Establishment Of The Methods For HMG-CoA Reductase Inhibitors Discovery And Mechanisms Study On IR Amelioration Effects Of Atorvastatin

The inhibitor of HMG-CoA reductase is a kind of statin drugs.It is clinically to applied for decreasing the cholesterol,triglyceride and low-density lipoprotein cholesterol as well as slightly increase high-density lipoprotein cholesterol.At the same time,it also has other functions besides decreasing lipoprotein,such as improving endothelial function,inhibiting smooth muscle cell proliferation,inhibiting inflammation,anti-thrombosis,immune suppression, anti-osteoporosis,control diabetes,kidney and Alzheimer's and multiple roles, such as anti-virus.Because of its wide range of pharmacological effects and low side effects,clinical experts have recommended person over the 50-year-old to take fewer daily doses of statin drugs in order to reduce the incidence of cardiovascular diseases and development.In order to develop independent intellectual property rights of the statin drugs in our country,we established a rapid screening model just like P-407-induced hyperlipidemia of hamster model in the first part of this dissertation. The results showed that P-407 hamster model was different from other hyperlipidemia model and was characterized as the following:1) High blood lipid levels,compared with the control P<0.01.Maintain a long time,the serum cholesterol levels are still higher than that after given P-407 120 hours.2) Hamster lipid metabolism is similar to the level of human and animal model achieves hyperlipidemia entirely through changing the enzyme pathway in the body,which is suitable for screening statins medicines.In the present study,we found that the blood cholesterol and triglyceride levels were significantly lower in P-407 hyperlipidemia hamster model after treated the mice with Fluvastatin isomers at 50 mg/kg.And this experiment takes the advantage of the reliability of Fluvastatin isomers model.Therefore,P-407 hamster hyperlipidemia model entirely suitable for the rapid screening and efficacy evaluation for statin drugs.In the current study,we established a method in vitro in the second part for screening the inhibitor of HMG-CoA reductase.We obtained recombinant HMG-CoA reductase catalytic domain protein after cloning and expressing the constructs in E.Coli.and evaluated the inhibitory effect by determining catalytic reaction before and after the change of NADPH coenzyme on the basis of the principle of Cholesterol synthesis.And we also monitored the stability and reliability in screening system.The result showed that the screening system is stable and reliable for screening.It showed good dose-effect relationship when the concentration of Atorvastatin ranges from 10^-5 to 10^-8M implied the screening method in vitro can be used to discover a novel HMG-CoA reductase inhibitor.The third part of papers focused on the relationship between Atorvastatin and type 2 diabetes insulin resistances.The current study shows that obesity is a low-inflammatory state,fat tissue can secrete large number of inflammatory factors,including TNF-α,IL-6,which can lead to organizations not sensitive to insulin as well as activate inflammatory pathway to further increase insulin resistance.Firstly,this paper used in vitro method to inspect the effect of the drugs on NF-K B,the results showed that Atorvastatin can inhibit NF-K B activity, and its role is equivalent to dexamethasone at 10^-5M.Secondly,we determined the effect of Atorvastatin on TNF-α-induced 3T3-L1 fat cells insulin resistance,the results showed that Atorvastatin can significantly increase insulin resistance cells to insulin sensibility.In vivo studies,we investigated Atorvastatin role in three kinds of fat insulin resistance animal models.The results showed that Atorvastatin can improve the oral glucose tolerance,insulin tolerance,lower serum TG,Chol and the FFA content,improve insulin sensitivity index(ISI) and lower resistance index(HOMA-IR) as well as reduce fat tissue TNF-αand IL-6. we found that drugs can lower TNF-α,IL-6,NF-K B and IKK-βgene in the fatty tissues and increase I K B gene expression and obviously decrease NF-KB,IKB protein level in fatty tissues.All in all,this study established HMG-CoA reductase inhibitor screening model in vivo and in vitro.At the same time we investigated the relationship between Atorvastatin and type 2 diabetes insulin resistance.We preliminarily draw the conclusion that Atorvastatin improve insulin resistance by inhibiting NF-K B activity,leading to inhibit TNF-α,IL-6 and other inflammatory factors.Inhibiting NF-K B activity may be through two ways:1) Atorvastatin inhibit it directly;2) Atorvastatin might inhibit NF-K B activity through enhancing IKB activity and the inhibiting of IKK-βactivity.