Study On Anti - Atherosclerotic Activity And Its Role In The Expression Of ABCA1, SR - BI / CLA - 1 Up - Regulators

Cardiovascular disease is a major killer of the human health in developed countries and most developing countries, and atherosclerosis (AS) is the pathological basis of many serious cardiovascular disease. The pathophysiology of AS is the damage of endothelial cells, and the deposition of macrophage cells or vascular smooth muscle cells in the vessel wall leading to the formation of foam cells. This makes the arterial wall thickening constantly, and ultimately causes atherosclerosis. ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) are the key proteins in reverse cholesterol transport (RCT),and they play an important role in in the process of removing the excess lipids. Human high-density lipoprotein receptors are called CLA-1. ABCA1 and SR-BI/CLA-1 is considered to be the potential targets to prevent or treat atherosclerosis.We have got the active compound Rutaecarpine (RUT) by a cell-based HTS method for up-regulators of ABCA1 and CLA-1, and further study have shown that RUT suppresses atherosclerosis through up-regulating ABCA1 and SR-BI within RCT. RUT is the agonist of the transient receptor potential cation channel subfamily V member1 (TRPV1). Recent studies have shown that the transient receptor potential cation channel subfamily V member 1 (TRPV1) is closely associated with cardiovascular diseases such as atherosclerosis, hypertension and so on. Activating TRPV1 causes the influx of calcium and a series of intracellular signaling, to play its role in cardiovascular protection. Ma and others found that activation of TRPV1 can upregulate the expression of ABCA1, and it is related with atherosclerosis.Because ABCA1 and SR-BI/CLA-1 play important roles in AS, we aimed at the two receptors to progress the following anti-atherosclerosis researches and its upstream target mechanism researches.1. The discovery of the upregulators of ABCA1 and CLA-1Firstly, we screened the compound library to search for active compounds that can increase ABCA1 and CLA-1 expressing level by a cell-based HTS method for up-regulators of ABCA1 and CLA-1. Among the compounds screened, E0869 and R20 were found as the positive hit.2. The anti-atherosclerotic activity of compound E0869 in vitroE0869 could upregulate the mRNA and protein levels of ABCA1、SR-BI/CLA-1 and ABCG1 genes in HepG2 and RAW264.7 cells by Real-Time Quantitative PCR and Western blotting analysis, but could not influence the expression of FAS、SREBP-1c and CD36. Foam cell assay showed that E0869 could inhibit lipids accumulation in mouse peritoneal macrophages RAW264.7. Cholesterol efflux assay showed that E0869 could induce HDL-mediated cholesterol efflux in mouse peritoneal macrophages RAW264.7.3. The anti-atherosclerotic activity and mechanism research of compound R20The active compound R20 was studied on cell level, and the results show that the active compound R20 could upregulate the mRNA and protein levels of ABCA1、CLA-1 and ABCG1 genes in HepG2, but could not influence the expression of FAS and SREBP-lc. R20 could inhibit lipids accumulation and induce HDL-mediated cholesterol efflux.R20 is the derivative of the RUT, and RUT is the activator of TRPV1. Recent studies shows that activation of TRPV1 can upregulate the expression of ABCA1 and it is closely related with atherosclerosis, hypertension and other cardiovascular diseases. Therefore, R20 is possible to upregulate the expression of ABCA1 through the activation of TRPV1,which plays a role in anti-atherosclerosis. For further identification, we established a TRPV1 agonist screening model for further screening and testing. We found that R20 showed good activity in the TRPV1 agonist screening model.Then, we conducted a study in vivo about the activity of R20.The apoE-/- mice results showed that R20 could upregulate the mRNA and protein levels of ABCA1、 SR-BI、ABCG1 and LDLR genes in liver, but could inhibit the expression of FAS and SREBP-1c. R20 can significantly reduce the content of TC, TG, LDL-C in serum and increase the content of HDL-C.The oil red staining experiment shows that reduce lipids accumulation in mouse liver.In conclusion, we obtained the active compounds E0869 and R20 through the screening model for upregulators of ABCA1 and CLA-1, and we identified the two active compounds in vitro. To find the target of the upregular of ABCA1 and CLA-1, we established a TRPV1 agonist screening model, and we found that R20 showed good activity in the TRPV1 agonist screening model.Then, we identified this active compound in vivo.Our researches made foundation for the discovery of drug candidates or lead compounds of new anti-atherosclerotic agents.