Hepatotoxicity Of Fructus Psoraleae Based On Depigmented Zebrafish Model

Objective:The hepatotoxicity of traditional Chinese medicine fructus psoraleae was studied using zebrafish model with disease state,and the hepatotoxicity difference of fructus psoraleae in zebrafish model with different body state(normal and depigmentation)was compared,so as to clarify the mechanism of enhanced hepatotoxicity of depigmented zebrafish caused by fructus psoraleae.Method:1.Phenylthiourea(PTU)was used to establish the model of depigmentation zebrafish,and the difference of hepatotoxicity of fructus psoralen ethanol extract(EEFP)on normal zebrafish and depigmented zebrafish was evaluated.The ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF/MS)was used to analysis the components of EEFP,and the hepatotoxic effects of different components on normal and depigmented zebrafish were compared.Two monomeric components of bavachalcone(Bava C)and isobavachalcone(IBC)in EEFP were selected for the mechanism study.2.In the study on the difference of hepatotoxicity between normal and depigmented zebrafish caused by Bava C,liver morphology,liver area,volume,degree of oil red O staining,pathological tissue sections and liver cell ultrastructure were used as the main indicators to evaluate the hepatotoxicity of Bava C on zebrafish in different states.RNA-Seq was used to analyze the effects of Bava C on gene expression profile of zebrafish.In addition,the changes of endogenous metabolites in zebrafish were investigated by mass spectrometry imaging technology.Finally,the expression level of metabolism-related genes was detected by RT-q PCR.3.In the study on the difference of hepatotoxicity between normal and depigmented zebrafish induced by IBC,liver morphology,liver area and volume,oil red O staining degree,hepatocyte pathological tissue and ultrastructure of zebrafish were used as the main indicators to evaluate the hepatotoxicity of IBC on zebrafish in different states.The gene expression profile of normal and depigmented zebrafish was analyzed by RNA-Seq.RT-q PCR results showed that IBC caused disorders in the expression of genes related to calcium signal,lipid metabolism,oxidative stress and apoptotic pathway in depigmented zebrafish,and to clarify the mechanism of IBC causing hepatotoxicity in depigmented zebrafish was stronger than that in normal zebrafish.Results:1.The hepatotoxicity of EEFP on depigmented zebrafish was significantly stronger than that on normal zebrafish.A total of 64 chemical compositions of EEFP were detected by UPLC-Q-TOF/MS,according to the size of the peak area and combined with the literature screening of 10 compositions to compare the hepatotoxicity,among which Bava C and IBC on the hepatotoxicity of depigmented zebrafish was the most serious.Therefore,the mechanism of enhanced hepatotoxicity induced by Bava C and IBC on depigmented zebrafish was studied in this thesis.2.Bava C can cause enhanced hepatotoxicity of depigmented zebrafish than normal zebrafish,which mainly shows that Bava C can cause liver hypoplasia in depigmented zebrafish,significant reduction in liver area and volume,deepening of oil red O staining,hepatocyte vacuolation and increased mitochondrial damage.The results of RNA-Seq showed that the pathway of Bava C on differential gene enrichment of normal and depigmented zebrafish was mainly related to metabolism,including drug metabolism,amino acid metabolism,glucose metabolism and so on.Meanwhile,mass spectrometry imaging detected changes in fatty acid and phosphatidylcholine contents in depigmented zebrafish caused by Bava C.The results of RT-q PCR showed that Bava C affected the expression of genes related to drug metabolism,amino acid metabolism,glucose metabolism and lipid metabolism pathways in depigmented zebrafish.3.In the study of enhanced hepatotoxicity of depigmented zebrafish caused by IBC,it was found that compared with normal zebrafish,IBC caused significant changes in liver morphology,decreased liver volume,deepened oil red O staining,and increased liver pathological tissue and ultrastructure damage on depigmented zebrafish.RNA-Seq showed that the difference between normal and depigmented zebrafish hepatotoxicity caused by IBC may be closely related to calcium signaling pathway,lipid metabolism and oxidative stress.The results of RT-q PCR showed that IBC caused the disturbance of the expression of calcium signal,lipid metabolism,oxidative stress and apoptotic pathways in depigmented zebrafish.Conclusions:In this study,it was found that the hepatotoxicity of EEFP on depigmented zebrafish was significantly stronger than that of normal zebrafish,and the mechanism of hepatotoxicity of Bava C and IBC,the main components of EEFP was further studied.It was found that Bava C may cause increased mitochondrial damage in depigmented zebrafish,affect the expression of genes related to drug metabolism,amino acid metabolism,sugar metabolism and lipid metabolism pathways,and thus lead to the hepatotoxicity of depigmented zebrafish was stronger than that normal zebrafish.IBC can increase hepatotoxicity in depigmented zebrafish by affecting calcium signal,lipid metabolism,oxidative stress and apoptosis.These results provide theoretical basis and experimental basis for the subsequent clinical application of fructus psoraleae.