Mellyely Carolina Basílio Soares¹, Cristiane Amorim de Paula¹, Thiago Verano - Braga¹

Emerging evidence highlight the role of the gut microbiota in the regulation of both behavior and metabolism has prompted research into the effects of gut dysbiosis in various pathophysiological contexts. To gain a deeper understanding of the functional axis encompassing gut microbiota, the renin-angiotensin system, and the heart, this study investigated the metabolic and behavioral effects of gut dysbiosis induced by chronic unpredictable mild stress (CUMS) for 21 days, and, in the last 14 days, they were subjected to antibiotic therapy (ATB). The aim was to conduct a comparative proteome analysis of the cardiac tissue among experimental groups and to quantify specific proteins. Male C57BL6/J mice aged 8–12 weeks were used. Dysbiosis was induced using an ATB protocol. Animals had free access to drinking water containing ampicillin for 14 days. On the 7^th day of treatment, animals were treated with an oral administration of streptomycin. Fecal samples were collected before and after the 21-day intervention for microbiome analysis. On day 22, animals underwent elevated plus maze and open field tests to assess anxiety-like behaviors. On day 23, glucose tolerance tests (GTT) were performed, followed by euthanasia on day 24 for subsequent analyses (CEUA: 69/2025). Cardiac tissues were harvested and cryogenically macerated with a mortar and pestle under liquid nitrogen, then lysed in buffer for protein extraction. Proteins were reduced and alkylated at room temperature, followed by enzymatic digestion with trypsin. Reactions were halted using 0.5% trifluoroacetic acid. A bottom-up label-free approach was employed, and proteomic analysis was conducted via LC-MS/MS (liquid chromatography–tandem mass spectrometry) using a nanoflow HPLC system (DIA; tims TOF MS, Bruker). Spectral data were processed with MaxQuant, and data mining was performed using DanteR. A subset of animals was also submitted to the Langendorff perfusion system to simulate myocardial infarction. Preliminary results indicated that induced dysbiosis was associated with improved glucose tolerance, evidenced by reduced glycemic curves compared to the control group: CTR (308.5 mg/dL ± 69.11); CUMS (267.1 mg/dL ± 58.38); ATB (267.3 mg/dL ± 44.28); CUMS+ATB (255.1 mg/dL ± 50.46). In contrast, CUMS did not significantly affect body weight: CTR (23.68 g ± 0.63); CUMS (23.68 g ± 0.63); ATB (23.40 g ± 0.52); CUMS + ATB (23.69 g ± 0.47). In the elevated plus maze, a reduction in time in the open arms was observed in the ATB (79.69 s ± 8.39) and CUMS+ATB (30.04 s ± 7.44) groups, indicating anxiety-like behavior. These findings suggest that induced gut dysbiosis improves glucose tolerance while simultaneously promoting anxiety-like behavioral changes. Ongoing molecular, histological, and cardiac proteomic analyses aim to elucidate the mechanisms underlying behavioral and metabolic changes triggered by gut dysbiosis. It is hypothesized that animals exposed to chronic stressors will exhibit higher levels of pro-inflammatory proteins, and potential biomarkers relevant to the brain–microbiota–heart axis. Funding: CNPq, CAPES, and FAPEMIG.

Agradecimentos: We gratefully acknowledge the financial support provided by the following funding agencies: the National Council for Scientific and Technological Development (CNPq), the Coordination for the Improvement of Higher Education Personnel (CAPES), and the Foundation for Research Support of the State of Minas Gerais (FAPEMIG).