| The proportion of fat in the diet has increaseed year by year with the continuous improvement of living standards,however,the long-term intake of high fat diet causes lipid metabolism disorder.Studies have reported that the occurrence of lipid metabolism disorder is often accompanied by hyperlipidemia,obesity,type II diabetes and cardiovascular diseases.In2019,the global death toll from cardiovascular diseases reached 17.9 million,and the death toll from diabetes reached 2 million,and there were 650 million obese adults in 2016,which more and more people were in sub-health due to the disorder of lipid metabolism.The long-term use of lipid-lowering drug such as statins have serious side effects.Probiotics as a kind of functional food have been reported that have lipid-lowing effect,among them,the mechanism of Lactobacillus and Bifidobacterium regulating lipid metabolism disorder is more in-depth.Pediococcus pentosaceus as a probiotic that widely used in the field of food fermentation,while the existing studies on mechanism of P.pentosaceus regulating lipid metabolism disorder are insufficient and not deep enough,which the specific mechanism is still unclear.Therefore,this study screened P.pentosaceus strain with potential lipid-lowering effect from naturally fermented foods,and revealed the specific mechanism of screened strain regulating lipid metabolism at the molecular level by combining in vivo and in vitro tests,providing a theoretical foundation for the development of probiotic functional foods.The main findings were as follows:(1)A strain of P.pentosaceus PP04 with cholesterol removal rate of 52.19 ± 2.8% and bile salt hydrolase activity of 1.73 ± 0.32 U/mg was screened from the traditional fermented cabbage in Northeastern China.The probiotic properties of PP04 such as bile acids conversion ability,antimicrobial activity,antibiotic sensitivity and intestinal colonization ability were analyzed,the results showed that PP04 could deconjugate bile acids including GCDCA,GCA,TCDCA and TCA to CDCA or CA,and it also converted CA to DCA at the same time.The survival rate of PP04 was 78.06% after continuous artificial simulation gastrointestinal fluid.The co-aggregation capacity of PP04 was 57.90 ± 0.64%.The surface hydrophobicity of PP04 measured by different hydrocarbons was more than 50%,and its adhesion rate to Caco-2 cells reached 79.06 ± 0.39%.In addition,PP04 showed different degrees of inhibition against three common pathogenic bacteria including Escherichia coli ATCC 25922,Staphylococcus aureus ATCC 25923 and Salmonella ATCC 13311,and PP04 was not sensitive to ampicillin,kanamycin and streptomycin.(2)The 8-weeks intervention of P.pentosaceus PP04 could significantly inhibit the increases of body weight,Lee′s index,epididymal,perirenal and mesenteric fat coefficients in high fat diet induced mice.The PP04 treatment reduced serum TC,TG,LDL-C,FFA and Leptin levels,which also inhibited the increase of TC,TG,LDL-C,AST and ALT levels caused by hepatic lipid accumulation of mice,and improving the glucose intolerance and insulin resistance induced by high fat diet.It confirmed that PP04 had the effect of lowering blood lipid.Western blot analysed hepatic proteins related to lipid metabolism of mice,it showed that PP04 activated AMPK/FXR signaling pathway,which down-regulated the hepatic expression of SREBP-1c,FAS and SCD1 proteins to inhibit lipogenesis,and up-regulated the expression of PPARα and CPT1 proteins to promote fatty acid oxidation,thus improving lipid metabolism disorder induced by high fat diet.