| BackgroundDrug-induced liver injury(DILI)refers to liver damage caused by the drug itself and/or its metabolites,which is one of the common adverse drug reactions and can lead to acute liver failure or even death.Psoraleae Fructus(PF)is a commonly used traditional Chinese herb in clinical practice.In recent years,with the expansion of the application of PF,reports of hepatotoxicity caysed by PF have increased,which seriously affects the safety of this herb in clinical use.As we all know,the safety of traditional Chinese medicine is affected by many factors.At present,the pharmacological factors of PF causing liver injury are still unclear,its hepatotoxicity mechanisms and toxic substance basis also remain to be revealed.Objective1 The uniform design method was assigned to study the hepatotoxicity of PF and clarify the drug factors causing hepatotoxicity of it.2 Metabolomic and proteomic analyses,combined with validation of key proteins were used to clarify the mechanism of hepatotoxicity caused by PF.3 The UHPLC-Q-Exactive MS method was used to identify and screen the components of the extracts of PF,together with in vitro cytotoxicity experiment and high-intensity screening to identify the main toxic components of PF-induced hepatotoxicity.Methods1 In the study of the drug factors of PF liver injury,Based on the factors of process,extraction technology,dosage and treatment period,each experimental group was arranged by uniform design method.220 SD rats and 220 Kunming(KM)mice with male and female in half were divided in tocontrol groups and drug groups of 1-8.The corresponding drugs(50%alcohol extract of salt PF in rats 2.57 g/kg,mice 5.14 g/kg,95%alcohol extract of PF in rats 0.51 g/kg,mice 1.02 g/kg,70%alcohol extract of salt PF in rats g/kg,mice 3.42 g/kg,water extract of PF in rats 1.03 g/kg,mice 2.06 g/kg,water extract of salt PF in rats 1.03 g/kg,mice 2.06 g/kg,70%alcohol extract of PF in rats 1.71 g/kg,mice 3.42 g/kg,95%alcohol extract of salt PF in rats 0.51 g/kg,mice 1.02 g/kg,50%alcohol extract of PF in rats 2.57 g/kg,mice 5.14 g/kg)were administered by gavage daily,the body weight and food intake of rats and mice were measured once a week.After each course of administration,rats were anesthetized with sodium pentobarbital,blood was taken from the abdominal aorta of the rats,and the mice were sacrificed by removing the eyeballs,and the liver and brain were taken to calculate the organ coefficient.Serum was taken to determine liver function-related indicators,and the liver was taken for hematoxylin-eosin(HE)staining for histopathological examination.Based on the liver pathology scores of rats and mice,multiple regression analysis was performed to evaluate the effects of various factors on hepatotoxicity.Based on the results of regression analysis,together with the biochemical indexes of liver function and liver weight,the combination of factor levels at the most significant hepatotoxicity was screened,and conducted long-term toxicity experiments to verify.2 In the preliminary toxicity mechanism study of hepatotoxicity caused by PF,non-targeted metabonomics and tandem mass tags-labeled quantitative proteomics methods were used to conduct omics determination and analysis on mouse liver tissue,and the differential proteins were validated by Western blot method to investigate the toxicity mechanism of PF-induced hepatotoxicity.3 In the study of the toxic components of hepatotoxicity caused by PF,the chemical compositions in water extracts of PF(PFW)and 70%ethanolic extracts of PF(PFE)were identified by UHPLC-Q-Exactive MS method,the differences in chemical compositions of the two extracts were analyzed,and the serum medicinal chemistry of the differential components were studied.Subsequently,two types of cells,L02 and HepG2,were used as in vitro models to screen for components with large differences in content that could be absorbed into the blood.In vitro experiments include cell viability determination,determination of alkaline phosphatase(ALP),alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels in cell culture fluid,apoptosis detection and high-content screening,in which the screening indexes included cell number,nuclear area,intracellular lipids,reactive oxygen species(ROS)and mitochondrial membrane potential(MMP).Through the above chemical composition analysis and indexes test to find the main toxic components.Results1 The results of long-term toxicity experiments showed that the liver visceral-brain ratio of female rats in 70%ethanol extract group were significantly increased(P<0.05),the liver quality,visceral-body ratio,and visceral-brain ratio of male mice in the 50%ethanol extract group,the 95%ethanol extract group and the 70%ethanol extract group were significantly increased(P<0.05,P<0.01).The liver histopathological results showed that the pathological changes of the liver tissues of mice were more obvious than those of rats,hepatocyte hypertrophy in the central of liver lobules in mice,and the mice in PFE group obvioused most.According to the multiple regression analysis,the extraction technology interacted with the process,dosage and treatment period,and the extraction technology was positively correlated with the pathological score of hepatotoxicity.And combined with the results of liver function index test and organ coefficient,the factor of extraction technology was most correlated with the hepatotoxicity of PF.Besides,the results of parallel long-term toxicity in rats and mice suggested that mice showed significant hepatotoxicity of PF than rats.The results of validation experiments confirmed that PFE caused significant hepatotoxicity in mice,and the liver histopathology existed Hepatocyte steatosis and central hypertrophy,while the hepatotoxicity in PFW group was slight,which confirmed that the extraction technology was the main influencing factor of hepatotoxicity caused by PF.2 The metabolomic results showed that compared with