Investigation On The Hepatotoxic Material Basis And Mechanism Of Polygoni Multiflori Radix With Zebrafish (Danio Rerio) Model

ObjectiveStudy the effect of different solvent extracts from polygoni multiflori radix on the liver of zebrafish larvae,and explore the hepatotoxic effect of the main 18 components in polygoni multiflori radix with zebrafish model to study the hepatotoxic material basis of polygoni multiflori radix and investigate the related hepatotoxicity mechanism.MethodsFirstly,raw,processed polygoni multiflori radix with different solvents(water,50% ethanol)were extracted for the liver fluorescent transgenic zebrafish and continuously administered for 72 h.Secondly,the liver fluorescent transgenic zebrafish were treated with 18 components of polygoni multiflori radix preliminarily screening of hepatotoxic constituents from polygoni multiflori radix.Then,the activity of ALT,AST,GSH and TBIL in zebrafish were measured after administration respectively.The liver sections were also analyzed to observe the histopathological changes of zebrafish liver,further screening of hepatotoxic components from polygoni multiflori radix.Next,select the points of NF-κB inflammatory pathway and P53 apoptotic pathway as research targets and the protein targets were docked with ligands and drug molecules respectively.Then,the mRNA relative expression of target gene of NF-κB inflammatory pathway and P53 apoptotic pathway was detected by qPCR method.The target protein expression was determined with Western blot method.Lastly,the staining of zebrafish in each group was observed under microscope and photographed after stainning with Sudan black B and TUNEL.ResultsPolygoni multiflori radix is hepatotoxic to the zebrafish larvae,meanwhile,the liver area and liver fluorescence intensity can be decreased significantly by aloe emodin in polygoni multiflori radix.Aloe emodin could significantly reduce the activity of ALT,AST,GSH,and increase the content of TBIL in zebrafish.And the zebrafish liver tissue of aloe emodin appeared large flaky necrosis,obvious morphologic changes in liver tissue cells,irregular arrangement of hepatocytes.The results of molecular docking showed that,aloe emodin could successfully dock with all the targets of NF-κB inflammation pathway and P53 apoptotic pathway.At the level of mRNA expression,the expression levels of all target genes in NF-κB and P53 pathway of aloe emodin group increased significantly.At the level of protein expression,the expression of P65,JNK and P53 was significantly increased in aloe emodin group.Aloe emodin could induce inflammatory cell infiltration in zebrafish liver and yolk sac and there were a large number of dense apoptotic signals were observed in the aloe emodin group,mainly distributed in the liver and yolk sac of zebrafish.ConclusionsPolygoni multiflori radix has toxic effects on liver of zebrafish larvae,and the hepatotoxicity of polygoni multiflori radix is mainly mediated by anthraquinones.Aloe emodin is the main hepatotoxic material basis of polygoni multiflori radix and its hepatotoxicity mechanism is to activate NF-κB inflammatory pathway and P53 apoptotic pathway,which can cause inflammation in the liver and lead to hepatocyte apoptosis.