| As an anti-tumor agent,Huachansu has been used clinically for more than 40 years in China.It has injections,tablets,oral liquids.Huachansu is mainly used in liver cancer,lung cancer,digestive cancer,and chronic hepatitis B.It is purified from the dried skin of Bufo bufo gargarizans Cantor by the water extraction and alcohol precipitation method.The current chemical findings include:peptides,amino acids,organic acids,amides,bases,nucleosides,alkaloids and bufadienolides.Studies have found that lipid-soluble components,bufadienolides,are the main active substances of Huachasu against tumors.In this study,we conducted a series of studies on the chemical separation,in vitro and in vivo metabolism,anti-glioblastoma activity and cardiotoxicity of bufadienolides,in order to provide beneficial data for the clinical application of Huachasu.1.Isolation and analysis of bufadienolides in HuachansuOn the basis of the previous work of the research group,the conventional chromatography technique was used to isolate and identify the chloroform part of Huachansu.20 bufadienolides were obtained,including 2 new compounds(3-epi-Ψ-bufarenogin and 3-epi-arenobufagin)and 2 new natural products(3-epi-gamabufotalin and 3-oxo-arenobufagin).Due to the high response of the hydroxy hydrogen signal in this experiment,the 1H NMR data were specified in more detail.The spectroscopic features were supplemented on the basis of the existing literature.All 30 bufadienolides reference substances in research group were integrated and LC-MS/MS technology was used to identify the components of bufadienolides in Huachansu.The results showed that bufadinolides with large polarity,such as gamabufotalin,arenobufagin,hellebrigenin and telocinobufagin,were main components.Their basic chemical composition of bufadinolides in Huachansu was basically clarified.This section laid the material foundation for subsequent research.2.Metabolism of bufadienolidesIn this part,the phase I metabolism of 10 bufadienolides in human liver microsomes(HLMs)and rat in vivo metabolism of arenobufagin(major bufadienolide in Huachansu)were studied.UHPLC-ESI-Orbitrap MS was used to elucidate the metabolic characteristics of bufadienolides.Firstly,Hellebrigenin(1),hellebrigenol(2),arenobufagin(3),gamabufotalin(4),bufotalin(5),bufalin(6),telocinobufagin(7),cinobufagin(8),cinobufotalin(9)and resibufagin(10),a total of 10 typical components was selected for HLMs · experiment.Based on the summary of the fragmentation pattern of bufadienolides,39 metabolites were detected and identified.Six of these metabolites were identified by the reference standards.The dehydrogenated metabolite of arenobufagin(3),the isomerized metabolite of gamabufotalin(4),the hydroxylated metabolite of bufalin(6)and the isomerized metabolite of telocinobufagin(7)were the first time to biotransformation generated in HLMs and have been accurately characterized.The results of the analysis showed that cross-metabolism exsited in phase I metabolism of the bufadienolides,and hydroxylation and dehydrogenation were the main metabolic types of these 10 compounds.The main metabolic pathways for compounds 1,3,4,5,6,8 and 10 were hydroxylation.No hydroxylation occurred in compounds 2,7 and 9,and their main metabolic pathway was dehydrogenation.The hydroxylation reaction most probably occurred at the A/B rings,especially the C-5,which was an easy substitution site.Besides,the dehydrogenation and isomerization always happened at C-3.After the recombinant human enzyme experiment,it was found that CYP3A4 was the main subtype of the reaction enzyme,and CYP2D6 specifically catalyzed the dehydrogenation reaction.Furtherly,hellebrigenin(1),hellebrigenol(2),arenobufagin(3),bufotalin(5)and bufalin(6),which hold higher metabolic rates in HLMs and had better activity was selected.Through the effective solid phase extraction(SPE)method to remove impurities and enrich the equal equivalents before and after HLMs metabolism,their anti-tumor activity was evaluated.This provided a new strategy for evaluating the activity of in vitro metabolites.The results showed that although the cytotoxicity of the bufadienolides metabolized by HLMs was reduced,its cell lethality was still significant.The in vivo distribution and metabolism experiments of arenobufagin,the major bufadinolide in Huachansu,showed that arenobufagin can rapidly enter into the blood after oral administration.Hydroxylated and reduced products were the major metabolites in liver,which were different from HLMs.In plasma and other tissues was mainly the prototype.By comparison with the metabolism in HLMs in the first section,the structures of the reduced products were analyzed which were thought to be 12-hydroxy-gamabufotalin and its isomers.The results of tissue distribution showed that apart from liver,heart,spleen,lung and kidney,arenobufagin was also found in cerebrospinal fluid,indicating that oral arenobufagin can also penetrate the blood-brain barrier(BBB),suggesting the potential of Huachasu for the treatment of brain tumors.3.Bufadienolides against human glioblastoma cell line U-87 in vitroIn order to explore the anti-brain tumor effect of bufadienolides,we studied the effects of arenobufagin and hellebrigenin on glioblastoma cell line U-87 in this part.After 48 hours of treatment with different concentrations of arenobufagin and hellebrigenin,cell viability was observed to be significantly and dose-dependently reduced in U-87 cells.Their IC50 values were 24.9±2.8ng/mL,and 23.5±2.4 ng/mL,respectively.At the concentration more than 100 ng/mL of arenobufagin and hellebrigenin for 48 h,a large number of U-87 cells were round up and became detached.At the concentration less than 40 ng/mL,although the number of cells in each area was observed to be significantly reduced,there was little effect on cell