Molecular Mechanism Of Inhibition Of Cholesterol Synthesis By Oleanolic Acid Through HMGCR Pathway

Cholesterol homeostasis imbalance can lead to metabolic diseases such as atherosclerosis(AS).3-hydroxy-3-methyl glutaryl coenzyme A reductase(HMGCR),as the first rate-limiting enzyme in the cholesterol synthesis pathway,is the target of many natural small molecule compounds to regulate cholesterol metabolism.Oleanolic acid can improve atherosclerosis in Apo E-/-mice,but the specific mechanism has not been fully elucidated.The purpose of this study was to investigate whether oleanolic acid,a small molecular compound present in a variety of natural herbs and foods,targets HMGCR to inhibit cholesterol synthesis in HepG2 cells.The research contents are as follows:Firstly,the toxicity of OA to HepG2 cells was detected by MTT experiment,and the results showed that OA concentration less than 20 μM had no significant effect on the viability of HepG2 cells.Then,bioinformatics was used to analyze the effect of OA on the transcriptome of HepG2 cells,and it was found that it significantly downregulated the gene expression of the PI3K-Akt signaling pathway and the PPAR signaling pathway related to the cholesterol synthesis pathway,suggesting that OA may have a certain effect on cellular cholesterol synthesis.In order to verify the transcriptome results,we performed experiments such as intracellular cholesterol content determination,cell production,and cell morphological observation,and the results showed that OA can inhibit cholesterol synthesis in HepG2 cells.Based on the importance of related rate-limiting enzymes to cholesterol synthesis,we investigated the effect of OA on the levels of HMGCR,ACLY,DHCR7,DHCR24 and SQLE proteins,some of the key enzymes of cholesterol synthesis in HepG2 cells,and found that OA significantly reduced the protein expression of HMGCR but had no significant effect on the protein expression of ACLY,DHCR7,DHCR24 and SQLE,so HMGCR was selected as a follow-up research target.The expression of OA in HMGCR and its upstream SREBP-2 was detected by Western blot and RT-PCR,and the results showed that OA inhibited the protein and m RNA levels of HMGCR and SREBP-2 in HepG2 cells.Then,the effect of OA on HMGCR enzyme activity was explored,and the results of Western blot and enzyme activity detection kit showed that OA can inhibit HMGCR activity by activating AMPK phosphorylation to enhance HMGCR phosphorylation.In order to further explore the mechanism of OA inhibition of HMGCR enzyme activity,we carried out molecular docking simulation and molecular interaction experiments.The results showed that OA and HMGCR could bind by hydrophobic action(KD=79.44 μM),thereby reducing the catalysis of substrate by HMGCR and inhibiting enzyme activity.In summary,OA can inhibit the protein and m RNA expression levels of HMGCR in HepG2 cells,and can directly bind to HMGCR and inhibit its enzyme activity,thereby inhibiting cholesterol synthesis,and the results can provide a basis for elucidating the mechanism of OA in regulating cholesterol metabolism and improving AS,and provide scientific basis for the development and application of OA-related functional foods.