Preliminary Pharmacodynamic Study On Ad-siDHCR24 Cholesterol-lowering Effect

Hyperlipidemia is a disease of abnormal lipoprotein metabolism of high cholesterol,high triglycerides,elevated low density lipoprotein and high density lipoprotein reduction.A number of clinical investigations have demonstrated that hyperlipidemia is a risk factor for cardiovascular diseases,such as atherosclerosis and coronary heart disease.Therefore,cholesterol-lowering drugs are considered to be an important and effective approach to prevent cardiovascular diseases.Although there are many kinds of blood lipid lowering drugs in clinic,the incidence of cardio cerebral vascular disease is still high because of the toxic side effects and individual differences of the patients.Thus,the development of new types of cholesterol-lowering drugs is still the the focus in the field of new drugs development.There are two sources to acquire cholesterol for the human body: exogenous intake and endogenous cholesterol biosynthesis,and the endogenous biosynthesis is the main way.So targeting cholesterol biosynthesis pathway can be a powerful strategy to control cholesterol level.DHCR24,the end enzyme in the cholesterol biosynthesis,is responsible for the conversion of desmosterol to cholesterol.The concentration of cholesterol was significantly reduced in embryonic fibroblasts derived from DHCR24 knockout mice,suggesting that DHCR24 can be used as a new target for cholesterol-lowering drugs.To investigate the possibility of DHCR24 as a target for lowering cholesterol,we used recombinant adenovirus as the interfering vectors and constructedfour kinds of vectors targeting to different regions of DHCR24 mRNA(Ad-siDHCR24)respectively,to investigate their efficiency of interfering with target gene DHCR24 expression and detect their effect on decreasing cholesterol level at the cellular and animal level.In order to test the efficiency of four kinds of Ad-siDHCR24(No.1,No.2,No.3 and No.4)to interfere with the expression of DHCR24 gene,we transfected them alone or mixture of four as ration 1:1:1:1(Mix)to human liver cell line HepG2..The result obtained from Real-time quantitative PCR showed that No.1,No.3,No.4 and AdsiDHCR24 Mix could significantly down-regulate the expression of DHCR24 mRNA.Western Blotting experiments also showed that Mix of Ad-siDHCR24 can efficiently interfere with the level of DHCR24 protein expression.These results demonstrated that the use of Ad-siDHCR24,constructed in the early stage,can reduce both levels of mRNA and protein expression of DHCR24,and also suggested that mixture of different RNA interfering vectors could reduce the off-target effect of siRNA on the target gene.To further explore the cholesterol-lowering effect of the recombinant adenovirus Ad-siDHCR24 at the cellular level,HepG2 cells were transfected with recombinant adenovirus mixture(Ad-siDHCR24 Mix)and then subjected to filipin staining.The results showed that the blue fluorescence intensity of Ad-siDHCR24 Mix cells were significantly lower than that of the control group,suggesting that the Ad-siDHCR24 Mix can reduce intracellular cholesterol level.Additionally,we used high-performance liquid chromatography to detect intracellular cholesterol and its precursor desmosterol levels.The results showed that the cholesterol level with the transfected Ad-siDHCR24 Mix group cells was significantly decreased,compared to that in the control group,which were consistent with the results of the cell cholesterol fluorescence staining method.These results demonstrated that Ad-siDHCR24 Mix achieves the function of cholesterol-lowering biosynthesis by silencing the expression of the target gene DHCR24.Finally,we investigated the effect of Ad-siDHCR24 Mix on lowering blood lipid levels at the animal level.We used high-fat diet to breed C57BL/6J mice and successfully established a hyperlipidemia model.Ad-siDHCR24 Mix adenovirus(drug group)was injected through the tail vein.The results of HE staining showed that,compared to the control group of the hyperlipidemia model group,the liver tissue cells of Ad-siDHCR24 Mix became smaller and the contained lipids were less,suggested by the reduced level of lipid’s vacuolization.At the same time,the results obtained from blood biochemical analysis showed there are significant decreases in serum cholesterol,triglyceride,and low-density lipoprotein sterol levels in the mice of the drug group,accompanied by an increase in high-density lipoprotein sterols.These results demonstrated that Ad-siDHCR24 Mix can successfully achieve lipid-lowering effects at the animal level.In summary,the preliminary pharmacodynamic study of the cholesterol-lowering effect of Ad-siDHCR24 demonstrated that Ad-siDHCR24 Mix can reduce blood lipids by interfering with the expression of DHCR24.This study indicated that DHCR24 can be used as a new target of lipid-lowering drugs,providing new ideas and theoretical basis for the development of new drugs anti-cardiovascular diseases.