| The number of hepatocellular carcinoma patients in the world was increasing.The mortality rate was also very high,especially in China,which posed a serious threat to human life and health.China was a large country with a long history of traditional Chinese medicine.Therefore,the quickest way to treat hepatocellular carcinoma was to find available anti-hepatocellular carcinoma drugs from natural compounds.Arenobufagin(ArBu)was a natural monomer compound with strong anti-hepatocarcinoma effect.Studies have shown that ArBu could not only inhibit the proliferation,invasion and metastasis of hepatocellular carcinoma cells,but also induce their apoptosis.Tumor cells mainly regulated their invasion process through the signal pathways of NF-κB and MAPK,and blocked the cell cycle at G2/M phase to inhibit their proliferation and growth.ArBu could induce autophagy and apoptosis of HepG2 cells by regulating the PI3K/Akt/mTOR pathway.However,the regulatory effects of ArBu on endogenous molecules such as proteins,lipids and neurotransmitters in vivo were still unclear,and further research was urgently needed.With the rapid development of liquid chromatography-tandem mass spectrometry(LC-MS/MS)and systems biology,it provided a strong guarantee for network association analysis based on multiomics and the mining of potential biomarkers.Sodium Norcantharidate(NCTD-Na)was also an animal traditional Chinese medicine.Similar to ArBu,it also had anti-hepatocarcinoma effect.Therefore,we would study the two monomers at the same time in order to compare the similarities and differences of their anti-hepatocellular carcinoma mechanisms.Zebrafish has been widely used in drug development and utilization because of its transparent embryos in the early stage,small size,high similarity with human genes and strong reproductive capacity.In the present study,a zebrafish HepG2 xenograft model was successfully established.The pharmacodynamics and lipidomics analysis were preliminarily studied.In addition,on the basis of removing the background of zebrafish HepG2 xenograft model,systematic study on the anti-hepatoma effect of ArBu in vitro(HepG2 cell)was carried out.It included proteomics research,validation of key differentially expressed proteins,lipidomics research,neurotransmitter research and joint analysis based on systems biology.Pharmacodynamic experiments in vivo based on HepG2 xenograft model in nude mice demonstrated once again the anti-hepatocellular carcinoma effect of ArBu.ArBu had good anti-hepatocarcinoma effect.Its narrow therapeutic window and intervention mechanism for endogenous substances severely restricted its application as an anti-hepatocarcinoma drug in clinic.Therefore,it was urgent to study the regulation mechanism of endogenous substances based on LC-MS/MS platform,especially the study of lipid homeostasis,which was more closely related to the occurrence and development of hepatocarcinoma.In the present study,a large number of differentially expressed lipids,proteins and neurotransmitters were found,which provided a scientific basis for further understanding the anti-hepatocellular carcinoma mechanism of ArBu.1.Evaluation of anti-hepatocellular carcinoma effect of ArBuThe zebrafish HepG2 xenograft model was used to evaluate the anti-hepatocarcinoma effect of ArBu.The inhibitory effect of ArBu on the zebrafish HepG2 xenograft model was studied by fluorescence quantitative method,which provided concentration basis and pharmacodynamic basis for the lipidomics study.The maximum tolerance concentration of ArBu was determined with normal zebrafish.Then,1/9 and 1/3 of the maximum tolerance concentration were selected as the research objects.The zebrafish HepG2 xenograft model was established by injecting HepG2 cells labeled with CM-Dil into yolk sac of 2 dpf zebrafish using a microinjector.After 3 dpf,zebrafish with the same fluorescence were screened under fluorescence microscope.After administration of cisplatin(positive drug)and ArBu,10 zebrafish in each group were randomly selected to observe,photograph and analyze the image data.The total fluorescence intensity of each zebrafish was quantitatively analyzed and the total relative fluorescence intensity was calculated.The results showed that the maximum tolerance concentration of ArBu on normal zebrafish was 0.01 μg·mL-1.The inhibition rate of 14.9 μg·mL-1 cisplatin,a positive drug,was 26%on zebrafish HepG2 xenograft model,while the inhibition rates of 0.0011 μg·mL-1-ArBu and 0.0033 μg·mL-1-ArBu were 29%and 46%,respectively.In order to further verify the anti-hepatocellular carcinoma effect of ArBu in vivo,a HepG2 xenograft model in nude mice was established.The anti-hepatocellular carcinoma effect of ArBu was evaluated in terms of tumor volume,tumor weight and anti-tumor efficacy.The aim was to determine the pharmacodynamics of ArBu on HepG2 xenograft model in nude mice in vivo,and to compare the pharmacodynamics of ArBu with that of HepG2 xenograft model in zebrafish to obtain more pharmacodynamic data of ArBu against hepatocellular carcinoma.The method was