 times the maximum amount used in adults),10 mice in each group,and the rats are fed for 4 weeks Serum testing for changes in blood biochemical indicators;Liver tissue is stained with HE to observe the pathological changes of liver tissue;The expression of CYP3A4 and Caspase-3 mRNA is aim to explore the toxicity mechanism of Polygonum multiflorum caused liver injury;WB is used to determine the effects of different components of Polygonum multiflorum on the expression of drug metabolism enzymes CYP1A1,CYP3A4 and cleaved Caspase-3 in liver tissue.Result 1.The dichloromethane extract and ethyl acetate extract of the four polar extracts of Polygonum multiflorum are highly cytotoxic;HPLC test results show that the dichloromethane extract mainly contains: emodin and stilbene;The ethyl acetate extract mainly contains: gallic acid,rhein,emodin.Among them,the different concentrations of emodin,rhein,and gallic acid all can inhibite the cells,but the inhibition of stilbene glycosides is not obvious.2.The results of CCK-8 and cell flow cytometry show that the administration group of emodin,rhein,and gallic acid have different degrees of damage to Hep G2 cells,and obvious cytotoxic effects occurre at high concentrations(P <0.01).As the concentration of each component administration group increased,the expression of Caspase-3 mRNA and cleaved Caspase-3 protein also increase significantly,suggesting that the cells had abnormal apoptosis.Emodin,rhein,and gallic acid dose-dependently induce CYP1A1,CYP3A4 mRNA and protein expression;3.In vivo experiment,raw Shouwu,Shouwu,emodin,rhein,gallic acid after gavage of the mice for 4 weeks,serum ALT,AST,ALP levels of Shouwu mice are prepared Compared with the blank group,there is no significant difference,indicating that the liver cells of the mice are not damaged;the serum AST and ALP levels of the Shouwuwu,emodin,rhein,and gallic acid groups are significantly increased(P <0.05),indicating that the rat liver the cell membrane is destroyed in large quantities,and the liver function show obvious abnormalities;Liver histopathological results show that the liver cells of Shengshouwu group,rhein group,emodin group and gallic acid group show different degrees of loosening and light staining,and focal necrosis is seen in the portal area.Gallic acid> rhein group> emodin group> Shengshouwu group> Shouwu group;Shengshouwu group,rhein and gallic acid group significantly increase Caspase-3 mRNA and protein expression in liver tissue of mice,among which Shengshouwu.The group and gallic acid group increase significantly(P<0.01),suggesting that hepatocytes begin to undergo apoptosis.The drug metabolic enzymes CYP1A1 and CYP3A4 protein expression of each administration group are up-regulated to varying degrees.The Shengshouwu group and gallic acid group could significantly induce the expression of CYP1A1 and CYP3A4 protein(P <0.01),and the difference has significant statistical significance.Conclusion 1.The dichloromethane extraction site and ethyl acetate extraction site of Radix Polygoni multiflorum are the main sites of liver injury.Dichloromethane extract mainly contains stilbene glycoside and emodin;ethyl acetate extract mainly contains gallic acid,rhein and emodin.2.Emodin,rhein,and gallic acid can inhibit the survival of HepG2 cells and induce apoptosis in a dose-dependent manner.Stilbene glycoside do not significantly inhibit cells at high concentrations.3.Emodin,rhein,and gallic acid can induce the expression of CYP1A1 and CYP3A4 enzymes in Hep G2 cells,which in turn increases the biotransformation rate of the corresponding enzyme substrate,thereby playing the role of activation and toxicity.4.The Shengshouwu and gallic acid groups caused significant damage to the liver function of mice by inducing the expression of CYP1A1,CYP3A4 mRNA and protein,and thereby activating the expression of the apoptosis factor Caspase-3.