(3)P.pentosaceus PP04 significantly increased the cecal and fecal Alpha diversity index of gut microbiota,and also changed the composition of intestinal microbial species in high fat diet induced mice.Specifically,PP04 could promote the abundance of short chain fatty acids producing bacteria including Akkermansia,Eubacterium,Clostridium and Pediococcus,as a result,the concentrations of acetic acid,propionic acid,isovaleric acid and valeric acid increased significantly,which further up-regulated the m RNA levels of GPR41 and GPR43 in ileum to promote the secretion of GLP-1.Meanwhile,PP04 also reduced the abundance of harmful bacteria including Proteobacteria,Pseudomonas and Oscillospira,which improved the imbalance of gut microbiota caused by high fat diet.Then,the treatment of PP04 increased the expression of intestinal tight junction proteins including Occludin,Claudin-1 and ZO-1 to repair the impaired intestinal barrier function and prevented the leakage of LPS from the intestine into the blood.Moreover,PP04 inhibited the intestinal TLR4/NF-κB signaling pathway to reduce the release of downstream inflammatory cytokines such as IL-6,IL-1β and TNF-α to ameliorate intestinal inflammatory response.(4)The treatment of P.pentosaceus PP04 decreased the ileal TCA,TCDCA and TUDCA levels of high fat diet induced mice significantly,while the concentrations of CA,CDCA and UDCA were increased remarkably.Spearman correlation analysis indicated that PP04 enriched cecal enterobacteria including Clostridium,Allobaculum,Parabacterodies and Ruminococcus,which resulted in the deconjugation of conjugated bile acids TCA and TCDCA in the ileum,leading to the increase of CA,CDCA and UDCA levels.Pediococcus was positively correlated with the concentrations of ileal T-α-MCA,T-β-MCA,CA and UDCA,therefore,PP04 intervention could influence the bile acids metabolism by changing the classification and distribution of intestinal flora.The PP04 treatment increased the ileal concentrations of FXR antagonists including T-α-MCA,T-β-MCA and UDCA to inhibit "Gut-liver axis" FXR/FGF15-FGFR4 signaling pathway,which significantly up-regulated the expression of CYP7A1,CYP27A1,CYP7B1 and CYP8B1 to promote bile acids de novo synthesis in the liver.This resulted in significantly higher hepatic CA and CDCA levels in HFD+PP04 group than HFD group,which activated hepatic FXR/SHP signaling pathway to avoid excessive de novo synthesis of bile acids.Compared with the HFD group,PP04 inhibited the expressions of ileal IBABP,ASBT and OSTα/OSTβ to prevent the reabsorption of bile acids in the small intestine,which promoted unconjugated bile acids(CA,CDCA,α-MCA,LCA and DCA)and conjugated bile acids(GCA,TCDCA,TCA,T-α-MCA,T-β-MCA,TUDCA,TDCA,TLCA and GDCA)excretion with feces to counteract the increased bile acids synthesis in the liver caused by high fat diet.In addition,PP04 inhibited the transcription of SREBP-2 to down-regulate expression of HMGCR protein in liver,which suppressed cholesterol biosynthesis.Meanwhile,compared with HFD group,PP04 also significantly increased the relative m RNA levels of LXR,ABCG5 and ABCG8 to promote the excretion of cholesterol with feces,which alleviated the occurrence of lipid metabolism disorder.(5)The treatment of 4%(v/v)PP04 not only down-regulated the expression of lipogenesis-related proteins including SREBP-1c and FAS,but also up-regulated the expression of fatty acid oxidation related protein PPARα by activating AMPK/ACC/FXR signaling pathway,which further significantly reduced the levels of TC,TG,LDL-C,AST and ALT and inhibited the excessive lipid accumulation in Hep G2 cells to alleviate hepatocyte injury in oleic acid induced Hep G2 cells.When AMPK inhibitor Compound C was administered to OA-induced Hep G2 cells at a dose of 10 μM,the expression of lipogenesis and lipolysis-related proteins were not influenced by PP04 intervention.The combined intervention of AMPK activator and PP04 could further activate the AMPK/ACC/FXR signaling pathway,which confirmed the mechanism of the improvement of lipid metabolism disorder by PP04 at the cellular level.In summary,P.pentosaceus PP04 was screened based on the cholesterol-lowering ability and bile salt hydrolase activity,which had favourable ability of intestinal colonization,tolerance to artificial gastroenteric fluid,inhibition of harmful bacteria and other probiotic properties.The PP04 alleviated high fat diet induced lipid metabolism disorder by activating "Gut-liver axis" FXR/FGFR4/NF-κB/AMPK signaling pathway and regulating intestinal flora disorders. |
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