the control group,significantly different metabolites in the of mice in the the PFW group were mainly involved in secondary bile acid biosynthesis,primary bile acid biosynthesis,bile secretion,biosynthesis of unsaturated fatty acids,cholesterol metabolism,linoleic acid metabolism and other biological processes related to hepatotoxicity.Compared with the control group,significantly different metabolites in the of mice in the the PFE group were mainly involved in bile secretion,primary bile acid biosynthesis,bile secretion,secondary bile acid biosynthesis,biosynthesis of unsaturated fatty acids,cholesterol metabolism,linoleic acid metabolism,PPAR signaling pathway,steroid degradation,degradation of aromatic compounds,C 5-Branched dibasic acid metabolism and other biological processes related to hepatotoxicity.The common pathways of the PFW group and the PFE group were involved in bile secretion,primary bile acid biosynthesis,secondary bile acid biosynthesis,biosynthesis of unsaturated fatty acids and cholesterol metabolism,linoleic acid metabolism.Besieds,PFE group also included in PPAR signaling pathway,purine metabolism and C5-Branched dibasic acid metabolism.Proteomic results showed that compared with the control group,PFW up-regulated metabolism of xenobiotics by cytochrome P450 and chemical carcinogenesis,down-regulated cholesterol metabolism,nonalcoholic fatty liver disease(NAFLD)and oxidative phosphorylation.Compared with the control group,PFW up-regulated glutathione metabolism,primary bile acid biosynthesis,a-linolenic acid metabolism,biosynthesis of unsaturated fatty acids,PPAR signaling pathway and chemical carcinogenesis,down-regulated bile secretion,cholesterol metabolism,NAFLD and oxidative phosphorylation.Both PFW and PFE down-regulated the NAFLD and oxidative phosphorylation pathways in mice.Compared with the PFW group,the PFE group had upregulated effects on glutathione metabolism,primary bile acid biosynthesis,?-linolenic acid metabolism,unsaturated fatty acid biosynthesis and PPAR signaling pathway in mice,while the PFW group was weakly enriched for the above pathways in mice.The results of the correlation analysis between metabolomics and proteomics showed that the common pathways in the PFW group and the PFE group included primary bile acid biosynthesis,oxidative phosphorylation,NAFLD,unsaturated fatty acid biosynthesis and chemical carcinogenesis.Besides,PFE group also had significant effects on bile secretion,biosynthesis of fatty acids,linoleic acid metabolism,arachidonic acid metabolism and PPAR signaling pathway.Western blot results showed that PFW and PFE group down-regulated the expressions of MRP2 and BSEP in mice liver(P<0.05,P<0.01),suggesting that PF blocked the bile transport in mice;the expressions of FXR1 and CPT1A were decreased(P<0.05,P<0.01),suggesting that that PF inhibited hepatic lipid oxidative decomposition in mice.Both PFW and PFE groups caused hepatotoxicity and also induced significant apoptosis in mouse liver tissue cells(P<0.01).The expression of Cyto C was up-regulated by PFE(P<0.05),suggesting that PFE damaged mitochondria.3 The results of UHPLC-Q-Exactive MS analysis showed that the PFE was enriched with coumarins(psoralen,isopsoralen,psoralidin),flavonoids(bavachin,bavachinin,neobavaisoflavone)and monoterpenes(bakuchiol)from PF.Serum medicinal chemistry results showed that bavachin,psoralidin,bavachinin,neobavaisoflavone and bakuchiol were present in plasma as prototypes.The results of CCK8 in vitro showed that bavachin,psoralidin,bavachinin,neobavaisoflavone and bakuchiol had large effect on cell viability.The results of biochemical indexes of liver function and apoptosis showed that bavachin,psoralidin,bavachinin,neobavaisoflavone and bakuchiol increased the levels of ALP,ALT and AST in the cell culture medium(P<0.05,P<0.01)and induced apoptosis of L02 and HepG2 cells(P<0.05,P<0.01).The results of high internal screening showed that bavachin,psoralidin,bavachinin,neobavaisoflavone and bakuchiol significantly reduced the number of living cells,caused an increase in nuclear area,increased intracellular lipid accumulation,ROS level and decreased MMP level(P<0.05,P<0.01),among which psoralidin and bakuchiol had a greater effect on the above detection index.In summary,bavachin,psoralidin,bavachinin,neobavaisoflavone and bakuchiol were the main toxic components of PF hepatotoxicity,among which psoralidin and bakuchiol were more toxic.Conclusions1 The extraction technology was the main influencing factor of PF-induced hepatotoxicity.The PFE was more toxic.In addition,there were species differences in hepatotoxicity,with mice showing significant hepatotoxicity than rats.2 The hepatotoxicity induced by PF may be accompanied by cholestasis and abnormal fat metabolism,the PFW and PFE groups produced significant effects on the pathways of primary bile acid biosynthesis,bile secretion,and biosynthesis of unsaturated fatty acids.The obstruction of bile synthesis and transport,impaired fat oxidation and catabolism and the apoptosis of hepatocytes caused by mitochondrial damage may be the potential toxic mechanisms of PF-induced hepatotoxicity.3 Bavachin,psoralidin,bavachinin,neobavaisoflavone and bakuchiol were the main toxic components of PF hepatotoxicity,among which psoralidin and bakuchiol were more toxic.This study firsty conducted a comprehensive and systematic study on the hepatotoxicity of PF from the aspects of pharmacological factors,hepatotoxicity mechanism and toxic substance basis,so as to scientifically evaluate the hepatotoxicity of PF.This study clarifies the pharmacological factors,hepatotoxicity mechanism and toxic substance basis of PF-induced hepatotoxicity,providing a basis for the safe clinical use of PF,the formulation and improvement of herbal standards,and also providing a reference for the development and drug evaluation of new drugs containing PF. |
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