Toxicity Evaluation Of Trichosantoside I, II And VII And Exploration Of The Mechanism Of Hepatotoxicity Of Trichosantoside I

Objective:Drug-induced liver injury is a serious public health problem.According to survey statistics,the incidence of acute drug-induced liver injury in China is increasing year by year.Paris polyphylla is a commonly used Chinese medicine in clinical practice,and it has received increasing attention due to its anti-tumor activity.Paris saponin Ⅰ,Ⅱ,and Ⅶ are three important saponins monomers in Paris polyphylla,and their killing effects on a variety of tumor cells have been studied and reported.However,while its anti-tumor effect,saponins of Paris polyphylla have little research on toxic and side effects,especially their monomeric components.In this study,the potential toxicity of Paris saponin Ⅰ,Ⅱ,and Ⅶ and their mechanism of toxicity were deeply explored.It hopes to provide reference for the clinical rational application of Chinese medicine-Paris polyphylla and the research and development of drugs based on Paris saponins monomer.Method:(1)The toxicity of Paris saponins monomer was evaluated from the cell level,the model organism-zebrafish level,and the animal level.Three kinds of liver cells including HepaRG,HL-7702 and WRL-68 cells,three kinds of kidney cells including HK-2,HKC and 293T cells,and three kinds of heart cells including H9c2,HUVEC and HCMEC cells were used to evaluate the cytotoxicity of Paris saponin I,II and VII;Secondly,the model organism-zebrafish model was used to investigate the effects of different concentrations of Paris saponin Ⅰ,Ⅱ,and Ⅶ on the survival rate of zebrafish,and the LCso,LC10 and the maximum non-lethal concentration(MNLC)were determined;Take pictures with a stereo fluorescence microscope,observe the liver,kidney,and heart morphology before and after administration of Paris saponin Ⅰ,Ⅱ,Ⅶ,count zebrafish kidney edema rate,count zebrafish heart rate,and count pericardial edema rate,Use Image-Pro Plus 5.1.0 software to count the changes in zebrafish liver area and fluorescence intensity,kidney area,kidney fluorescence intensity,and heart SV-BA distance after Paris saponin Ⅰ,Ⅱ,and Ⅶ are given respectively.The above results are used to evaluate the potential liver,kidney,and cardiotoxicity of Paris saponin Ⅰ,Ⅱ,and Ⅶ.Then observe the liver tissue pathology and hepatocyte apoptosis of zebrafish before and after administration of Paris saponin I by HE staining and TUNEL staining;Next,through the acute toxicity experiment in mice,the LC50 was obtained,and then the biochemical test of mouse serum and histopathological section analysis were performed.Data at all levels support each other,complement each other and verify each other,making the research foundation more comprehensive and reliable,and the conclusions more reasonable.(2)HepaRG cells were used to study the mechanism of hepatotoxicity of Paris saponin I.Using microplate reader,flow cytometer,inverted fluorescence microscope and other instruments,the release of lactate dehydrogenase(LDH),DAPI staining,reactive oxygen species(ROS),mitochondrial membrane potential(MMP),cell apoptosis and cell cycle were measured;On the other hand,HUVEC cells were selected to study the cardiovascular toxicity mechanism of Paris saponin I.The cardiovascular toxicity mechanism of Paris saponin I was evaluated from two aspects:inhibiting the formation of new blood vessels and promoting the apoptosis of cardiovascular cells.The release of LDH,cell migration,cell apoptosis,DAPI staining,cell cycle,and angiogenesis in vitro were measured,and the expression of nearly 30 proteins that cardiovascular toxicity-related was determined.