| With the continuous progress of human society and the combined effects of various factors such as obesity,diabetes and cancer,hyperlipidemia has gradually become a great concern to human beings,which is mainly characterized by abnormal lipid metabolism and is a serious threat to human life.In addition,studies have shown that cholesterol imbalance may lead to cardiovascular disease(Cardiovascular diseases,CVD),or be associated with diseases such as Alzheimer’s disease(Alzheimer’s disease,AD)and various cancers.Patients can treat hyperlipidemia and related diseases by regulating their cholesterol levels through diet and exercise,and by taking lipidlowering medications.For example,the homeostasis of cholesterol metabolism is of great importance in maintaining cellular physiological functions,and studies have shown that abnormal metabolism in tumor cells affects the ability of cells to migrate and proliferate,which also provides new inspiration for the treatment of cancer by regulating cholesterol metabolism.Since about 80% of cholesterol in the human body is synthesized by the body itself,most of the current research and development of lipidlowering drugs are based on the idea of inhibiting the endogenous synthesis of cholesterol.24-dehydrocholesterol reductase(DHCR24),a key enzyme in the cholesterol biosynthesis pathway,serves to catalyze the reduction of streptolysin to cholesterol,Therefore,it is theoretically possible to inhibit the enzymatic activity of DHCR24 protein to lower the cellular cholesterol level and thus achieve the anti-cancer effect by killing cancer cells.In this study,we firstly screened DHCR24,a key gene in the cholesterol metabolism process,and then used DHCR24 protein as a target to screen DHCR24 inhibitors from the Chinese natural product database using molecular docking,followed by molecular dynamics simulations to assess its binding to DHCR24 protein in the simulated natural state,and used human liver cancer cell line Hep G2 cells were used to validate the cholesterol-lowering and anti-cancer biological activities of DHCR24 natural product small molecule inhibitors and to preliminarily explore the molecular mechanisms of their effects.Firstly,we screened the adipose tissue samples from the GEO database for differentially expressed genes and performed GO and KEGG analyses on them;the genes related to hyperlipidemia and cholesterol metabolism were collected from the Gene Cards database and Pharm GKB database,respectively,and the genes collected from the three databases were intersected to obtain one of the key genes DHCR24.This result suggests that DHCR24 is a key gene for hyperlipidemia and cholesterol metabolism,and has great potential to be a new target for cholesterol-lowering drug development.Then,we used DHCR24 protein as the target and used computerized molecular docking to screen the Chinese natural product database containing more than 50,000 small molecules of traditional Chinese medicine monomers for small molecule inhibitors that may inhibit DHCR24 enzyme activity.The binding energy values of the docked small molecules were ranked by comparing them with those of desmosterol,a substrate of DHCR24,and 533 small molecules were initially screened as the topranked natural product candidates.In order to evaluate the stability of the docking system and to study the interaction pattern of the three candidate natural product small molecules with In order to evaluate the stability of the docking system and to investigate the interaction pattern between the three candidate natural product small molecules and DHCR24 protein,molecular dynamics simulations were used to assess the binding of the three candidate natural product small molecules to DHCR24 protein under the simulated natural state.The root mean square deviation results showed that the three candidate natural product small molecules tended to be stable in the 100 ns regime.The free energy of ligand-receptor binding was calculated according to the MM-PBSA algorithm,and the results showed that the binding ability of the three candidate natural product small molecules was stronger than that of the substrate chain sterol,and the binding positions of the kinetic simulated small molecules and DHCR24 protein were analyzed,and the results showed that the three candidate natural product small molecules all bound to the chain sterol region of the DHCR24 receptor,and all the above results indicated that the three candidate These results indicated that the three candidate natural product small molecules could competitively inhibit the binding of chain sterol to the DHCR24 receptor.Finally,we validated the cholesterol-lowering and anticancer effects of the three candidate small molecules at the cellular level using human hepatoma cell line Hep G2 cells.For the cholesterol-lowering effect,the cell membrane cholesterol was stained by the cholesterol fluorescent dye felipid;the lipid droplet accumulation was observed by cellular oil red O staining;the intracellular cholesterol and triglyceride levels were measured by the assay kit;and the intracellular cholesterol was detected by high performance liquid chromatography instrument.The results showed that the cholesterol-lowering effects of candidate small molecules 36066 and 40441 were superior to those of the DHCR24 inhibitor U18666 A,while the cholesterol-lowering effect of 19990 was inferior.In terms of anticancer effect,the cells were cultured for scratch assay to observe the cell migration ability;colony formation assay to detect their proliferation activity;oil red O staining to observe the accumulation of lipid droplets after the cells were administered in the presence of serum;and Western Blot assay to detect the effect on AKT protein activity.The results showed that drugs 19990,36066 and 40441 all inhibited the migration and proliferation of Hep G2 cells,and intracellular cholesterol levels decreased in the presence of serum,and Western Blot assay was performed on the most effective drugs,36066 and 40441,and it was observed that the levels of p-AKT in cells could be down-regulated,which is presumed to be the main mechanism for its anti-cancer effect.The Western Blot assay observed that the level of p-AKT could be down-regulated in cells,which was presumed to be one of the main mechanisms of their anti-cancer effects.In summary,this experiment obtained the key gene DHCR24 in cholesterol metabolism through histological analysis and clarified the importance of DHCR24 gene in the regulation of cholesterol metabolism,and then conducted a computer virtual screening using DHCR24 protein as the target to initially obtain the competitive inhibitor candidate natural product small molecules of DHCR24 enzyme.The cholesterol-lowering and anti-cancer effects of the candidate natural product small molecules were then initially explored and confirmed by cellular experiments.Due to the structural differences of the natural product small molecules,our research has identified candidate natural product small molecule inhibitors of DHCR24,laying a solid foundation for the development of a series of new drugs using it as a lead compound and the rapid development of new lipid-lowering drugs with our own intellectual property rights,as well as the research of new anti-tumor approaches using DHCR24 as a target. |
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