| Polygonum(P.)multiflorum is the dry root of Polygonum multiflorum Thunb.,which had a tonic function to liver and kidney,furthermore,it also benefit to blood and could black hair.P.multiflorum is commonly used in clinical medicine.It has been chemically investigated,which resulted in the isolation of many secondary metabolites,including anthraquinones,stilbenes,phenolic acid,phospholipids,flavones,and so on.However the adverse reactions of P.multiflorum and its preparation are reported increasingly in recent years,especially the liver injury.Although the research on this issue is extensive,the mechanism of hepatotoxicity and toxic substances caused by P.multiflorum remains controversial.This topic starts from the clinical features of hepatotoxicity caused by P.multiflorum,that is,abnormal elevation of bilirubin and symptoms of jaundice after long-term oral administration of P.multiflorum preparations,and most patients recover spontaneously after discontinuation.Since UGT1A1 enzyme is the only metabolic enzyme of bilirubin that can metabolize bilirubin to bilirubin glucuronide conjugates which is excreted in the bile to complete the detoxification process.Therefore,based on the in vivo metabolism of bilirubin it is proposed that the mechanism of hepatotoxicity caused by P.multiflorum probably was the inhibition of the UGT1A1-mediated metabolism of bilirubin.In this research it firstly investigated the effects of P.multiflorum and monomer components on the UGT1A1 enzyme through the in vitro(liver microsomes)experiment and preliminarily speculated its possible hepatotoxicity mechanism and potential toxic components.Secondly,we used 2D,3D cells to confirm the hepatotoxicity of the test substance from the perspective of human body.Thirdly,the monomer components with strong inhibitory effect in vitro were selected to performance toxicity experiments in vivo to further verify the subject hypothesis.Finally,from the point of structure computer molecular docking was used to clear the target and interaction mode in order to explain the hepatic toxicity mechanism and toxic substances.This paper mainly includes the following aspects:Investigated the inhibitory effect of the compositions and its metabolites of P.multiflorum on UGT1A1 enzyme in rat liver microsomes(RLM)in vitro.Firstly we established an in vitro liver microsome incubation system for different species,and finally determined the screening model as RLM.Then we investigated the effect of extracts and components(Stilbene glycosides,anthraquinones,anthrones)of P.multiflorum on UGT1A1 enzyme(using Ki as an index)to estimate its potential hepatotoxicity.Through the phase Ⅱ metabolic reaction,the direct effect of the original components on the enzyme was examined.By the phase Ⅰ and Ⅱ metabolism,the effects of the original form and its metabolites on UGT1A1 were detected and the change of inhibition was observed.On the basic of changes in inhibition with phase Ⅰ metabolites,it was suggested potential hepatotoxicity.The results showed:(1)Stilbene glycosides had no obvious inhibitory effect on UGT1A1-mediated bilirubin metabolism.(2)Anthraquinones:emodin-8-O-β-D-glucoside strongly inhibits the UGT1A1 enzyme regardless of whether it is in the form of a prototype or a metabolite.Physcione only produces a large amount of emodin-8-O-β-D-glucoside after the metabolism of phase Ⅰ and Ⅱ,which has potential hepatotoxicity risk.(3)Monanthone with emodin type have strong inhibitory effects on either the prototype or its metabolites,and its metabolites are emodin-emodin dianthrones,dianthrone glycosides,and cis-emodin dianthrones,which also have strong inhibitory effects.Therefore,it is inferred that the monanthone is an important potential hepatotoxic component in P.multiflorum.(4)dianthrones:polygonumnolide C2 and trans-emodin dianthrones have strong inhibitory effects on either the prototype or its metabolites.The inhibiton of polygonumnolide C1,polygonumnolide C3,and polygonumnolide C4 were significantly increased after two-phase metabolism.Metabolites were analyzed and polygonumnolide C1 and C3 were metabolized to polygonumnolide C2 while polygonumnolide C4 was metabolized to emodin-8-O-β-D-glucoside.All the results suggested that these components had hepatotoxicity risk.(5)the extract of P.multiflorum:The inhibitory effect of the extract of P.multiflorum by two-phase metabolism was weakened,and the reason was related to the decrease of the inhibitory effect of emodin,chrysophanol,and citreorosein,which were high content in P.multiflorum.Furthermore the extract of prepared P.multiflorum after two-phase metabolism was from weak inhibition to no inhibition,proved that processed P.multiflorum has a smaller risk of hepatotoxicity.The experiments in liver microsomes in vitro preliminary confirmed that P.multiflorum and related monomer components may inhibit the UGT1A1-mediated bilirubin metabolism,that could induce a risk of abnormal bilirubin metabolism,leading to hepatotoxicity.Used human 3D liver model to study the effect of P.multiflorum and monomer components on human hepatocytesBased on the experiment of liver microsomes in vitro,70%of P.multiflorum extract and monomer components(monanthone with emodin type,emodin 8-O-β-D-glucoside,rhein,physcione)were selected to performance liver cell experiments with 2D and 3D liver model to study the liver toxicity.The hepatotoxicity evaluation was performed on the test substances,and the results showed a significant dose dependent relationship.P.multiflorum extract and monanthone with emodin type,emodin 8-O-β-D-glucoside and physcione were confirmed their hepatotoxicity.In vivo study of hepatotoxicity of P.multiflorum and monomeric Components in ratsOn the basis of the experiments in vitro,rats were orally administered with P.multiflorum extract and components of monanthone with emodin type,emodin 8-O-β-D-glucoside and physcione,all of them had a significant inhibitory effect.It was demonstrated that:(1)Physiological pathology:emodin-8-O-β-D-glucoside could induce mild vacuolar degeneration of hepatocytes/Kupffer cells with hemorrhage.After administration of physcione it showed moderate hepatocyte degeneration and necrosis.Simultaneously monanthone with emodin type caused a significant decrease in CK and LDH in rats.It suggested the three monomeric components had a certain risk of hepatotoxicity in vivo.