| Objective:There are reports about hepatotoxicity of traditional Chinese medicine in clinical application,such as Polygonum multiflorum,Rheum palmatum L.and Cassia occidentalis.It is presumed that the aloe-emodin might be one of the major components of hepatotoxicity.Other studies have confirmed that aloe-emodin could induce DNA damage in mice,but its underlying mechanism is not well clarified.The aim of this study was to determine the hepatotoxicity and its mechanism induced by aloe-emodin on two hepatocyte models combined with metabonomics and proteomics in vivo and in vitro.The zebrafish model was utilized to verify the hepatotoxicity and its mechanism in order to underscore the need for risk assessment of human exposure to aloe-emodin and provide suggestions for the clinical management and the safety of drug development.Method:1)The cytotoxicity of HL-7702 and HepaRG cells,incubated with a series of aloe-emodin,was investigated at different time points.The level of MTT,LDH,DAPI,ROS,GSH,MMP,cell cycle and cell apoptosis was detected by microplate reader,flow cytometry and inverted fluorescence microscope.Moreover,the expression of apoptosis-related proteins was determined by Western blot analysis in order to underscore the hepatotoxicity induced by aloeemodin and its mechanism.2)SD rats were orally administered with different doses of aloe-emodin for 10 weeks and were sacrificed.The serum was used for the analysis of the level of biochemical index including ALT,AST,ALP,TP,TBIL,GLU,TG and so on.The liver tissues were used for the analysis of pathological conditions and the level of antioxidant index(SOD and GSH-Px.)3)iTRAQ quantitative proteomics technology was used to detect the differential proteins in the liver tissues of rats in different groups.The differential proteins were analyzed by GO analysis and pathway pathway enrichment analysis.The phenotypic related protein markers were screened by using the IPA commercial database,and the mechanism of hepatotoxicity was deduced.4)GC-MS/MS technique was used to detect endogenous small molecule metabolites in serum samples of rats in different groups.Based on the method of combination of single dimension and multidimensional statistics,we compared the difference of of rats serum samples metabolic profiling in different doses treatment in order to search the metabolic difference and metabolic regulation network.In this study,technology was used to detect the differential proteins in liver tissues of each group.GO analysis and differential analysis of Pathway pathway were used to analyze the differential proteins.The IPA commercial database was used to screen out the phenotypic related protein markers and to infer the mechanism of its effect on the liver.5)The zebrafish model was used to observe the survival rate of zebrafish treated with different concentrations of aloe emodin.The LC10 and MNLC were determined.Moreover,the liver area,the mean value of liver opacity and the delayed absorption area of yolk sac were detected.The pathological changes of liver tissue were observed by HE staining.The level of ROS in zebrafish was observed by inverted fluorescence microscope.The CYP450 enzyme and apoptosis-related proteins in zebrafish were measured by Elisa kit in order to investigate and the mechanism of hepatotoxicity induced by aloe-emodin.Results:1)Treatment with aloe-emodin significantly reduced cell viability and induced apoptosis in HL-7702 and HepaRG cells in a dose-and time-dependent manner.When compared with controls,it provoked ROS generation and depolarization of MMP in HepaRG cells.Aloeemodin dose-dependently increased release of mitochondrial cytochrome c,and levels of Fas,p53,p21,as well as activation of caspase-3,caspase-8,caspase-9,and subsequent cleavage of poly(ADP-ribose)polymerase(PARP).It also induced cell cycle arrest by increasing the expression of p21 and cyclin E proteins while significantly decreasing the expression of cyclin A and CDK2.2)The results of biochemical indexes showed that the serum levels of AST,ALT,ALP and LDH in high dose group significantly increased compared with the blank control group,indicating that hepatocytes injury might occur in rats of high dose group.The level of biochemical indexes,such as GLU,TG,CHOL,were significantly increased in high dose of male group,indicating that there might be abnormal glucose or lipid metabolism.However,the level of TBIL significantly were increased in high dose of female group,suggesting that