| [BACKGROUND]Drug- drug interaction plays a more and more important role these years. A lot of lethal drug-drug interactions have been found. As many studies showed, when different drugs are applied together, interactions based on hepatocyte cytochrome P450 account for the main part and leads to severe consequences. Cytochrome P450, so called mixed function oxidase, monooxygenase or drug-metabolizing enzyme, is a super family which contains many isoenzymes widely located in animal, plants and microbes. Cytochrome P450 which metabolizes exogenous drug mainly exists in the microsomes of animal liver.When metabolizing exogenous drugs is mentioned, Cytochrome P450 has two functions. One is to lower the activity and deactivate the compounds. Most drugs has the character of lipotropy which is a disadvantage for the elimination of drugs in the body, so the density of the drugs accumulates to a toxic level. Fortunately, cytochrome P450 has can eliminate the drugs by catalytic oxidation which increases the drugs metabolic products' water-solubility and make it easy to be excreted through the kidney. Only the subtype CYP3A can catalize and metabolize 60 percent of the clinical drugs. This oxidization usually lowers the biologic activity of the substrates and the toxicity of the drugs. The other function is that it can activate the compounds through oxidization. Some substrate's water-solubility does increase a lot after metabolized, but after group modification, the biologic activity is reinforced. Arylamine and heterocyclic amine, which were found in fried or baked fish and meat,are indirect carcinogen and have no carcinogenicity, but with the N-hydroxylation and oxidization by CYP1A2 they acquired strong carcinogenicity. Since it was found in 1955, cytochrome P450 has been an attractive field in pharmacology and toxicology research because of its elimination of drugs and activation of pre-carcinogen.In vivo, there are many factors affecting the activities of cytochrome P450. Many drugs are the metabolic substrates of cytochrome P450, which are also enzyme's inducer and inhibitor. Since such drug exists, if given combinedly, one drug may regulate the cytochrome P450 which is related to the other drug, and as a result, the second drug's metabolizing will be affected, which will make the effect and toxicity of the second drug enhanced or weakened. That is to say, the drug interaction bases on metabolic enzyme, which involves two situations, one is that competitive saturation mechanism may enhance the toxicity by slowering elimination of drugs. For example, carbamazepine is metabolized by CYP3A, and carbamazepine's plasma concentration and toxicity will increase while drugs metabolized by CYP3A, such as cimetidine and isoniazid, are given at the same time. The second situation is that the drug's metabolic velocity will speed up or slow down, leading to the changes of drug effect or toxicity when one drug is metabolized by cytochrome P450, the other drug is induced or inhibited by it. For instance, cyclosponne A is one of acceptable CYP3A4's substrates, and its main metabolic path is hydroxylation and N-methylation. The combination of berberine hydrochloride and cyclosporine A can inhibit apparently CYP3A's activity, decreasing cyclosporine A's metabolizing in liver, increasing its plasma concentration, enhancing its therapeutic efficiency. Because of the importance of cytochrome P450 in pharmacology, the research on drug metabolic path and drug interaction is not only a pure theory question but also an important rule of research and development. Therefore, Food and Drug Administration (FDA) demands that every new drug's subtypes of cytochrome P450 must be assured before its further research. [OBJECTS]To study the effects on animals and induction-inhibitory action on cytochrome P450 with application of Euphorbia and Glycyrrhiza which are one pair of eighteen imcompatible pairs. Then find the effective subtype of cytochrome P450 and make further qualitative and quantitative investigation hoping to reveal part of the moleculemechanism of eighteen imcompatible pairs to accumulate evidence for clinical drug application. [METHODS]l.Make it sure weather Euphorbia and Glycyrrhiza play converse roles by observation of the existence of the rats and their liver tissue's pathological changes.2.Identify the subtypes of P450 protein by comparing the mass spectra of pr otein gained through Two-dimensional gel electrophoresis and One-dimensional gel electrophoresis and establish the method of isolating and identifying P450 isozymes in rat's liver microsomes.3.Make semi quantitative study of the identified P450 isozymes by RT-PCR and protein immunoblot assay.4.incubate the enzyme and substrate in vitro and compare the P450 isozymes' activity according to their specific products' absolute quantitation measured by high-performance liquid chromatography and nuclearic acid and protein analyzer. [RESULTS]1 .we found the combined use of Euphorbia and Glycyrrhiza may enhance the pathological damage of liver and render the survival condition worse by the comparison of pathological changes of liver and the survival condition of rats.2.By one way gel electrophoresis and biology mass spectrogran technique, we confirmed there were at least twelve protein subtypes of P450 in the liver of rats,and determined to study three isozymes, CYP1A2> CYP2E1 and CYP3A2 which are significant in human clinical trials.3.Undertaking the semiquantitative study of CYP1A2, CYP2E1 and CYP3A2 by RT-PCR and protein immunoblot assay technique, we concluded the combined use of Euphorbia and Glycyrrhiza has a various effect on contents of the three isozymes.4.We infered from the statistical analysis of CYP1A2, CYP2E1 and CYP3A2 activity that both combined Use and single use of Euphorbia and Glycyrrhiza may have different effect on the function of the three isozymes. [CONCLUSIONS]l.We established the methods isolating and identifying P450 isozymes based on the gel electrophoresis and biology mass spectrogram,which were more than the result identifying seven subtypes in 2002.2.Euphorbia and Glycyrrhiza played converse roles in terms of animals experiment and its result is to enhance the toxicity of medicine in Liver.3.Euphorbia and Glycyrrhiza played converse roles drawing from the element and function of CYP1A2,CYP2E1 and CYP3A2 based on the drug-metabolizing enzyme.4.CYP1A2 and CYP2E1 activated pro-carcinogen and pro-toxicant.Some literature reported that Euphorbia contained these components and that CYP1A2 and CYP2E1 beared correlation with the pathogenesis of carcinomas.The research showed that activity and content of CYP1A2 and CYP2E1 increased in the combined use group than in the single use group,which mean that the possitility of carcinogenesis increase.5.CYP3A2 metabolize sixty percent of medicine, enhance its hydrophilicity and make it easily to be eliminated .The researchs showed that the combined use of Euphorbia inhibit its activity much more than the single use of it .It meaned that CYP3A2 metabolization become slower ,CYP3A2 was accumulate and its toxicity was enhanced after the combined use of it.6.We introduced drug-metabolizing enzyme to raditional Chinese medicine imcompatibility study for the first time. |
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