(3)TMT labeled quantitative proteomics technology was used to detect the differential proteins in the liver tissues of mice in the administration group and the control group,and perform GO and Pathway enrichment analysis on the differential proteins.Next,protein-protein interaction(PPI)network was constructed by string 11.0 and IPA software used to further screen out the protein markers related to phenotype of liver damage.On the other hand,Q300 targeted metabolomics was used to study the changes of endogenous metabolites in serum samples of mice in each group.PCA,OPLS-DA and other analytical methods were used to analyze the changes in the profile of mouse serum metabolites.Objective to compare the differences of serum metabolic profiles before and after administration,and to find out the metabolic differences and metabolic regulatory network related to Paris saponin I-induced hepatotoxicity.Furthermore,the Cytoscape software was used to perform correlation analysis on proteomics and metabolomics,and PCR and Western blot techniques were used to further verify the key proteins in the liver toxicity mechanism induced by Paris saponin Ⅰ.Results:(1)The results of the toxicity test of Paris saponin Ⅰ,Ⅱ and Ⅶ:Paris saponin Ⅰ,Ⅱ,and VII have obvious cytotoxicity,can significantly promote the apoptosis of 8 kinds of cells except H9c2 cells,and it is dose-dependent and time-dependent.For H9c2 cells,all three saponins showed the effects of low-dose to promote proliferation,and high-dose to inhibit proliferation;Results of zebrafish experiment:The LC50 of Paris saponin Ⅰ,Ⅱ,and Ⅶ against zebrafish were 121,109,99 ng/mL,LC10 were 213,178,146 ng/mL,and MNLC were 570,456,357 ng/mL,respectively.Regarding the evaluation of toxic organs,Paris saponin Ⅰ,Ⅱ,and Ⅶ have significant hepatotoxicity to zebrafish,which can significantly reduce liver area and fluorescence intensity.In addition,Paris saponin Ⅰ affect heart rate,suggesting cardiovascular toxicity.On the other hand,Paris saponin Ⅰ and VII can reduce kidney area and fluorescence intensity,and have certain nephrotoxicity.However,none of the three saponins caused renal edema.Next,the zebrafish pathological section showed vacuoles and severe hepatocyte necrosis in the liver tissue of administration group of Paris saponin Ⅰ.The results of TUNEL staining showed that hepatocyte apoptosis occurred in the administration group;Animal experiment results:It is shown that the Paris saponin Ⅰ have in vivo toxicity,and its median lethal concentration is 24.5 mg/kg.The serum ALT and AST in the administration group increased significantly,while SOD decreased.The pathological section of the administration group showed significant liver cell damage and steatosis,as well as certain cardiotoxicity.(2)The hepatotoxicity mechanism of Paris saponin I:Paris saponin Ⅰ significantly increased the level of ROS in HepaRG cells,which caused the decrease of MMP,the release of Cyt c from mitochondria to the cytoplasm,the increase of early and late apoptotic cells,and the increase of LDH release.Simultaneous administration obviously activated the expression of apoptosis pathway proteins contained Fas,P53,P21,caspase-3,caspase-8,and caspase-9,which were dose-dependent.And Paris saponin Ⅰ changed the expression of cyclin P21,cyclin E,cyclin A and CDK 2,and induced cell cycle arrest;The vasotoxicity mechanism of Paris saponin Ⅰ:It significantly promotes the release of cardiovascular cells LDH,inhibits cell migration and angiogenesis,changes the cycle distribution,and induces cell apoptosis.At the same time,the expression of nearly 30 proteins changed such as VEGFR2,p-VEGFR2,Src,p-Src,PI3K,Src,P38,PLCy and so on in HUVEC cells after administration.(3)Proteomics results:A total of 302 differential proteins were screened,including 182 up-regulated proteins and 120 down-regulated proteins.Differential proteins have molecular functions such as oxidoreductase activity and catalytic activity,and participate in biological processes such as fatty acid metabolism,organic acid metabolism,drug metabolism,and redox processes.These are mainly distributed in the cytoplasm and intracellular membrane-bound organelles including mitochondria and endoplasmic reticulum.Pathway results showed that differential proteins are highly enriched in pathways of drug metabolism-cytochrome P450,cytochrome P450 metabolism of foreign substances,peroxisome proliferator-activated receptor-y(PPARy)and so on.Next,the PPI network analysis found that the differential proteins aggregated in