Study On Cholesterol-lowering Mechanism Of Sterol Compounds In Ganoderma Lucidum Based On Cell Models And Metabolomic Analysis

Ganoderma Lucidum has been playing a prominent role in the long edible and medicinal history of China,sterol compounds are one kind of the ingredients with high content and rich functional activities in Ganoderma Lucidum.In recent years,the cholesterol-lowering effect of sterol compounds has attracted extensive attention,and the mechanism of its cholesterol-lowering effect has become a research hotspot.Studies have shown that the cholesterol-lowering activity of sterol compounds is closely related to their structure.However,present studies on the regulation of cholesterol level by sterol compounds in fungi are generally limited to the shallow description,which cannot reflect the status of its effect on cholesterol metabolism,and there are relatively few studies on its mechanism.Therefore,this study intends to explore the absorption and metabolism of cholesterol in vitro by HepG2 cell cholesterol metabolism model and Transwell model(Caco-2 cells),aim to investigate the effects of sterol compounds in Ganoderma lucidum(ergosterol,ergosterol acetate,(22E)-Ergosta-4,6,8(14),22-tetrean-3-one and stellasterol)on cholesterol absorption and metabolism by direct action,their mechanism and structur-activity relationship.Furthmore,the regulation mechanism of sterols in Ganoderma lucidum on cholesterol uptake and transport in vitro was analyzed and signal transduction pathway of sterols in ganoderma lucidum was proposed,and the mechanism of cholesterol metabolism regulated by sterol compounds in ganoderma lucidum was studied by metabolomics.The main results are as follows:1.High cholesterol cell model(HepG2 cells)was constructed to investigate the effect of sterol compounds on cholesterol metabolism.The results showed that the total cellular cholesterol and triglycerides were reduced by four kinds of sterol compounds(25,50,and 100 μM)in the high-cholesterol model,and the lipid increase and disorder caused by the addition of 2 5-hydroxycholesterol and cholesterol were also improved.In addition,sterol compounds promote the excretion of cholesterol into the intestinal lumen(up-regulating ABCG5 and ABCG8),lower the absorption of cholesterol(down-regulating NPC1L1),while lowering the biosynthetic of cholesterol in the liver(down-regulating HMGCS1 and SREBP2)and affecting the conversion of cholesterol into cholesterol esters in the liver and into the blood circulation(down-regulating ACAT2).Further discussion on sterol compounds struct-activity relationship revealed that:(1)Ergosterol had better cholesterol-lowering effect than ergosterol acetate in most cases(except the expression of NPC1L1),indicating that their cholesterol-lowering activity might be related to the functional group on C3(hydroxyl or ester)of sterol compounds.(2)The cholesterol-lowering activity of ergosterol was significantly stronger than that of(22E)-Ergosta-4,6,8(14),22-tetrean-3-one,but further experimental exploration was needed to determine the specific structural association.(3)The cholesterol-lowering effect of ergosterol(a double bond on C5)was higher than that of stellasterol(no double bond on C5),indicating that their cholesterol-lowering activity was also related to the double bond on C5.2.Transwell model was used to investigate the effect of sterol compounds on cholesterol absorption and transport in Caco-2 cells.The results showed that the effects of high cholesterol on monolayer integrity and compactness of Caco-2 cells were improved and cellular cholesterol absorption in Transwell high cholesterol model was reduced by the four sterols(25,50 and 100μM).At the same time,sterol conpounds promote the excretion of cholesterol into the intestine lumen by lowering the absorption of cholesterol(down-regulating NPCILland up-regulating ABCG5,ABCG8),lowering the activity of cholesterol regulatory protein(down-regulating SREBP2),and affecting the conversion of cholesterol into cholesterol esters into the blood circulation(down-regulating ACAT2).Further discusses the structure-activity relationship of sterol compounds found that(1)Different results were obtained on comparison,of ergosterol and ergosterol acetate of the various indicators,indicating that their cholesterol-lowering activity may be related to the functional group on C3(hydroxyl or ester)of the sterol compounds,but further exploration and study are needed.(2)The cholesterol-lowering activity of ergosterol was significantly stronger than that of(22E)-Ergosta-4,6,8(14),22-tetrean-3-one,but further experimental exploration was needed to determine the specific structural association.(3)The cholesterol-lowering effect of ergosterol(a double bond on C5)was not consistent with that of stellasterol(no double bond on C5).3.Differential metabolites were screened by GC-MS,there were 14 metabolites were selected,including lactic acid,oxalic acid,citric acid,palmitic acid,succinic acid,malic acid,oleic acid,stearic acid,alanine,β-alanine,glycerin,inositol,L5 oxyproline and glycine.According to these metabolites,6 metabolic pathways were used to interfere with lipid metabolism by sterol compounds,including glycolysis pathway,TCA cycle,alanine metabolism,glycerophospholipid metabolism,proline metabolism and bile acid metabolism.Cholesterol can stimulate the energy metabolism of cells,which will lead to disorders of TCA cycle and amino acid cycle in cells.Ezetimibe can relieve the TCA cycle disorder,but has no obvious effect on energy metabolism and amino acid metabolism disorder.Among the four kinds of sterol compounds,only stellasterol can relieve the energy metabolism disorder of cells.Ergosterol,ergosterol acetate and stellasterol can relieve TCA circulation disorder of cells,both them have no effect on amino acid metabolism disorders.It can be concluded that the four selected sterol compounds mainly affect lipid metabolism by affecting the TCA cycle of cells,and that the(22E)-Ergosta-4,6,8(14),22-tetrean-3-one have no effect on lipid metabolism disorder in cells.