(2)Effect of UGT1A1 Enzyme Activity in vivo:After the administration of the extract of P.and its monomers,all the four analytes could reduce the affinity for the substrate of UGT1A1 and reduced its clearance rate in order to inhibit the enzyme activity significantly.The experiments in vitro and in vivo provided data support with the hypothesis that P.multiflorum inducing the hepatic toxicity may be the reseason of inhibiting bilirubin metabolism UGT1A1 enzyme activity.(3)Toxicokinetics:after administration the AUCall concentration of the three monomers were higher in rats,suggesting that the high concentration of the original form exists in rats and there is a risk of hepatotoxicity.(4)Gene regulatory effects:it was found that emodin-8-O-β-D-glucoside inhibited the expression of UGT1A1 enzyme,down-regulated the uptake transporter Oatp1a1,upregulated the efflux transporter MRP2,that may cause bilirubin accumulation then induced hepatotoxicity.Moreover,it had not been found significant changes in the gene expression of UGT1A1 after administrating monanthone with emodin type and physcione but the transporters were up-or down-regulated,suggesting increasing drug delivery cycle for further studied.The results of toxicity experiments in vivo were basically consistent with the previous inhibition results in vitro.From another aspect,we verified the hepatotoxicity hypothesis of P.multiflorum that P.multiflorum may cause liver toxicity by inhibiting UGT1A1 enzyme activity,and further clarified potential hepatotoxic substances.Molecular docking technology to explore the mechanism of interaction between UGT1A1 and analytesTaking the chemical structure of the potential hepatotoxic components of P.multiflorum as a starting point and combining the Ki results in vitro,the structure-activity relationship analysis was performed.It was found that emodin-type anthraquinones,when the increase of-OH or glucose molecules on the nuclear parent,the inhibitory effect was significantly enhanced,at the same time,for the dianthrones and its glycoside compounds,10 and 10 ’ H configuration and additional glucose molecules could affect the exposure of-OH,which may affect their inhibition.Therefore,it is presumed that-OH and glucose molecules were beneficial to the binding of molecules and enzymes.Moreover molecular modeling studies were performed to investigate the mechanism underlying the UGT1A1 inhibitory activities of the P.multiflorum compounds.All compounds docked into active sites C and F depending on their mode of inhibition.The action mode suggested that hydrophobic interactions and hydrogen bonds are important for the affinity of the tested compounds for UGT1A1.Therefore,the specific mother nucleus structure facilitates the binding with the enzyme,at the same time,whether it has a functional group capable of hydrogen bonding with amino acid residues such as-OH and an additional glucose molecule also affects its affinity with the UGT1A1 enzyme.Furthermore different configurations of 10 and 10’ affect their space configurations,and the increased molecular volume enhances rigidity,which alters their docking orientations.From the interaction energy,we preliminarily speculated that the affinity of UGT1A1 with UGT1A1 is site F:trans-emodin dianthrones(-42.4254 kcal·mol-1)>cis-emodin dianthrones(-39.8091 kcal·mol-1)>emodin-8-O-β-D-glucoside(-34.7005 kcal·mol-1)>citreorosein(-29.6367 kcal·mol-1)>emodin(-25.9932 kcal·mol-1)>monanthone with emodin type(-23.4174 kcal·mol-1)>chrysophanol(-18.3957 kcal·mol-1).Site C:Polygonumnolides C1(-43.0276 kcal·mol-1)>polygonumnolides C2(-36.9558 kcal·mol-1)>polygonumnolides C3(-37.2953 kcal·mol-1)>polygonumnolides C4(-31.1608 kcal·mol-1)>rhein(-20.6433 kcal·mol-1)>physcione(-19.7442 kcal·mol-1).In summary,this research has initially demonstrated that the extracts of P.multiflorum,its main monomer components and metabolites have inhibitory effects on UGT1A1 enzyme,and that may induce bilirubin accumulation and lead to hepatotoxicity in vivo.At the same time,starting from the structure of the enzyme protein,the target of UGT1A1 and the interaction between the analyte and UGT1A1 were further clarified.On the basic of all the experiments in vivo and in vitro we speculated that the mechanism of hepatotoxicity induced by P.multiflorum may be the inhibition of UGT1A1-mediated bilirubin metabolism and we found that the potential toxic substances probably are:trans-emodin dianthrones,cis-emodin dianthrones,Polygonumnolides C1-4,monanthone with emodin type,emodin-8-O-β-D-glucoside and physcione.The hepatotoxicity assessment method established in this study provides a new idea for the in vitro and in vivo studies of toxic traditional chinese medicine,and it is also an important exploration for establishing a research system for hepatotoxic components. |
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