| Objective:To establishment and optimization of NMR-based cell metabonomics study protocols for neonatal Sprague-Dawley rat cardiomyocytes.Investigate the toxical changed rule and toxical mechanism of aconitum alkaloids(aconitine,hypaconitine and mesaconitine),and explore the attenuated mechanism of the compatibility of aconitine with 6-gingerol.Method:1.In order to optimize the sample preparation method.we conducted the comparison of metabolic profiles and PCA analysis to evaluate the metabolite extraction efficiency of different cell collection methods and different extraction solvents.2.By using toxicology experimental methods to investigate the toxicity of aconitum alkaloids and attenuation effect of 6-gingerol and glycyrrhetinic acid after compatibility.3.Metabonomics study of the mechanism of toxicity of aconitum alkaloids on primary neonatal SD rat ventricular cardiomyocytes by using the established method as above.4.Metabolomics study the attenuated mechanism of 6-gingerol and aconitine compatibility by using the established method as above.Result:1.In order to investigate the extraction efficiency of sample preparation methods,for collecting cells,trypsin digestion and direct scraping method were used,methanol and acetonitrile were used as the solvent extraction.We selected 19 metabolites in the spectrum,which were less overlaps in 1H NMR spectrum,and compared the metabolite concentration.We compared the extraction efficiency of cell collection method and comparison of different extracting solutions using the PCA analysis method,the results show that using trypsin digestion method collecting the cells can destroy cell membrane and cause choline phosphate dissociate to the cytoplasm.As the cell membrane damage,low molecular metabolites in cells will leak into the culture medium,and cause the in intracellular metabolite concentrations changed,at the same time the metabolic profiling of cells are different.The metabolites concentration of collected by trypsin digestion method is only 39.1%comparing with the directing method,the relative concentration of trypsin digestion were collected and choline phosphate method is higher than that of direct scraping group.In addition,the consuming time by trypsin digestion method was more than the direct collecting method.For the comparative on cell extraction was found in the reagent,the extraction efficiency of acetonitrile is 8.06%higher than methanol.In the selected 19 metabolites,the concentration of 17 metabolites in acetonitrile extraction group was high.The optimized sample preparation method were harvested directly by using ACN scraping the cells,and then extracted with 50%ACN.2.Using MTS and LDH methods to investigate the toxic and attenuated effect of aconitum alkaloids and the compatibility with 6-gingerol.The results showed that the toxicity of three kinds of aconitum alkaloids on myocardial cells enhanced with increasing of dose and time.The cell viability using ITS(serum substitute)instead of 1%FBS was higher.Attenuated role in the study of the compatibility of discovery of 6-gingerol compatibility of aconitum alkaloids are attenuated effect of certain compatibility.3.To investigate the toxicity of aconitum alkaloids on primary neonatal Sprague-Dawley rat cardiomyocytes using metabonomics,two doses of aconitum alkaloids(3?M and 12?M)was used to incubate with cells.The result showed that,the metabolites concentrations between each group were tyrosine,lactic acid,alanine,glutamate,glutamine,citric acid,taurine,glycine and serine etc..The toxicity of three different kinds of aconitum alkaloids also shows a different intensity.Toxicity of hypaconitine,mesaconitine,aconitine are increased to myocardial cells,which had significant difference between aconitine and the control group(p=3.62E-05),and have obvious toxicity.A separate analysis of aconitum alkaloids between different groups were found:the metabolites of aconitum alkaloids of different concentration on cell metabolites influence is slightly different,but the concentration of metabolites of aconitum alkaloids on cell reduced metabolites are basically the same,wherein the differences in taurine,choline and phosphocholine metabolites were changing mostly,at the same time,the concentration of acetate in aconitum alkaloids groups was lower.According to the concentration of various metabolites obtained information analysis of hypaconitine and mesaconitine in the data,we conducted metabolic pathway analysis and enrichment analysis.According to the results of the analysis found the significant differences metabolites were mainly exists in the taurine and hypotaurine metabolism pathway and glutathione metabolism metabolism pathway.Suggested that the metabolic pathway and the role of the main mechanism of the effects of aconitum alkaloids on myocardial cells.Metabolic pathway analysis found that aconitine alkaloids have a variety of metabolic pathways of cells.Some antioxidant substances used for intracellular metabolites have also changed,such as taurine and glutathione.Aconitum alkaloids on the possible impact of arrhythmic cardiac toxicity of aconitum alkaloids on the phenomenon and phospholipid metabolism pathway related.Aconitum alkaloids influenced by phospholipid metabolism and related effects of metabolic.pathways of intracellular acetylcholine synthesis pathway and glycerophosphocholine.Aconitum alkaloids may affect glycerophosphocholine metabolism into choline and acetylcholine synthesis influence,resulting in the occurrence of arrhythmia.4.In the analysis of 6-gingerol and aconitine compatibility data,the score plot of 6-gingerol compatibility of aconitine shows three groups were separated,and the combination group is more close to the control group indicated that the metabolism of cells on the compatibility of more close to the control group.This result suggesting that 6-gingerol compatibility of aconitine and produce a certain the attenuated effect.Especially in the combination with 12pM aconitine,detoxification of 6-gingerol is more obvious.Metabolism related the attenuated mechanism may be related to myocardial intracellular taurine-hypotaurine metabolism pathway,threonine/lactic acid and other metabolites.The specific mechanism needs further study reveals.Conclusions:In this work,we first established and optimized the nuclear magnetic resonance metabonomic in primary neonatal rat cardiomyocytes sample processing method,and first applied to the metabolic study of primary mammalian cells.The establishment of the method for metabonomic study provides a new research material,widens the applicable range of metabonomic studies,deepens the metabonomics study of theoretical,and provides a new way for the study of biological metabolic responses at the cellular level.At the same time,the application of this method was used to study the toxic effect of aconitum alkaloids on myocardial cells.Metabolism in reducing the toxicity of aconitine,hypaconitine and mesaconitine toxicity and 6-gingerol compatibility after aconitine has conducted the system research.The results of the study on toxicity of elucidating the mechanism of action of aconitum alkaloids metabolic effects on cell are discussed,a metabolic pathway map of aconitum alkaloids on primary neonatal SD rat myocardial cells was described,and laid the theoretical foundation for elucidating the mechanism of aconitum alkaloids provides methods and basis for the use of metabonomics method in Chinese Medicine and pharmacology,toxicology and safety evaluation. |
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