that a certain number of nude mice were selected in advance.HepG2 cells were inoculated subcutaneously into the right back of nude mice.When tumors grew to a certain volume,they were cut into small pieces and inoculated them into the axilla of new nude mice to establish a nude mice HepG2 xenograft model.After 7 days,nude mice without tumors were excluded.The nude mice with qualified tumors were randomly divided into multiple groups and administered with ArBu for 21 days.The tumor volume,tumor weight and anti-tumour effect were detected on time.The research result indicated that the tumor volume of mice in model group reached 1361.97 mm3 after 21 days of administration.The volume of tumors in 15 mg/kg-ArBu group and 45 mg/kg-ArBu group were 756.16 mm3 and 736.85 mm3 respectively(P<0.05).The TGI values were 47%and 51%respectively,which indicated that ArBu could significantly inhibit the growth of tumors in vivo.2.The anti-hepatocarcinoma mechanism study of ArBu based on lipid homeostasis regulationTaking the zebrafish HepG2 xenograft model as the research object and LC-MS/MS as the technical support,a comprehensive lipidomics study combining targeted with untargeted methodes was carried out to elucidate the regulation of ArBu on lipid homeostasis in the process of anti-hepatocellular carcinoma.The combined use of the two analytical methods was conducive to find more and more reliable potential biomarkers.Targeted analysis was mainly accomplished by extracting retention time and characteristic ion parameters from Skyline software based on lipid standard database.Untargeted analysis mainly was completed by using Progenesis QI software for data processing,SIMCA 14.1 software for statistical analysis,HMDB and LIPIDMAP database for identification.In the zebrafish HepG2 xenograft model,16 differentially expressed lipids were revealed in the targeted lipidomic analysis,including 8 PCs,7 PEs and 1 SM.13 differentially expressed lipids were found in untargeted lipidomic analysis,including 8 PCs,4 TGs and 1 PE.There were 5 common lipids obtained by two different analytical methods.They were PC(22:6/16:0),PC(18:2/16:0),PC(20:4/20:3),PC(20:3/16:0)and PE(22:6/16:0),respectively.Therefore,24 potential lipid biomarkers have been identified in the present study.Analysis of differential lipids on the MetaboAnalyst website revealed that the glycerophospholipid metabolic pathway was the primarily affected metabolic pathway by ArBu.In order to further explore the mechanism of ArBu against hepatocellular carcinoma,HepG2 cells was used as the research object to carry out lipidomics verification and proteomics research at the same time,so as to make a multiomic network association analysis between the obtained differentially expressed lipids and proteins,to find a breakthrough in the mechanism of ArBu against hepatocellular carcinoma as a whole,and to explain the possible metabolic pathway of ArBu.Metabolism of small molecules such as neurotransmitters was closely related to the occurrence and development of hepatocellular carcinoma.At the same time,the changes of neurotransmitters in HepG2 cells before and after the administration of ArBu have been determined when the multiomics studies were performed,which further explain the regulatory effect of ArBu against hepatocellular carcinoma on endogenous substances.HepG2 cells were more pure,without the complex biological background of the zebrafish HepG2 xenograft model,which were more suitable for the study of ArBu agaist anti-hepatoma mechanism.In order to further explore the regulation of ArBu on macromolecule proteins,small molecule lipids and neurotransmitters in cells,lipidomics and proteomics analysis were studied on the LC-MS/MS platform.The differential proteins,lipids and neurotransmitters screened by the above methods could be compared and analyzed at a single or multiple level.At the same time,these endogenous differential metabolites could be further validated as potential biomarkers.In the lipidomics study of HepG2 cells for ArBu,the raw data were analyzed by OPLS-DA model to obtain correlation load maps(S-plots).Screening between two groups was performed with P<0.05,VIP(Variable Importance in the Projection)>1 and intra-group RSD<30%.It was found that there were 470 differential lipids between the control group and the low-dose group.There were 167 between the control group and the high dose group.When the control group,the ArBu low-dose group and the ArBu high-dose group were compared,81 differentially expressed lipids were excavated with VIP>1,P<0.05,RSD<30%and in a ArBu-dependent manner.Finally,24 differential lipids with MFC>1.5 or<0.7 were selected as potential biomarkers,including 12 PCs,4 DGs,2 PEs,2 SMs,2 TGs,1 Cer and 1 PA.The differential lipids rising in a ArBu-dependent manner were polyunsaturated PC,PE,SM,TG and Cer.The dose-dependent decrease of lipids was saturated/monounsaturated PC,DG and PA.The glycerophospholipid metabolic pathway was also the main pathway for the influence of ArBu on HepG2 cells.The non-standard quantitative proteomics study