Preliminary Study On The Antihyperlipidemic Effect And Mechanism Of A New Type Of Lipid-lowering Drug DHCR24 Inhibitors

Hyperlipidemia is a disease caused by elevated levels of low-density lipoprotein cholesterol?LDL-C?,total cholesterol?TC?,triglyceride?TG?or total cholesterol?TC?and triglyceride?TG?.Recent clinical studies have shown that hyperlipidemia is a major risk factor for cardiovascular and cerebrovascular diseases,atherosclerosis and coronary heart disease.Although there are many hypolipidemic drugs in clinic,some patients with the disease caused by hyperlipidemia is not well treated and controlled due to individual difference.Moreover,the research on development of new drugs takes a long time and the side effects are uncertain.Therefore,the use of conventional drug in new has become the good way to develop lipid-lowering drugs.The sources of cholesterol in human body are mainly two ways,exogenous intake and endogenous synthesis.24-dehydrocholesterol reductase?DHCR24?is the last-step catalytic enzyme in the cholesterol synthesis pathway,which reduces the desmosterol to cholesterol.In the early stage of the laboratory,a virtual screening based on DHCR24using the existing drug database as a source of small molecule compounds was carried out,and four new lipid-lowering drug candidates DHCR24 inhibitors were initially identified,followed by using the confirmation of their cholesterol-lowering effect at cellular level.The four inhibitors are irbesartan,risperidone,tolvaptan,and conivaptan.On this basis,in this study,we further studied the hypolipidemic effect and preliminary molecular mechanism of these four drug candidates using hyperlipidemia animal models,and verified inhibitory effect of the irbesartan on the activity of DHCR24 enzyme by in vitro enzymatic assay experiments.To validate the effects of four novel DHCR24 inhibitors on blood lipid levels at the animal level,we successfully established a hyperlipidemia animal model using C57BL/6J mice through high-fat diet feeding.The mice were treated with four new lipid-lowering drug candidates,followed by HE staining analysis of liver tissue and blood lipid analysis to determine the cholesterol-lowering effect.The results of HE staining showed that,in the experimental group treated with the four novel drug candidates,the liver tissue of the mice showed small cells and a significant decrease in vacuolization,if compared to that in the hyperlipidemic model group.At the same time,the results of blood biochemical indicators showed that the serum levels of cholesterol,triglyceride and low-density lipoprotein cholesterol in the irbesartan group showed a significant decrease,accompanied by an increase in high-density lipoprotein cholesterols.And the other three drug-groups also showed the similar effect.This result demonstrates that the four hypolipidemic drug candidates can significantly improve the symptoms of hyperlipidemia in animal level.To study the molecular mechanism of this type of DHCR24 inhibitor to lower blood lipids,we performed Western Blot experiments on mouse liver tissues of the control and experimental groups.The results showed that the irbesartan group showed a significant increase in the level of LDL-R?low density lipoprotein receptor?compared to the control group.This result suggests that the effect of irbesartan on lowering blood lipids may be achieved by blocking endogenous cholesterol synthesis and feedback-adjusting LDL-R levels.Finally,we used the in vitro experimental system of immune complex enzyme activity assay to study the inhibitory effect of irbesartan on the cholesterol synthesis by DHCR24 enzyme.We first infected liver HepG2 cells by the recombinant adenovirus Ad-cmv-DHCR24 driving DHCR24 overexpression,and then obtained a large amount of antibody-DHCR24 immune complex by immunoprecipitation.The complex product was added to a DHCR24 enzyme-catalyzed reaction system using desmosterol as a substrate,and the content of substrate desmosterol and product cholesterol after the reaction was detected by high performance liquid chromatography.The experimental results showed that compared with the IgG control group,the desmosterol content in the experimental system with DHCR24immune complex decreased,and the cholesterol content increased significantly.The addition of the positive control reagent DHCR24 inhibitor U18666A significantly inhibited the enzyme catalytic activity of DHCR24.These experimental results demonstrate that the DHCR24 immune complex enzyme activity detection system was successful constructed.Then we added different concentrations of Irbesartan to the DHCR24 immune complex enzyme activity detection system.The results obtained from high performance liquid chromatography showed that irbesartan significantly reduced the cholesterol content of the reaction product in the reaction system,and increased the contents of the substrate desmosterol.Finally,we determined that the IC₅₀0 for Irbesartan to inhibit DHCR24 enzyme activity is598.64nM.In summary,this study demonstrated that the four novel lipid-lowering drug candidate DHCR24 inhibitors could achieve the effect of lowering blood lipids through the preliminary pharmacodynamic study on the cholesterol-lowering effect of the novel DHCR24 inhibitor.The molecular mechanism of the drug irbesartan to lower blood lipids and its enzyme reaction kinetics for inhibiting DHCR24 enzyme activity were studied.Our research using DHCR24 as a target to screen DHCR24inhibitors provides a new strategy for the development of lipid-lowering drugs,laying a solid foundation for the further development of new lipid-lowering drugs.