The Mechanism Of Aspirin On The Inhibition Of The Growth Of Saccharomyces Cerevisiae

Aspirin,also known as acetylsalicylic acid,has been used as antipyretic and analgesia for centuries.It is one of the three classical drugs in the history of medicine and is still the most widely used as antipyretic,analgesic as well as anti-inflammatory drug in the world.According to some reports,approximately 40,000 tons of aspirin were consumed to treat various diseases globally every year.As further study on the drug,other potential pharmacological effects of aspirin,such as anti-cancer,antibacterial activity and prolonging life expectancy,were discovered by researchers.Nevertheless,clinical adverse reactions such as intestinal bleeding,Reye's syndrome,liver and kidney impairment have gradually been reported when aspirin was widely applied.Hence,study on the specific molecular mechanism of action of aspirin is not only conducive to the development of its new pharmacodynamic functions,but also to guide the clinical use of drugs and avoid the side effects during drug administration.Saccharomyces cerevisiae is widely used in the study of genetics and cell biology,and it is also an excellent unicellular model for exploring the mechanism of drugs or compounds action.Compared to mammals and other pathogenic fungi,yeast genes are easily mutated,knocked out,over-expressed,and marked,providing a wealth of information on the molecular mechanisms of small molecule compounds.In order to deeply and systematically explore the molecular mechanisms of action of aspirin and provide theoretical basis for clinical drug use,this article utilizes the advantages of S.cerevisiae as a model organism in gene function and metabolic regulation to study the intracellular targeting genes or signaling system that was inhibited by aspirin.The research content of this paper mainly contains the following four parts:Part ?:Screening of targeting genes or signaling pathway of aspirin in yeast based on chemical genetic analysisIn order to find potential molecular targets for the aspirin,we took advantage of yeast knockout(YKO)mutants collection and gene-expression level of fitness-based assays.Homozygous diploid mutants that including 4200 non-essentials genes were screened using high-throughput gene chip technology for aspirin drug susceptibility screening.35 genes which cell show more sensitivity to aspirin after they were deleted in cells were first selected.These genes can be divided into four groups:The first was the non-specific drug resistance genes such as RPL27A,SSO2,RAD33,ATG39,etc.;The second is the genes that related to the regulation of the synthesis of saturated and unsaturated fatty acids such as MGA2,ECI1,etc.;The third is the genes that relate to the regulation of chitin synthesis and maintain cell wall integrity such as CDA1,WSC3,SLG1,etc.;The fourth is ATP and other energy synthesis and its related regulatory genes such as COR1,PMA2,ECM31,etc.;The monoploid and heterozygous diploid strains were further confirmed by sensitive test.The discovery that aspirin interfered with genes belonging to the second and third group in yeast is novel to the study of the medicinal mechanism of aspirin.Furthermore,MGA2 was identified as a hypersensitive gene.We hypothesized that aspirin interfere into fatty acid metabolism of yeast and the unsaturated fatty metabolism pathway MGA2 involved may be a specific target to aspirin in yeast.We hope to further explore the molecular mechanism of aspirin by applying genetics and biochemical techniques.Part ?:The accumulation of intracellular saturated fatty acid caused by aspirin leads to the damage on the stability of cell membraneWe found that MGA2 mutations exhibited hypersensitivity to aspirin after the screening of targeting genes.MGA2 is an important transcription factor that regulates the OLE1 gene.OLE1 encodes a ?(9)fatty acid desaturase that regulates the production of oleic acid and palmitic acid which promote the formation of unsaturated fatty acids that are essential for maintaining cell membrane stress and integrity.The gene DCI1 is functionally opposite to OLE1 and encodes a ?(3,5)-?(2,4)-dienyl-CoA isomerase that converts unsaturated fatty acids into saturated fatty acids to promote ?