there might be existed the case of capillary bile duct hepatitis.The levels of oxidative damage index,such as SOD and GSH-Px,were decreased,indicating that the mechanism of hepatotoxicity might be associated with oxidative damage.HE results demonstrated that fatty degeneration,inflammatory cell infiltration accompanied by balloon degeneration were observed in high dose group.3)Compared the high group with the control group,the protein expression profiles were investigated by iTRAQ quantitative proteome technology.The results showed that a total of 114 differential proteins were screened out,including 77 up-regulated proteins and 37 downregulated proteins.GO results showed that these differential proteins had the functions of iron ion binding,homologous dimerization,heme binding and receptor binding,were involved in the process of redox,reaction to drug and metabolism of fatty acid,were mainly distributed in mitochondria and cell membrane.Pathway analysis showed that retinol metabolism,fatty acid degradation,steroid hormone biosynthesis,linoleic acid metabolism and arachidonic acid metabolism are well enriched.There were 16 differentiated proteins associated with phenotypes were screened by the commercial database of IPA.4)The PCA and OPLS-DA analysis of GC-MS/MS metabonomics showed that there was a trend of separation between the blank group and the different dose groups,and the metabolic spectrum of each group had a regular clustering.A total of 38 endogenous metabolites,mainly amino acids and carbohydrates,were identified in the different groups.The MetPA results showed that lysine degradation,alanine,aspartate and glutamate metabolism,and pentose phosphoric acid pathway had relatively higher metabolic effects,which were 0.21,0.21 and 0.13,respectively,indicating that the abnormal metabolism of amino acids was closely related to the hepatotoxicity induced by aloe-emodin.5)The results showed that the LC10 and MNLC concentrations of zebrafish,treated with aloe-emodin,were 312 M and 229 M,respectively,showing a dose-response relationship.Compared with the blank group,the liver area of zebrafish did not change obviously,but the liver impermeability and the delayed absorption area of yolk sac increased in a dose-dependent manner.HE staining showed that the liver nuclei were clear,the structure of the liver cells was normal and the edges were clear in the control group.However,in MNLC group,the structure of zebrafish hepatocytes was blurred,the edges were not clear,the gap of hepatocytes was increased and the hepatocyte nucleus was enlarged obviously.Aloe-emodin significantly increased the production of ROS in zebrafish in a dose-dependent manner.Furthermore,this drug also inhibited the expression of cyp450 3A4 and cyp450 2E1 in zebrafish,but significantly upregulated the protein levels of bax,caspase-3 and 9.Conclusion:These results suggested that aloe-emodin inhibits cell proliferation and induces apoptosis in HL-7702 and HepaRG cells,most probably through a mechanism involving both Fas death pathway and the mitochondrial pathway by generation of ROS.The differentially expressed proteins were identified by itraq proteomics technique including apoptosis-promoting protein(Bax),phase Ⅰ,phase Ⅱ metabolic enzymes(UGT 1A1,CYP450 2E1 and CYP450 2C9)and oxidative stress-related antioxidant protein(SOD1,SOD2).The results indicated that aloeemodin may cause oxidative stress reaction in rat hepatocytes and then activate apoptotic protein,which is consistent with the results in vitro.Moreover,the drug may also affect the levels of phase Ⅰ and Ⅱ metabolic enzymes in rats.The joint action induced the occurrence of hepatic toxicity.The abnormal metabolism pathway of amino acid was closely related to the hepatotoxicity induced by aloe-emodin.Alanine,aspartic acid and glutamate could be considered as potential biomarkers.The liver toxicity index and liver histopathology of zebrafish results further confirmed that aloe-emodin had the potential liver toxicity.The mechanism might be that this drug could not only increase the level of ROS in zebrafish,destroy the structure of mitochondria,initiate a series of apoptotic pathways,induce the expression of apoptosis-related proteins,and induce hepatocyte apoptosis.Moreover,it could also induce the abnormal expression of the zebrafish phase Ⅰ metabolic enzyme,and the combined action of the two resulted in the occurrence of hepatic toxicity. |
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