Gut Microbiota and Metabolomic Analyses of Polysaccharide from Processed Typhonii Rhizoma against PTZ-Induced Epilepsy in Mice
Description
Through metagenomic sequencing, the changes in gut microbiota in PTZ induced mouse epilepsy model improved by PRTP were analyzed at the level of species composition and functional genes. Non targeted metabolomics was also used to analyze metabolites in brain tissue and colon contents.
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Total DNA was extracted from colon content samples, assessed by agarose gel electrophoresis, and quantified using a Qubit fluorometer. High-quality DNA was fragmented to ~400 Bioruptor, with a Covaris ultrasonicator, followed by end repair, A-tailing, adapter ligation, and PCR amplification to construct libraries. Sequencing was performed on an Illumina NovaSeq platform (2 × 150 bp). After quality filtering and host read removal, clean reads were assembled into contigs using metagenomic assembly software. Gene prediction was performed, and predicted genes were clustered into a non-redundant gene set. Gene profiling, taxonomic analysis and functional annotation (KEGG, KO, CAZy) were then performed. Non-targeted metabolomic analysis was performed on intestinal contents and brain tissue samples from the control, model, PRTP-H, FMT-Model, and FMT-PRTP groups. Metabolites were extracted with pre-cooled methanol containing 5 ppm 2-chlorophenylalanine. The extracts were analyzed using an ultra high performance liquid chromatography system (UHPLC, Vanquish, Thermo Fisher Scientific) coupled to an Orbitrap Exploris 120 mass spectrometer. Chromatographic separation was carried out on an ACQUITY UPLC HSS T3 column with gradient elution using 0.1% formic acid aqueous solution and acetonitrile containing 0.1% formic acid. Data were acquired in positive and negative electrospray ionization modes with data-dependent acquisition. Raw data were processed using MS-DIAL for peak extraction, alignment, and metabolite identification against the mzCloud and local databases.