-oxidation of fatty acids.Mutant strains lacking DCI1(dcil?)or down-regulating OLE1(mga2?)gene showed resistance and sensitivity to aspirin,respectively.Overexpression of DCI1 made wide type sensitive to aspirin and also restored the sensitivity of dci1? to aspirin.Overexpression of OLE1 weakened resistance of dci1? to aspirin,The unsaturated fatty acid index of the cells treated with aspirin was significantly decreased based on the result of GC-MS,and the unsaturated fatty acid index was significantly decreased in dci1?.Real-time quantitative PCR analysis on the gene expression of OLE1,DCI1 in cells showed that the inhibition of aspirin on these genes are in a dose-dependent manner,consistent with the result of analysis on fatty acid composition,aspirin induced the accumulation of saturated fatty acids in cells by promoting the expression of DCI1 and inhibiting the expression of OLE1.Changes in the proportion of fatty acids in cells can impact on composition,fluidity,and stability of cell membrane.Through the analysis on cell membrane integrity(PI staining),we found that the stability of membranes treated with aspirin was decreased,and the damage on plasma membrane become seriously.This may be a major mechanism for aspirin to inhibit the growth of fungi.Part ?:Aspirin inhibits cell growth by interfering with the mannoprotein synthesis of the cell wall in yeastAlthough the mutants lacking the third type of genes such as CDA2 and WSC3,which maintain cell wall integrity,have no apparent sensitivity to aspirin,we have further determined that aspirin cause damage on the cell wall in dose-dependent manner through scanning electron microscopy and cell hydrophobicity.Mutants lacking key genes in the pathways of chitin(chs1?,chs2? or chs3?)or cells with overexpression genes(ALG7,SEC53,DPM1,etc.)in pathway of mannoproteins were employed to aspirin sensitivity test.We found that only overexpressing esstential gene DPM1 in yeast cells can improve the resistance to aspirin.The result of RT-PCR and Western Blot showed that aspirin significantly down-regulated DPM1 expression levels.Observations of scanning electron microscope confirmed that overexpression of DPM1 can largely restore damage caused by aspirin on cell wall.Further studies have shown that exogenously increasing the substrate of DPMlp catalyzes enable yeast cells to exhibit stronger aspirin resistance.Thus,we speculated that DPMlp may be a potential target protein of aspirin.By bioinformatic analysisof simulating the interaction between drug and protein molecules,8 potential aspirin binding sites on DMP1p were discovered.In addition,it was found that aspirin can also down-regulate DPM1 gene expression in HCT116 cells,suggesting that this mechanism of aspirin may have the conservation of eukaryotes.Part ?:Analysis on the energy metabolites in yeast cells based on chromatography and the effect of aspirin on cell energy metabolismAccording to our previous result of screening and some research reports,aspirin can cause changes in the content of energy metabolites in cells such as ATP,NAPDH and so on.However,its biological significance is unclear.Proposal on measuring the content of energy metabolites ATP,ADP and AMP in cells,and the analysis of the dynamic state of the energy in vivo can be of significance for the study of metabonomics and the mechanism of drugs.On this purpose,we have established two simple,effective methods with high-accuracy that can simultaneously detect ATP,ADP,AMP and cAMP based on high performance liquid chromatography and capillary zone electrophoresis.The recoveries of four nucleotides in the methods are all above 90%,indicating that they are suitable for dynamically analyzing the changes of energy metabolism in the yeast.Compared with HPLC,CE has advantages such as higher sensitivity and less consumption on sample.It was applied to analyze the effect of aspirin on yeast energy metabolism.Through statically analysis,it was found that aspirin can reduce the energy metabolism of yeast and inhibit the growth of yeast.Aspirin is widely used and its mechanism of action is complex.In this dissertation,model yeast was used to study the molecular mechanism of aspirin on cell process through systemic biology.It was found that aspirin specifically interfered with fatty acid metabolism in cells and the down-regulation of unsaturated fatty acid index decreased the stability of the plasma membrane.Its inhibition of the synthesis of the mannoglycoprotein of the cell wall impaired the integrity of the cell wall.Additionally,aspirin reduced the energy load,leading to the inhibition of yeast growth.Our investigation provides basic data for further study of molecular mechanism of aspirin and guidance for clinical medication.