Identification Of Hepatotoxic Anthraquinones In Cassiae Semen And Study On The Underlying Mechanisms Of Hepatotoxicity

BackgroundAs one of the 101 homologous varieties of medicine and food published by the National Health Commission of the People’s Republic of China,cassiae semen has a long history of medicinal use in China.In recent years,there have been reports of cassiae semen causing liver injury,and its hepatotoxicity has attracted widespread attention.However,the existing research conclusions on hepatotoxicity of cassiae semen are not unified,and whether it could cause liver injury,the possible major hepatotoxic components and the mechanism of action are still unclear.As a new model organism,which possesses the characteristics of high throughput,high efficiency and easy observation,zebrafish larvae fits perfectly in the evaluation of liver toxicity of cassiae semen and the identification of liver toxicity components in cassiae semen.ObjectivesBased on the above scientific questions,this study intends to study and elaborate the above problems through zebrafish and three kinds of liver cells,so as to provide further reference for the safe use and management of cassiae semen.Methods1.Experimental subjectsWild-type zebrafish larvae（AB strain）,WRL 68、L 02、HepG 2 cells.2.MethodsAccording to the main ingestion method of cassiae semen,an aqueous cassiae semen extract was prepared which was used as the test substance to administer zebrafish larvae.At the same time,liver changes of zebrafish larvae were observed and compared to evaluate the hepatotoxicity induced by aqueous cassiae semen extract.On this basis,the anthraquinone components in aqueous cassiae semen extract were detected,and the hepatotoxicity test of the detected anthraquinone components was carried out with zebrafish larvae and three kinds of hepatocytes.Combined with the liver toxicity of each anthraquinone component and its content in aqueous cassiae semen extract,the main hepatotoxic anthraquinone components in aqueous cassiae semen extract was inferred.Finally,zebrafish larvae and hepatocytes were used to evaluate the hepatotoxicity of the main hepatotoxic anthraquinones in the aqueous cassiae semen extract,and the mechanism of hepatotoxicity was also discussed using hepatic cells.Results1.The aqueous cassiae semen extract induced hepatomegaly,delayed the absorption of yolk sac,and increased the leakage rate of alanine aminotransferase.Histopathological examination showed hepatocyte enlargement and obvious vacuolation on zebrafish liver after the treatment of aqueous cassiae semen extract.2.There were mainly six anthraquinones in aqueous cassiae semen extract,among which the content of aurantio-obtusin was the highest.Among the six anthraquinone compounds,only aurantio-obtusin,emodin and rhein showed hepatotoxicity.3.Aurantio-obtusin induced hepatomegaly,delayed absorption of yolk sac and abnormal liver biochemical indexes in zebrafish.Cell experiments showed that the cell activity was impaired and the leakage rate of alanine aminotransferase,aspartate aminotransferase and lactate dehydrogenase was increased.4.Aurantio-obtusin leaded to hepatocyte apoptosis,decrease of mitochondrial membrane potential and generation of reactive oxygen species（ROS）,which was specifically manifested by increased expression of its corresponding apoptotic characteristic protein cleaved Caspase-3,mitochondrial damage protein Bax and cytoplasmic Cytochrome C protein while the protein expression of Bcl-2 and mitochondrial Cytochrome C protein decreased.However,after the ROS scavenger N-Acetyl-L-cysteine（NAC）was used to clear ROS in the aurantio-obtusin-administered group,cell viability,mitochondrial damage and apoptosis did not recover.ConclusionsThe main hepatotoxic component of aqueous cassiae semen extract was aurantio-obtusin which induced hepatocyte apoptosis and mitochondrial damage by a ROS-independent toxic mechanism.The specific molecular mechanism needs to be further studied.