Bacterial Supplementation Reverses Gut‒Brain Axis Disruption in Pelvic Irradiation by Restoring Bacteria-Derived Neuroactive Metabolites

Description

Pelvic radiotherapy induces gut dysbiosis that propagates neurotoxicity via the microbiota-gut-brain axis. In Sprague-Dawley rats exposed to 6 Gy pelvic irradiation, prophylactic/therapeutic multi-strain probiotics restored depleted beneficial taxa (Lactobacillaceae, Butyricicoccaceae, Ruminococcaceae) and reduced opportunistic pathogens. Untargeted LC-MS brain metabolomics (3,535 metabolites; 84 microbial-derived) revealed radiation-altered compounds from Proteobacteria, Firmicutes, Ascomycota, Actinobacteria affecting xenobiotic degradation, oxidative stress, lipid signaling, and neurotransmitter pathways. Prophylactic-therapeutic supplementation recovered allantoin, PA(18:0/15:0), and reactivated neuroprotective cascades, linking microbiome restoration to brain metabolome normalization. Keywords: gut dysbiosis, pelvic radiation, microbiota-gut-brain axis, probiotics, LC-MS metabolomics, neuroprotection Organism: Rattus norvegicus (Sprague-Dawley rats, male, 3-4 months) Experimental Factors: Treatment Variant (Control, Radiation, Bacterial supplements, Combination) Technology Type: 16S rRNA sequencing; Untargeted LC-MS metabolomics Study Design: Multi-omics analysis of radiation-induced brain metabolome changes and probiotic mitigation.

Steps to reproduce

16S rRNA Profiling (Gut Microbiota) Collect fecal samples; extract DNA per ref 17. PCR amplify V3-V4 region (341F: CCTACGGGNGGCWGCAG; 806R: GACTACHVGGGTATCTAATCC); exclude PCR failures. Quantify successful amplicons (Qubit dsDNA BR); dilute to 1 ng/µL in low-TE. Prepare libraries (Fadrosh phased primer strategy ref 12); pool at 10 nM; validate (Agilent TapeStation/Qubit HS). Sequence on Illumina MiSeq (2x250 v2 kit); demultiplex to .fastq. Raw Data Processing FastQC v0.11.9 → MultiQC report. fastp v0.12.4 (adapter removal). QIIME2/DADA2 pipeline: alpha diversity (Observed, Chao1, Shannon, Simpson). PICRUSt2 for functional prediction. Brain Metabolomics (Untargeted LC-MS) Homogenize 50 mg brain in 1 mL 50% MeOH (35 Hz, 60 s, 4°C); vortex; precipitate (-20°C, 20 min). Centrifuge (6,200×g, 10 min, 4°C); dry supernatant (N2); reconstitute in 200 µL 80% MeOH. LC-MS: Xevo QTOF ACQUITY UPLC H-Class PLUS (ESI+); BEH C18 column (2.1×50 mm, 1.7 µm). Gradient: 0-3.06 min 5% B; 3.06-3.20 min 5-35% B; 3.20-3.54 min 75% B; hold; return 5% B (0.306 mL/min, 0.1% FA). Process in MS-Dial v4.9 (±15 ppm; adducts: [M+H]+, [M+NH4]+, [M+Na]+, [M+K]+, [M+CH3]+). Annotate vs HMDB/ChEBI/COCONUT/FooDB; microbial metabolites via MiMeDB. Analyze: MetaboAnalyst 6.0 (PCA, heatmaps, pathways); QEA (globaltest v3).

Institutions

• Manipal Academy of Higher Education

  Karnataka, Manipal

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