Mechanism Of Enterococcus Faecium GEFA01 And Lactiplantibacillus Plantarum GLPL01 Alleviating Hypercholesterolemia In Mice

Hypercholesterolemia,a pathological condition characterized by an exaggerated rise in serum cholesterol,plays a causal role in the development of atherosclerosis and is one of the main risk factors for cardiovascular diseases(CVDs),CVDs are the leading cause of death worldwide especially in developed countries.Statins are the fist-line drugs in the clinical treatment of hypercholesterolemia,however,there are some side effects accompanied with elevation of serum transaminase levels and some muscle diseases.So,there is urgent need to find novel drugs for the prevention and treatment of hypercholesterolemia.Probiotics are a kind of live culture which have beneficial effects on the host.Research on cholesterol-lowering and alleviating hypercholesterolemia has become a hot topic.The purpose of this study was to screen out probiotics with cholesterol-lowering effect,and to explore the effect of probiotics on cholesterol-lowering and the mechanism of regulating lipid metabolism in mice fed with high cholesterol and fat diet.Cholesterol-lowering Enterococcus faecium GEFA01 and Lactiplantibacillus plantarum GLPL01 were screened from the feces of healthy people,and their probiotics(cholesterol-lowering,bile salt tolerance,stomach and intestinal fluid tolerance,antioxidant,adhesion to Cacao-2 cells,antagonistic bacteria and antibiotic susceptibility tests)were evaluated in vitro.The results showed that the in vitro cholesterol removal rates of the two strains were E.faecium GEFA01(46.13%)and L.plantarum GLPL01(50.62%),and the cholesterol degradation rates were E.faecium GEFA01 GEFA01(35.84%)and L.plantarum GLPL01(35.72%),respectively.Both strains could survive under low acid environment(p H = 2.5),high concentration of bile salt(0.3%)and high concentration of hydrogen peroxide(1.0 mmol/L).The two strains possessed inhibitory effects on Staphylococcus aureus,Salmonella,Bacillus cereus,Listeria monocytogenes,Escherichia coli and Enterobacter sakazakii,and with the strongest inhibitory activity on Listeria monocytogenes(inhibition diameter of 20 mm).Both strains showed high adhesion ability to Caco-2 cells(more than 30%)and were sensitive to most of 13 broad-spectrum antibiotics(penicillin,ampicillin,amoxicillin,cefoperazone,clindamycin,erythromycin,tetracycline,chloramphenicol and rifampicin).The two strains possess good benefit and stress resistance,and can be further used in the study of hypercholesterolemic mice.Hypercholesterolemic mice were constructed with a high cholesterol diet to further investigate the cholesterol-lowering effects of E.faecium GEFA01 and L.plantarum GLPL01 in vivo.The results showed that the two strains could effectively reduce TC,TG and LDL-C,body weight and visceral fat accumulation in mice fed a high cholesterol and fat diet;reduce blood sugar and insulin secretion,improve insulin resistance.Compared with the HCD group,the expressions of Hmgcr,Npc1l1,Fxr,Shp,Srebp1-c,Fas and Scd1 in GEFA01 and GLPL01 intervention groups were significantly down-regulated,while the expressions of Ldlr,Abcg5/8,Abca1,Cyp7a1 and Lxr were significantly up-regulated.The results showed that the two strains of Lactobacillus had a good regulatory effect on the synthesis,transport and decomposition of cholesterol and the synthesis genes of fatty acids.16 S r RNA gene high-throughput sequencing analysis showed that the two strains of Lactobacillus could increase the diversity of intestinal microbiota of mice fed with high cholesterol and fat diet and change their intestinal microbiota closer to that of the chow diet group.Specifically,the abundance of Lactobacillus,Akkermansia,Bifidobacterium and Roseburia of mice in HCDGEFA01 group significantly increased;the abundance of Acetatifactor and Lactobacillus increased significantly in HCD-GLPL01 group(P＜0.05).At the same time,fecal acetate content was significantly increased in mice after administrated with E.faecium GEFA01 and L.plantarum GLPL01,and acetate and total bile acid contents of GEFA01 and GLPL01 mice were significantly increased(P＜0.05).The effects of L.plantarum GLPL01 on alleviating hypercholesterolemia in mice via intestinal microbiota were verified by fecal microbiota transplantation.The results showed that the microbiota of donor mice was transferred to recipient mice by fecal bacterial transplantation,which significantly decreased the levels of TC,TG and LDLC,body weight,visceral fat accumulation,blood glucose and insulin secretion in serum and liver of recipient mice(P＜0.05);reversed liver damage,liver lipid accumulation and adipocyte degeneration in recipient mice;Compared with FMT-HCD group,the expressions of Hmgcr,Npc1l1,Fxr and Shp in FMT-GLPL01 group were downregulated,while the expressions of Ldlr,Abcg5/8,Abca1,Cyp7a1 and Lxr were upregulated.In vivo experiments confirmed that the fecal short-chain fatty acids increased significantly after the intervention of lactic acid bacteria in hypercholesterolemic mice.In order to explain the effects of short-chain fatty acids on lipid metabolism,acetate,propionate,butyrate and mixed acids(acetate: propionate: butyrate 3:1:1)were used to intervene high lipid cells,and the effects of short-chain fatty acids on lipid metabolism were evaluated at the cellular level.The results showed that acetate could significantly reduce the total cholesterol level of high fat cells,acetate and mixed acid could significantly reduce the triglyceride level of high fat cells(P＜0.05).From the cellular gene level,acetate significantly increased the expression of Cyp7a1,Ldlr and Ppar-g(P＜0.05),reduced the expression of Hmgcr,Scd1,Fas and Srebp-1c,propionate and butyrate significantly reduced the expression of Hmgcr(P＜0.05).In conclusion,two Lactobacillus with development and application value were isolated and screened from the feces of healthy lean individuals in this experiment.The results suggested that E.faecium GEFA01 and L.plantarum GLPL01 exhibited cholesterol-lowering effects partially through assimilation,coprecipitation,and degradation of cholesterol and partially through modulation of the gut microbiotaSCFA axis.Oral administration with the strain GEFA01 and GLPL01 increased the abundance of Lactobacillus,Akkermansia,Bifidobacterium,Acetatifactor,Butyricicoccus and Roseburia,and decreased the relative abundance of Helicobacter.The increased abundance of Lactobacillus,Akkermansia,Acetatifactor,Butyricicoccus and Roseburia promoted the production of SCFAs,which downregulated the expression of Hmgcr,Srebp-2,Npc1l1,Fxr,and Shp and upregulated the expression of Ldlr,Abcg5/8,Abca1,Cyp7a1,and Lxr,thereby promoting reverse cholesterol transport and bile acid excretion.To provide theoretical basis for the clinical application of Enterococcus and Lactiplantibacillus plantarum probiotics in the prevention and treatment of hypercholesterolemia,and provide reference of biological treatment for improving atherosclerosis and coronary heart disease.