Study Of Hypolipidemic Mechanisms And Efficacy Of The Compound Prescription Of FTZ Based On The HMG-CoA Reductase

Compound prescription consisted of Chinese herbs embodies the concept of wholism specifically in clinical practicing. In accordance with the rule of "sovereign drug,minister drug,adjwvant drug,courier drug", the compound prescription is made up of a combination of appropriate herbs with exact amount after the therapeutic method is determined according to the cause of the disease.And the effective ingredients will work on different parts of the human body in order to regulate the integral functions.The key points of compound prescription include material base, mechanism,and rules of combination, which are acknowledged by the academic circles, and also hot and difficult points in the research of Chinese medicine fundamentaltheories.The compound prescription of Fufang zhenshu tiaozhi fang(FZT) is for many years experience in clinical practice made by professor Guojiao. It has been demonstrated that has the effect of nourishing spleen and kidney, regulating liver qi, resolving phlegm ,cooling blood and removing toxin. Preliminary studies showed that the compound FTZ to have the function that lower blood TC,TG,LDL-C and increased HDL-C, such as the role of the lipid metabolism and can increase two key enzyme-LPL,ACOmRNA expression levels, regulating lipid metabolism better than Zhibituo, and Xuezhikang, similar to the role of fenofibrate, and no adverse reactions.this subject will further reearch the mechansim of multi-target effects of FTZ, which has a function of antiatherosclerosis and regulating the metabolism of blood lipid.Objective: Hyperlipidemia is a complex and multi-factor multi-target disease, which closed related to many factors such as genetic and environmental of diet and so on. The subject using gene chip technology to observe the changes in gene expression profiles of liver tissues of hyperlipidemia rats before and after administer FTZ. And found the impact of the key genes for lipid metabolism, and then study the gene expression, activity and function. Secondly,competitive inhibition experiments (receptor - ligand binding assay) will be used to found the active ingredients of the target molecule, then the active ingredient of FTZ and part of the mechanism will be confirmed combined with modern separation, purification and analysis of FTZ operation generated. The subject is expected to be completed which will lay the foundation for the development of lipid-lowering drugs.Methods: (1) The gene chip technology was used to observe Observe the changes in gene expression profiles of liver tissues of hyperlipidemia rats before and after administer FTZ. Then seek the differentially expressed genes and analyze their possible affect targets. (2 ) The target gene expression level was detected by using RT-PCR, then impact for the target gene on FTZ was inferred from this way. (3) Research the effect of FTZ on the target activity using (enzyme) receptor function assay and Kit; (4) Screening the active ingredients of FTZ by classical receptor-ligand binding assay, in order to complete the active components and effective mechanism of FTZ.RESULT:(1)FTZ's influence on gene expression profiles in liver of the Hyperlipidemic rat.This study showed significant changes in their gene expression of FTZ Hyperlipemia model rats, including 128 upregulated genes in which NM.130741,NM.199208,NM.013105 were particularly significant and 120 downregulated genes in which NM.001009663,NM.017136,NM.012941 were particularly significant. With particular attention, gene NM.017136 which encoded Squalene epoxidase was another indispensable enzyme in the cholesterol biosynthesis pathway. That indicated that the regulation of Squalene epoxidase might be another target of metabolitic regulation of cholesterol by FTZ and Squalene epoxidase might be a new target of development of lipid-lowing drugs.(2)FTZ's influence on the expression of HMG-CoA reducase mRNA in liver of the Hyperlipidemic rat.HMG-CoA reductase is the key rate-limiting enzyme of endogenous cholesterol synthesis, thus inhibiting its gene expression can reduce cholesterol synthesis, thereby regulating cholesterol metabolism. This study shows that, FTZ can significantly suppress HMG-CoA reductase mRNA expression in hyperlipidemic rat liver, and existing a dose-dependent matter. In the animal experiments, the middle dose group(3.2g/kg) could inhibite the mRNA expression of HMG-CoA reductase. It indicated that FTZ can be effective regulation of cholesterol synthesis in the body.(3)FTZ's influence on the active of HMG-CoA reductase. The results showed that, When the concentration of FTZ are 1.5,3.0,6.0,12,24μg / ml, it can dose-dependently inhibit HMG-CoA reductase activity. These studies show that, FTZ can regulation of all liver esterase to play a role in the effect of regulate Hyperlipidemic.(4) FTZ's effects of material composition based on HMG-CoA reductase target. Using receptor-ligand binding technology and molecular imprinting technology, pravastatin as positive drugs in high concentrations(500-1000 times), the experimental sub - as follow groups: 1) FTZ + HMG-CoA reductase group; 2) FTZ + HMG-CoA reductase + pravastatin group; 3) Methanol + HMG-CoA reductase Group; At the same time, three groups were incubated in the water bath for 1 hour with oscillation, Then filteing with GF/B glass fiber membranes, washing by Tris-HCl. Finally the membrane (including the HMG-CoA reductase and the FTZ) extracted with anhydrous methanol, then ultracentrifugation to remove the protein. Three treatment groups analyze by HPLC at the same time. At last the differences in composition peak analyze by MS and the preparation HPLC to character and identify the components.Conclusion: (1) Identified the changes in gene expression profiles of hyperlipoidemia rat model administer FTZ by gene chip technology, and determine 128 significant up-regulation genes and 120 significant down-regulation genes. (2) In the gene expression level, it confirmed FTZ could down regulate the mRNA of HMG-CoA reductase using RT-PCR technology; (3) It confirmed FTZ could inhibite the activity of HMG-CoA reductase by using enzymatic reaction. (4) It identified the differences materias in FTZ through competitive experiment, pravastatin as positive drugs, using receptor-ligand binding technology and molecular imprinting technology. It will clarify lipid-lowering effect of the FTZ by the research the characterization and lipid-lowering efficacy of this material.