three functional areas,including cytochrome P450 enzymes,lipid metabolism,and redox reactions.IPA commercial database screened 9 differential proteins related to liver toxicity as potential biomarkers;Results of metabolomics:the cluster distribution of metabolites in the administration group deviated significantly from the control group.Through the intersection of multi-dimensional and single-dimensional differential metabolites,97 differential metabolites and 25 metabolic pathways were screened and preliminarily identified.Through generalization,it is concluded that Paris saponin I caused five aspects of metabolic abnormalities in the body,including amino acid metabolism,fatty acid metabolism,glutathione metabolism,energy metabolism,and protein synthesis.Among them,D-glutamine and D-glutamate metabolism,alanine,aspartic acid and glutamate metabolism,arachidonic acid metabolism,aminoacyl tRNA biosynthesis pathways have relatively high influence values.Representative differential metabolites are arachidonic acid,pyroglutamic acid,glycolic acid,acylcarnitine and so on.The hepatotoxicity mechanism of Paris saponin I studied in the full text was comprehensively analyzed,and the specific mechanism of hepatotoxicity caused by Paris saponin I was summarized and speculated.The expressions of key proteins Bax,CYP1A2,P53,and Fas were verified by qPCR and Western blot,so as to preliminarily verify the presumed mechanism of liver injury.Conclusion:(1)It was found that Paris saponin Ⅰ,Ⅱ and Ⅶ had hepatotoxicity,nephrotoxicity and cardiovascular toxicity;zebrafish experiments determined that the three most toxic saponin were Paris saponin Ⅰ,and the most definite target of toxicity were the liver and cardiovascular;Zebrafish pathological sections,TUNEL staining,detection of biochemical indicators in mouse serum,and pathological sections further verified the liver and cardiotoxicity of Paris saponin Ⅰ.(2)The mechanism of Paris saponin I hepatotoxicity is cell apoptosis mediated by ROS stress pathway and death receptor pathway.In addition,the main pathways of cardiovascular toxicity are VEGFR2-PI3K/AKT/mTOR,Src/eNOS,PLCγ/ERK/MEK,P38 signaling pathway,JAK2/STAT3 signaling pathway and Bcl-2 family-mediated apoptosis pathway,which produces cardiovascular toxicity from two aspects:inducing cardiovascular cell apoptosis and inhibiting new blood vessels.(3)The results of proteomics and metabonomics combined with the full text data suggest that the mechanism of hepatotoxicity induced by Paris saponin I may be hepatocye damage mainly caused by CYP450 enzyme and lipid metabolism mediated mitochondrial dysfunction.The drug affected:① A variety of CYP450 enzymes,mainly including phase Ⅰ metabolic enzymes including CYP1A1,CYP1A2 and phase Ⅱ metabolic enzymes including GSTA3 and UGT1A1,which are expressed disorderly,showing a downward trend.The hydrolysis and metabolism of the drug in the body are slowed down.With the extension of the action time,the drug toxicity reaction increases.Drugs that have not been metabolized may act on mitochondria and cause mitochondrial dysfunction;it is also very likely that the drug metabolizing enzymes activate drugs to produce toxic metabolic intermediates,which may cause liver damage;②Lipid metabolism.After mitochondrial damage,the function of β-oxidation is affected.Free fatty acids in the body cannot be oxidized and converted into triglycerides,which leads to the accumulation of lipids in the body,the reduction of ATP synthesis,and the impaired energy supply.Lipid accumulation can also cause lipotoxicity to further affect mitochondrial function and produce excessive ROS;③ Oxidative stress.Excessive ROS production induces mitochondrial oxidative stress,releases Cyt c,increases Bax,and decreases Bcl-2,which in turn activates the caspase family and leads to hepatocyte apoptosis.The verification results showed that the drug metabolizing enzyme CYP1A2 was significantly down-regulated and the expression of key apoptosis proteins Bax,P53 and Fas were significantly up-regulated,supporting the rationality of the mechanism inference.