Study On Hepatotoxicity Of Gynura Japonica Based On Bile Acid Metabolism

Gynura japonica?Thunb.?Juel.is a commonly used Chinese herbal medicine in China.It is also known as Tusanqi.It has the effects of stopping bleeding,promoting blood circulation and removing blood stasis.It is also due to its rhizome and the genus Panax notoginseng?Burk.?F.H.Chenis very similar and is often misused or misused.In particular,in recent years,the frequent occurrence of Hepatic sinusoidal obstruction syndrome?HSOS?caused by the misuse of Gynura japonica has caused widespread international concern about the safety of traditional Chinese medicine.Studies have shown that pyrrolidine alkaloids?PAs?,such as senecionine,are the major toxic components of Chrysanthemum.After metabolic activation of PAs in the liver,a series of biotransformation occurs,causing serious damage to the liver.The PAs are widely distributed and exist in more than 6,000 plants in the world.There are also a large number of reports of severe liver damage caused by PAs exposure.It is not only an urgent problem to be solved in China,but also a great concern in the world.However,due to the complicated mechanism of hepatotoxicity caused by PAs and numerous symptoms,studies in recent decades have failed to find effective treatment methods.It has been found that PAs cause HSOS to be associated with mitochondrial apoptosis,apoptosis,NO depletion,oxidative stress,and blood clotting pathways.Studies have found that PAs are associated with bile acid metabolism from genomics and metabolomics^[1],and a large number of clinical cases have found that HSs caused by HSOS with cholestasis symptoms,but there is no in-depth study of its related mechanisms.Therefore,this study uses analytical techniques,pharmacology,molecular biology and other technical means to study the association between hepatotoxicity and bile acid metabolism in vitro and in vivo from multi-faceted and in vivo,and to elucidate the hepatotoxic mechanism of Gynura japonica in order to clinical HSOS.Research provides a reference.The main research contents are as follows:1 Study on the amount-toxic relationship of total alkaloids in Gynura japonicaMale mice were given different doses of total alkaloids?TA,30 mg/kg,60 mg/kg,120mg/kg?in a single dose,and the amount-toxic relationship was analyzed.The results showed that low dose TA?30 mg/kg?can cause elevated serum ALT,AST,TBA and liver pathological changes.Medium dose?60 mg/kg?and high dose?120 mg/kg?TA can cause a decrease in diet and activity in mice,and abnormalities in behavioral appearance;hepatocyte necrosis and extensive hepatic congestion in the liver.When the dose of TA120 mg/kg was given,the mice began to die after 24 h,and the survival rate was zero after48 h.The bile acid in the liver was detected by HPLC/MS.It was found that the bile acid profile of the liver in the drug-administered group changed significantly,and the bile acid content increased significantly with the dose.Therefore,PAs in Gynura Rhizoma could cause severe liver injury in a dose-dependent manner.2 Study on the time-to-toxic relationship study of senecionineMale mice were given a single gavage of 50 mg/kg of sulphate,and samples were collected at different times within 10 min-72 h after dosing to study the time-toxic relationship of senecionine hepatotoxicity.The results showed that serum ALT and AST peaked at 24 h and then decreased gradually.HE staining for hepatic sinus congestion and necrosis was also most obvious at 24h,suggesting that there may be self-recovery mechanism.However,serum TBA continued to increase within 24h-72h,suggesting an important role of bile acid metabolism disorder for senecionine hepatotoxicity.The content of bile acids in the liver,serum,bile and ileum of mice was further determined by HPLC/MS technique.The changes of bile acid pool in mice at different time after the administration of senecionine were investigated.In the liver,the total free bile acid content of the mice was significantly increased 10 min after the administration of senecionine?especially primary bile acids CDCA,?MCA,?MCA?,while the total bound bile acid content showed a downward trend.At this time,both free bile acids and bound bile acids in bile showed a downward trend.It indicates that the bile acid and synthesis in the liver increase,while the bile acid biotransformation pathway is decreasing,and the bile acid toxicity is increased.At this time,the bile acid in the ileum also slightly increased.After 10 minutes of administration of senecionineline,the free bile acid in the liver gradually decreased until 2 hours,and the bile acid content in the serum and ileum generally changed with the liver.It may be that after the bile acid is elevated,the body's compensatory protective mechanism regulates the reduction of bile acid synthesis.After 12hours,the bile acid bile acid free bile acid and the combined bile acid in the liver and serum increased significantly,and only the free bile acid increased in the ileum.These results indicate that senecionine stimulates the synthesis of bile acids at 10 min.At this time,the biotransformation in the liver is insufficient to solve the increase in free bile acids.The free bile acids are discharged into the serum,and the free bile acids in the liver are in the ileum.Absorption is weakened.As the increase in bile acid content triggers the body's compensatory protection mechanism,the synthesis of bile acids is gradually reduced,and the bile acid content gradually declines.However,due to long-term celery alkali caused by hepatocyte necrosis,hepatic sinusoidal endothelial injury,occlusion of the sinusoidal lumen,long-term accumulation of bile acid,resulting in severe damage to the liver bile acid metabolism,liver and intestinal circulation imbalance,after 24h Bile acid free bile acids increase,efflux is reduced,causing severe bile acid deposition,giving the liver a heavier blow.In order to explore the mechanism of bile acid changes in hepatointestinal circulation,we examined the enzymes and transporters involved in bile acid synthesis,transport and excretion in the liver.The results showed that Fxr,the nucleic receptor of bile acid,was inhibited to varying degrees after administration of senecionine.Ten minutes after administration of senecionine,bile acid synthase Cyp7a1,Cyp8b1,Cyp27a1 were up-regulated,and bile acid-related transporters Mdr2,Bsep and Mrp2 were also up-regulated.Bile acid,as an important signal molecule,activated the negative feedback regulation mechanism mediated by Fxr,resulting in the gradual weakening of the genes Cyp7a1related to bile acid synthesis between 20 minutes and 2 hours,while the transporters Bsep,Mrp2.Up-regulation of gene transcription results in the decrease of bile acid content.After that,the expression of Cyp7a1 increased significantly from 6h to 12h,and bile acid synthesis increased.However,after 24 hours,due to extensive liver damage,most of the liver metabolic function was lost.The expression of bile acid synthase Cyp7a1,Cyp8b1,Cyp27a1 and transporter genes Mdr2,Bsep and Mrp2 remained at a low level without significant changes.At this time,a large amount of bile acid is deposited in the liver.3 Inhibition of nuclear receptor FXR activity by senecionine and its effect on primary hepatocytesUsing 293T cells as a tool,the effect of senecionine on bile acid receptor Fxr was detected by double enzyme fluorescence reporter gene system.The results showed that senecionine could directly inhibit the transcriptional activity of Fxr.In addition,primary mouse hepatocytes were used to analyze the cytotoxicity of senecionine and its effects on related bile acid synthase and transporters.The results showed that senecionine could directly reduce the survival rate of primary hepatocytes in a dose-dependent manner,IC50=5.72?M;and senecionine induced the expression of bile acid synthase Cyp7a1,Cyp8b1,Cyp27a1 in primary hepatocytes in a dose-and time-dependent manner.