of HepG2 cells treated with ArBu was performed using iBAQ software.A total of 921 differentially expressed proteins were found with RSD<30%and in a ArBu-dependent manner.After the analysis of MV.4.9.0 software,ANOVA was used for statistical test.When the ArBu-administered groups were compared with the control group respectively,521 differentially expressed proteins were found with P<0.05.Among them,41 differentially expressed proteins were obtained by functional annotation with UniProt and GO databases.The ratio of the largest differentially expressed proteins was more than 2.0 less than 0.5.It was found that differentially expressed proteins were mainly involved in autophagy,regulation of apoptotic signaling pathway,cell development,signal transduction,cell cycle,cell surface receptor signaling pathway,cell metabolism,enzyme linked receptor protein signaling pathway,apoptosis,transmembrane receptor protein tyro sine kinase signaling pathway,protein phosphorylation,regulation of cell proliferation,DNA repair,regulation of phosphorylation,cellular component organization,RNA synthesis and metabolism,regulation of necrosis process,programmed cell death regulation,cell response to tumor necrosis factor and other biological processes.In this study,KEGG database was used to explore the signaling pathways that differentially expressed proteins might participate in.The results showed that it was mainly involved in cancer pathway,endoplasmic reticulum protein processing,endocytosis,p53 signaling pathway,cysteine,methionine and glutathione metabolism,amino acid-related metabolism such as arginine and threonine,and amino acid biosynthesis.Based on the network analysis,it was found that the differential proteins of TSPO,STAT3,RPS27 and LCLAT1 were closely related to the progress of liver cancer.The validation results were consistent with proteomics and decreased in a dose-dependent manner by RT-PCR.29 neurotransmitters in HepG2 cells were detected using Agilent 1290 LC-Agilent 6490 QQQ-MS instrument.The results showed that 7 amino acids in HepG2 cells were significantly overexpressed,including arginine,glutamine,isoleucine,tyrosine,leucine,glutamic acid and threonine.When ArBu was administered to HepG2 cells for 24 h5 the contents of 7 amino acids decreased by about half(48%-58%).But there was no dose-dependent relationship except glutamic acid.3.The activity evaluation and anti-hepatocarcinoma mechanism study of NCTD-Na based on lipid homeostasis regulationWhen the pharmacodynamics and anti-hepatocellular carcinoma mechanism of ArBu were carried out,some researches on sodium norcantharidinate(NCTD-Na)were also performed.In order to compare the pharmacodynamics and mechanism of ArBu and NCTD-Na against hepatocellular carcinoma and excavate the similarities and differences,the inhibitory effect,lipidomics and proteomics(only in HepG2 cells)of HepG2 cells and L02 cells were preliminarily investigated.Lipidomics and proteomics analysis of NCTD-Na in HepG2 cells were studied by LC-MS/MS technology.Meanwhile,lipidomics study of NCTD-Na in L02 cells was also studied.The differential lipids and the metabolic pathways in the two cells were analyzed to compare the similarities and differences between the anti-hepatocellular carcinoma mechanism.In the lipidomic research of NCTD-Na in HepG2 cells,40 significant differential lipids were screened and identified,including 18 DGs,8 MGs,6 PCs,and 3 Cers,2 SMs and 1 PE,1 TG and 1 PA.In the lipidomic study of NCTD-Na in L02 cells,32 potential biomarkers were also screened and identified,which included 10 LysoPEs,5 LysoPCs,4 DGs,and 3 PCs,3 Cers,1 SM,1 PE,1 PA,1 LysoPA,1 PG,1 FAHFA and 1 PS.The metabolic pathways affected by both were the glycerophospholipid metabolic pathways.It also suggested that the metabolic pathways that NCTD-Na and ArBu mainly influence were the same.In summary,ArBu showed a certain anti-hepatoma effect on the two liver cancer models,including the zebrafish HepG2 xenograft model and the nude mice HepG2 xenograft model.In the present study,the lipidomics research of the zebrafish HepG2 xenograft model in vivo and HepG2 cells in vitro were analyzed.The effect of ArBu on lipid homeostasis in vivo was basically elucidated.Among them,PCs,PEs,TGs,SMs and Cers were the main lipid types affected by ArBu.The predominantly affected lipid metabolism pathway was the glycerophospholipid metabolic pathway.In addition,the proteomic analysis in HepG2 cells and its association with lipidomics,key proteins mainly affected by ArBu,such as TSPO,STAT3,RPS27 and LCLAT1,were identified.At the same time,7 kinds of intracellular amino acids mainly affected by ArBu in the anti-hepatocellular carcinoma process,namely arginine,glutamine,isoleucine,tyrosine,leucine,glutamic acid and threonine were also found.Both ArBu and NCTD-Na could regulate the lipid homeostasis of HepG2 cells,which has similarities and differences.The metabolic pathways mainly affected by both ArBu and NCTD-Na were the glycerophospholipid metabolic pathways. |
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