Lactobacillus paragasseri HM018 Derived from Breast Milk Ameliorates Hyperlipidemia in High-Cholesterol Rats by Modulating Bile Acid Metabolism
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Hyperlipidemia is a prevalent metabolic disorder with rising incidence, becoming a major worldwidehealth concern. Probiotics have gained attention as mild interventions for improving hyperlipidemia.In this study, fifty specific pathogen-free Sprague-Dawley rats were randomly divided into fivegroups: normal diet, high-fat diet (HFD) model, and HFD low-dose, medium-dose, and high-doseintervention groups. After six weeks of intervention with varying concentrations of Lactobacillusparagasseri HM018, fecal samples (for microbiota analysis and non-targeted metabolomics), serumsamples (for biochemical analysis), ileum and liver tissues (for transcriptomic analysis) werecollected. The results showed that HlM018 significantly reduced the serum levels of total cholesteroltotal triglycerides, and low-density lipoproteins induced by the HFD, HM018 also ameliorated gutmicrobiota dysbiosis.'Transcriptomic analysis of the liver and ileum revealed significant upregulationof the bile acid efflux genes Abcg5 and Abcg8 and the high-density lipoprotein-related gene Apoal in the ileum after intervention, Hepatic glucose metabolism and fatty acid metabolism were alsoimproved. Metabolomic results showed that the probiotic significantly altered the composition of thefecal bile acid pool. In conclusion, HM018 may ameliorate hyperlipidemia by modulating the gutmicrobiota, regulating hepatic lipid, glucose, and bile acid metabolism, and increasing the expressionof efflux genes in the ileal tissues.
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2.5 Gene sequencing analysis of 16S rRNA Using the OMEGA Mag-bind DNA Kit, genomic DNA was isolated from samples. The V3-V4 region of the 16S rRNA gene was then amplified with specific barcoded primers 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT). Polymerase chain reaction (PCR) products were analyzed using 2% agarose gel electrophoresis and subsequently purified using a Quant-iT PicoGreen dsDNA Assay Kit. Based on the preliminary quantification results from electrophoresis, the recovered PCR products were quantified using a fluorescence quantification system on a microplate reader (BioTek, FLx800). Library construction was performed using an Illumina TruSeq Nano DNA LT Library Prep Kit. The quality of the constructed libraries was assessed using an Agilent Bioanalyzer 2100 and Promega QuantiFluor, followed by sequencing. Raw sequencing data were processed to remove primer sequences using Cutadapt, and quality control and annotation were performed using QIIME 2 with the SILVA database (version 138). Alpha and beta diversity indices were calculated using QIIME 2. 2.6 HPLC analysis Fecal samples were collected from 10 rats per group (ND, HFD, and LPH groups; n = 30 total). Samples underwent liquid nitrogen flash-freezing and were maintained at −80 °C Untargeted metabolomic analysis was performed at Shanghai Applied Protein Technology Co., Ltd. (Shanghai, China) using an ultra-high-performance liquid chromatography (UHPLC) system (1290 Infinity II, Agilent Technologies) coupled with a quadrupole time-of-flight mass spectrometer (AB Sciex TripleTOF 6600). 2.7 Transcriptome analysis Total RNA was extracted from the liver tissues of rats in the three groups (ND, HFD, and LPH) using the TRIzol reagent. RNA purity was evaluated by measuring the A260/A280 ratio using a Nanodrop ND-2000 spectrophotometer (Thermo Scientific, USA), and RNA integrity was assessed using an Agilent Bioanalyzer 4150 system (Agilent Technologies, CA, USA). Subsequently, paired-end libraries were prepared using the ABclonal mRNA-seq Lib Prep Kit (ABclonal, China) following manufacturer protocols. Sequencing was conducted across dual platforms: Illumina Novaseq 6000 and MGISEQ-T7 systems.