Research On Proteomic Approach To Analysis On Liver Protection Mechanism Using Dimethyl-4, 4'-dimethoxy-5, 6, 5', 6'-dimethylenedioxybiphenyl-2, 2'-dica Rboxylate Regulated Cytochrome P450 Isozyme

Dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylenedioxybiphenyl-2,2'-dicarboxylate (DDB) is an intermediate process of synthesizing Schizandrin C,a natural compound isolated from Fructus schizandrae chinensis.Clinical experience has shown that DDB is effective in lowering the serum glutamie pyruvic transaminase activity in patients with chronic viral hepatitis.DDB protects against carbon tetrachloride-, D-galaetosamine-,and thioacetamide-induced hepatic injury.Moreover,the enhanced effectiveness in combination with garlic oil and the protective effect of DDB against ethanol-induced fatty liver were reported.Currently,this drug is widely used in Asian countries.Cytochrome P450(CYP450) is one of the most important representatives of drug metabolism enzymes,and plays a key role in the metabolic inactivationlactivation of numerous exogenous and endogenous.CYP exists mainly in liver microsomes.CYP superfamily shows wide range,poor selectivity,large structure difference, inter-individual variation,overlapping substrate specificities in exogenous metabolism and can be easily induced and inhibited by a number of chemical compounds.There is, therefore,a great probability of competition between drugs and endogenous compounds for the same enzyme,between different enzymes for the same substrate and between two drugs for the same enzyme.Meanwhile,many drugs are not only the substrate of the enzyme,but also the inducer or inhibitor of the enzyme or others.This may change the pharmacological and toxicological effects of drug itself or other drugs, which is one of the reasons resulting in drug interaction in pharmacokinetics.Testing for drug-drug interactions has been the subject of a FDA guidance document in 1997. At present,in order to decrease the risk in the development of new drugs,the international pharmaceutical factories have enhanced the research of absorption, distribution,metabolism,excretion and toxicology.Commonly adopted techniques for the eytochrome P450 identification are based on protein immunodetection,enzyme activity assays and detection of P450 mRNA levels. Some of these techniques have inherent limitations.Immunoblotting is a very sensitive method but it requires the preparation of cytochrome P450 forms-specific antibodies.A PCR-based approach is not reliable enough as there is no precise correlation between mRNA levels and protein expression.Enzyme activity assays may also produce confusing results due to the cross-reactivity of marker substrates.In view of the above-mentioned difficulties,protein separation methods coupled with mass-spectrometric protein identification provides an attractive approach for the analysis of expressed proteins.Proteomics is a new and rapidly evolving scientific discipline with many promising applications.This technology enables us to detect low concentrations of multiple proteins in a single analytical run.After the accomplishment of the Human Genome Project,the emphasis of the life science research has shifted from the structural genomics to the functional genomics. As one of the most important areas of functional genomics,proteomics mainly focuses on studying the genes' products and their functions.Recently,on the basis of the mature protein identification,a lot of researchers have been more interested in quantitative proteomics because it can help know the function of proteins,the network of the protein-protein interaction,the target of drug discovery and the protein markers for diagnosis.In the present,the method for quantitative proteomics can be divided into two kinds.One is the method that combines two dimensional electrophoresis with the image analysis,the other is that that combines the stable isotopic label with the mass spectrometry analysis.Among the various methods of the latter,the isotope affinity coded tag(ICAT) is the most widely used method for its high accuracy,the capacity to be directly applied to the sample in vitro and the ability to decrease the complexity of the sample.The necessities for large scale of proteome research are separation techniques with high resolution and proteins identification techniques with high throughput and high sensitivity.So in the first part of this research,SDS-PAGE gels was established and optimized.An optimal in-gel digestion procedure for SDS-PAGE separated proteins acquired via additional reduction and alkylation of the protein in gel slice.As the linkage of 2DE separation and MS identification,in-gel digestion remains the primary procedure for peptide coverage.The original protocol of manual in-gel digestion has been re-adjusted to some extent,and was applied to the semi-automatic analysis, resulting in high speed and throughput;Many factors during the semi-automatic in-gel digestion,such as trypsin quantity,digestion duration,pre-desaRing or not prior to MS, et al,which would interfere with the peptide coverage and definition of MS identification severely are tested.The stable-isotope quantification in proteomics is based on the fact that proteins can be normally characterized through their tryptic peptide fragments.It is designable to determine differential protein concentrations of two samples under different conditions by labeling proteins or peptides with isotopicaUy distinct forms of a protein dedvatizing agent.A wide variety of coding strategies has been investigated.Among so many isotope labeling techniques,the acetylation technique provides an easy way to label all peptides universally in a system,by using d0/d3 isotopic distinct forms of N-acetyl-succinimide(d0/d3-NAS) at primary amino groups in tryptic peptides;1-D PAGE is able to separate all kinds of proteins including extremely acidic,extremely basic proteins and hydrophobic proteins,as the detergent SDS can be used to extract proteins from bio-samples,and owns excellent reproducibility than 2-D PAGE.Sample pre-fractionation is essential to the proteomic investigation because of the complexity of a certain proteome,and sequential extraction holds a great promise on protein fractionation prior to further analysis.Reasonable selection of the extraction solution is the key factor to the successive analysis of proteomics study.As the linkage of 2DE separation and MS identification,in-gel digestion remains the primary procedure for peptide coverage.The original protocol of manual in-gel digestion has been re-adjusted to some extent,and was applied to the semi-automatic analysis, resulting in high speed and th111ghput;Many factors during the semi-automatic in-gel digestion,such as trypsin quantity,digestion duration,pre-desalting or not prior to MS, et al,which would interfere with the peptide coverage and definition of MS identification severely are tested.A strategy for quantitative proteomics was established through H₆/D₆-acetic anhydride stable isotopic labeling.Some major influencing factors,such as reactive buffer system,acetic anhydride concentration,pH,temperature and reaction time,were optimized.The results showed that a full acetic anhydride labeling was realized in a pH 8.0 buffer composed of Na₂B₄O₇/H₃BO₄ after 30 minutes of reaction at 22℃,with a 25-fold molar excess of acetic anhydride over amino groups in peptides.Under the optimized conditions,the efficiency of acetic anhydride labeling for the tryptic peptides was up to 100%.The investigation on the dynamic range and quantitative accuracy were performed by observing and calculating the ratio of the peak intensity in mass spectra with several d₀/d₃-acetylated peptide couples from tryptic digestion of myoglobin and good linear correlations were obtained in the dynamic ranges of 1:10 and 1:30,with the R² value of 0.99 and 0.98 and RSD of 5%and 20%,respectively.By combining 1-D PAGE separation with acetylation isotope labeling,almost all proteins in a complex proteome can be quantified as an important complement to the other quantitative proteomic methods,such as 2DE or ICAT stragety,as shown in this work.We investigated the CYP450 protein expression in the in vivo hepatotoxicity of rats induced by CCl₄.With this approach,17 cytochrome P450 proteins were identified and quantified with high confidence.Among them,the expression amount of 2Cll,3A2,and 2 E1 were down-regulated,while that of 2C6,2B2,and 2B1 were up-regulated.Note:CCL₄ caused liver damage is directly related to CYP2E1.Besides, CYP2E1 is also related to liver protection function of DDB,which helps us to understand the work of the induced CYP2B1 which is another way to provide liver protection.The result of our work has for the first time discovered the function of target enzyme in DDB induced liver protection and its biological implications,through a CYP450 quantitative analysis using this method.In summary,results of this study have founded a solid experimental basis,also exploited a promising new prospect of proteomics study.