morphology.Similar cytotoxicity was not detected in mouse primary astrocytes.A mechanistic study was conducted and found that necrotic-like cell death was only observed in the cells treated with a relatively high concentration(>100 ng/mL).Both drugs down-regulated the expression of Cdc25C,cyclin B1 and survivin,accompanied by G2/M arrest.These results indicated that both drugs have strong cytotoxicity and selectivity for U-87 cells,and related to G2/M phase arrest and/or necrosis.To investigate whether the PI3K/Akt and/or MAPK signaling pathways were involved in the cytotoxicity of arenobufagin and hellebrigenin,some experiments were conducted.Western blot results showed that the activation of p38MAPK was closely related to the cell killing effect of the two components.Akt and Erk might partial participated.In addition,the level of phosphorylated JNK was not detected regardless of the presence or absence of drugs,indicating that JNK has no correlation with the cytocidal effects of the two drugs.SB203580,a specific inhibitor of p38MAPK,caused a significant decrease in cell viability and further enhanced the cytotoxicity of both drugs,indicating that p38MAPK plays an important growth-promoting role in U-87 cells.In addition,p38MAPK inhibitors experiments on LDH release,DNA ladder,and cell cycle showed that activation of p38MAPK was not associated with cell membrane damage,apoptosis,and cell cycle arrest.These results combined with the characteristics of bufadienolides that which can through the BBB,suggesting the possibility of clinical application of Huachansu in patients with glioblastoma.4.Cardiotoxicity of bufadienolidesConsidering that the bufadienolides belong to the B-type cardiac aglycone structure and have potential cardiotoxicity,this part of the study investigated the cardiotoxicity of arenobufagin to provide reference for the safety of Huachansu.Our previous studies showed that the LD50 value was 16.2 mg/kg after the mice injected intraperitoneally with arenobufagin.In this study rats were selected as the subjects.The oral administration of arenobufagin might cause cardiotoxicity were set at high(120 mg/kg)and low(60 mg/kg)dose according to the pre-test.First of all,through the traditional toxicity research methods,including the detection of heart rate,pathological sections and biochemical indicators,the cardiac effects caused by arenobufagin were investigated.The results showed that the low dose of arenobufagin increased the heart rate,while the high dose mainly reduced the heart rate,whch showed a two-way adjustment.The detection results of myocardial enzymes AST,CK,LDH,HBDH,CKMB,heart failure index BNP and cardiac pathological sections showed that arenobufagin might have a certain inhibitory effect on heart function in rats.Further,a rapid and sensitive LC-MS/MS method was established to determine the pharmacokinetics of arenobufagin in rat plasma at toxic doses.The method validation proved that the method was stable and feasible.Arenobufagin can be detected in 2 min in the rat blood.The Tmax of the low-dose group and the high-dose group was approximately at 0.6 h,and the Cmax and the in vivo retention time(MRT)increased with the increase of the dose.The changes in heart rate of rats after administration of arenobufagin were all around Tmax· After the drug was quickly eliminated,the heart rate returned to be normal when drug was under certain concentration(After 1 h in the low-dose group,and after 110 min the high-dose group).After that,ultra-high performance liquid chromatography-quadrupole orbitrap high resolution mass spectrometry(UHPLC/QE-MS)was used to establish a non-target lipid metabolic fingerprints of rat serum and hearts.Principal component analysis(PCA)and orthogonal partial least squares-discrimant analysis(OPLS-DA)were used for data processing and screening for differential compounds.The qualitative analysis of differential metabolites was performed by searching online database HMDB.The differential lipid molecules in serum are mainly cholesteryl ester(CE),glycerol phosphatidylcholine(PC)and sphingomyelin(SM).The main differential lipid molecules in the hearts were glycerophosphatidylcholine(PC).phosphatidylethanolamine(PE)and triglycerides(TG).A total of 20 dose-dependent differential lipid molecules were analyzed for biological significance.It was found that lipid perturbation may be related to fatty acid metabolism,sphingomyelin metabolism,phosphatidylcholine biosynthesis synthesis,and glycerophospholipids metabolism.The metaboanalist analysis showed that it might have a greater impact on glycerophospholipid metabolic pathways.Simultaneously,proteomics analysis of heart tissue was performed using a label-free protein quantification method,and relative quantitation of protein hydrolyzed peptides was performed by a liquid chromatography and mass spectrometry technique.95 different proteins were screened from more than 3,000 proteins,of which there were 29 differential proteins in a dose-dependent manner(up or down).According to the analysis of UniProt database and Go database,the function of down-regulated proteins tended to be related to coagulation,hemostasis,and wound healing functions.Most of the up-regulated proteins were involved in the synthesis and metabolis,and were mostly found in mitochondria.Down-regulation of coagulation and hemostasis and wound healing-related proteins may be related to red blood cell infiltration in the drug delivery group.Mitochondria-related protein changes may affect the energy metabolism process.Some protein perturbations are associated with changes in calcium concentration and may be the cause of heart rate imbalance.Proteomics should be correlated with lipidomics results for further exploration of targets and pathways. |
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