Alleviating Effects Of Probiotics On Hypercholesterolemia And Potential Mechanisms

Hypercholesterolemia is a metabolic disease caused by abnormal fat metabolism which makes the serum lipid levels exceed the normal range,and is colsely related to cardiovascular diseases.Drug therapy has always been the primary way of clinical treatment to hypercholesterolemia,but it has some side effects such as damage to liver function and kidney function.Probiotics are a kind of living microorganisms which are widely recognized to promote human health and have been proved to play an important role in regulating lipid metabolism.Therefore,it is of great significance to use probiotics in preventing and treating hypercholesterolemia.At present,the selection of probiotics with cholesterol-lowering effect is mainly throgh two ways:one is the high ability of bile salt deconjugation,the other is the high ability of cholesterol assimilation.However,the differences in the hypercholesterolemia-alleviating effects and potential mechanisms of probiotics with different abilities of bile salt deconjugation and cholesterol assimilation remain unknown.Therefore,we tried to explore the alleviating effects and mechanisms of probiotics with different abilities in bile salt deconjugation and cholesterol assimilation.The main results are listed as follows:At first,54 strains of probiotics were isolated using selective media MRS and LAWAB.Bile salt deconjugation ability and cholesterol assimilation ability of probiotics were used as the indicators in vitro,then four groups consisted of fourteen probiotics were obtained.Group No.1,which contains both high abilities of bile salt deconjugation and cholesterol assimilation,was composed of Bifidobacterium longum CCFM1077,I5,I7 and Lactobacillus reuteri A9,Lactobacillus mucosae A13.Group No.2,which contains only high ability of bile salt deconjugation,was composed of Bifidobacterium longum I3,J2 and J5.Group No.3,which contains only high ability of cholesterol assimilation,was composed of Bifidobacterium longum J3,I1 and J8.Group No.4,which contains neither high ability of bile salt deconjugation nor cholesterol assimilation,was composed of Bifidobacterium longum B3,B8and B10.Then,the results of tolerance to simulated gastrointestinal fluid showed that all these14 strains could survive well in the simulated gastrointestinal environment.Furthermore,the results of growth characteristics of these 14 probiotic strains indicated that they could reach stable period within 24 hours.All these showed that the 14 probiotics with different abilities of bile salt deconjugation and cholesterol assimilation could have the potential for further animal and human experiments.Secondly,the alleviating effects of Bifidobacterium longum strains with different abilities in bile salt deconjugation and cholesterol assimilation on hypercholesterolemia in rats were investigated when different doses were ingested.The results showed that when the amount of probiotics intake reached 1×10⁸ CFU/d,only Bifidobacterium longum strinas(CCFM 1077,I5 and I7)in group No.1 with the above two abilities could significantly reduce serum cholesterol(p<0.0001)and LDL cholesterol(p<0.0001).When the amount of probiotics reached 1×10¹⁰ CFU/d),Bifidobacterium longum strains with only one of the above abilities could significantly reduce serum cholesterol(p<0.0001)and LDL cholesterol(p<0.0001).However,Bifidobacterium longum strains with neither abilities couldn't significantly reduce serum cholesterol nor LDL cholesterol.In order to verify this result,Lactobacillus strains(L.reuteri A9,L.mucosae A13 and the positive strain L.plantarum ST-III)with both high abilities in bile salt deconjugation and cholesterol assimilation were used to investigate the alleviating effects on hypercholesterolemia rats.The results showed that these three Lactobacillus strains could significantly reduce serum cholesterol and low density lipoprotein cholesterol when ingested at low doses.These results suggested that probiotics with different abilities of bile salt deconjugation and cholesterol assimilation could exert different effects on cholesterolemia.Based on the above results,we further tried to explore the potential alleviating mechanisms of probiotic on hypercholesterolemia.(1)By ultra high performance liquid chromatography tandem mass spectrometry,we found that probiotics with high ability of bile salt deconjugation could deconjugate conjugated bile acids to free bile acids,which was represented by glyco-conjugated bile acids,to exert the cholesterol-lowering effect.(2)By16s metagenomics analysis,we found that probiotics with both high abilities of bile salt deconjugation and cholesterol assimilation could significantly improve the diversity of intestinal microorganisms and increase the relative abundance of beneficial bacteria.(3)By molecular biological techniques,we found that probiotics with both high aboved two abilities could regulate cholesterol metabolism in vivo by upregulating the expressions of FXR,CYP7A1,LXR,SREBP2 and LDLR,and downregulating the expression of SHP.Finally,the alleviating effect of B.longum CCFM1077 on hypercholesterolemia was evaluated in a randomized,placebo-controlled clinical trial.The results showed that B.longum CCFM1077 alleviated hypercholesterolemia by reducing serum cholesterol and low density lipoprotein cholesterol.The results of metagenomic analysis showed that B.longum CCFM1077 could change the composition of gut microbiota by improving the diversity of intestinal microorganism in hypercholesterolemic patients.In addition,B.longum CCFM1077could improve the relative abundance of Bacteroidetes?Fusobacterium,reduce the relative abundance of Actinomycete.Particularly,B.longum CCFM1077 could increase the relative abundance of Butyricicoccus,which is good for producing short chain fatty acid,and improve the relative abundance of Muribaculaceae and Parasutterell,which are good for producing polysaccharides and get involved in the bile acid and cholesterol metabolsim.The results of untargeted metabolome showed that B.longum CCFM1077 could affect the metabolic pathways such as bile acid biosynthesis,ammonia circulation,propionate metabolism,especially increasing the excretion of bile acids in the feces of patients with hypercholesterolemia.