| Purpose Polygonum multiflorum Thunb,the root of Polygonaceae,was first recorded in the history book Kai Bao Ben Cao.It is lukewarm,bitter,sweet and astringent,which is divided into Radix Polygonum multiflorum（RPM）and Radix Polygoni multiflori preparata（RPMP）in clinical application.RPM is widely used for detoxification,eliminate phlegm and laxative,as well as RPMA is used for protecting liver and kidney,strong bones,benefit essence blood and black hair.Emodin as the main anthraquinone component in Polygonum multiflorum Thunb has a wide range of pharmacological effects,including anti-cancer,anti-bacterial and inflammatory effects,which also has therapeutic effects on liver cancer,lung cancer,nasopharyngeal cancer and breast cancer.With the widespread use of Polygonum multiflorum and the strengthening of the understanding and supervision of the safety of Chinese medicine,clinical reports on hepatic injury caused by Polygonum multiflorum have emerged at home and abroad,which had attracted the attention of scholars on the safety issue.At present,it is generally believed that the liver injury effect of Polygonum multiflorum is mainly related to its anthraquinone components,such as the direct toxic effects of emodin and rhein on hepatocytes.Cytochrome P450（CYP）,an important human drug metabolizing enzyme,is involved in the metabolism of more than 90%of clinical drugs.When its activity is inhibited or induced,drug interactions and adverse reactions occur easily,and there is no report in the relationship between hepatotoxicity and CYP450 caused by Polygonum multiflorum.Therefore,to explore the effect of Polygonum multiflorum on CYP450 has a great significance in elucidating its toxic effects and mechanisms.This study will explore the mechanism of toxicity of emodin-induced liver injury in vivo and in vitro levels from the perspective of drug metabolizing enzymesMethods（1）L02 cells were treated with different concentrations of emodin and cell toxicity was examined by MTS.qRT-PCR and Western blot were used to detect mRNA and protein expression of CYP450 isoforms respectively.（2）The interaction of between AhR and CYP1A1 was detected by EMSA.（3）qRT-PCR and Western-blot were used to detect the effect of emodin in L02cells before and after knocking down the expression of AhR by siRNA;Ca2+in L02 intracellular were used to detect by being labeled with Fluo/4-AM fluorescent probe cytokines and flow cytometry.GSH,MMP and ROS were detected by HCS.（4）CYP1A1 induced and inhibitory animal models were established on the whole animals,and the effects of emodin on hepatic injury of mice under the conditions of CYP induction or inhibition were examined.Results（1）1100μM emodin caused damage to L02 cells in different degrees,which showed significant hepatotoxicity at high concentrations.（2）When emodin was applied to L02 cells at low（5μM）,medium（10μM）,and high（20μM）concentrations,CYP1A1 mRNA and protein expression were induced in a concentration-dependent manner.（3）The results of EMSA and reporter genes showed that the expression of CYP1A1 induced by emodin was related to the activation of its transcription factor AhR.（4）Knockdown effect in CYP1A1 expression could reverse the emodin-induced apoptosis of L02 cells and the decrease of MMP.（5）Emodin can significantly aggravate hepatic injury in CYP1A1 induced C57 mice,and enhance the expression of ATF-4and CHOP,a molecule involved in ER stress,which further activate caspase3and induce the expression of caspase9.Conclusion Cytochrome P450,a major metabolic enzyme,participated in the metabolism of endogenous and exogenous substances in the human body.In the screening process for the effects of emodin on CYP450 isoforms,it was the first time found that emodin could time and concentration-dependent induce the mRNA and protein expression in CYP1A1 and CYP1B1.It was further confirmed that the induction effect started from the binding phase of AhR and CYP1A1 regulatory regions,that is,emodin could activate AhR and enhance its transcription activity to CYP1A1.Whether the emodin-induced effect of CYP1A1 is related to liver damage caused by Polygonum multiflorum,this study is sequentially used the cell apoptosis,high content screening（ROS,MMP,ER stress）,calcium transients,western blot technology binding molecules biological verification techniques to examine various indicators closely related to hepatotoxicity in combination with in vitro and in vivo experiments.The results showed that emodin induced L02 cells apoptosis in a dose-dependent manner,induced intracellular ER stress,decreased MMP and GSH content;induced intracellular ROS production and calcium accumulation.At the same time,we found that emodin can activate caspase9and caspase3 activity in mouse liver and induce apoptosis.In order to verify the relationship between CYP1A1 induction and hepatotoxicity,comparative inhibitors or inducers were used for comparison studies.It was found that when emodin was co-treated with the AhR specific inhibitor CH223191,cell viability was significantly improved compared to the non-added CH223191.The reduction of MMP was significantly improved,and the production of ROS was reversed.Animals also significantly reduced emodin-induced liver injury after administration of CYP1A1 inhibitors.According to the experimental results of cells and animals,it is presumed that emodin activation of AhR and induction of CYP1A1 is an important finding for aggravating liver injury.The possible mechanisms may be related to the significant induction of intracellular calcium accumulation after activation of AhR,and sustained oxidative stress and internal Reticulocyte stress,which in turn activates the caspase pathway to induce apoptosis,is also relevant to whether emodin is involved in its own metabolic transformation and leads to hepatotoxicity in response to CYP1A autoinduction.Therefore,we believe that CYP1A1 may be an important regulatory target of emodin aggravate hepatic injury in Hedysarum multiflorum,that is,emodin can induce intracellular oxidative stress and ER stress through the AhR-CYP1A1 pathway and then induce apoptosis pathways in hepatocytes.The injury suggests that the long-term use of Polygonum multiflorum to induce CYP1A or in combination with CYP1A-inducing drugs（including Chinese medicine）may have potential hepatotoxicity risk. |
