Effects of sodium butyrate on the inflammatory response in enteric glial cells in vitro

• Grant number: 25/12448-3
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: December 01, 2025
• End date: May 31, 2026
• Field of knowledge: Biological Sciences - Morphology - Anatomy
• Principal Investigator: Patricia Castelucci
• Grantee: Marcos Antônio Ferreira Caetano
• Supervisor: Brian Gulbransen
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: Michigan State University (MSU), United States
• Associated to the scholarship: 22/14406-8 - Characterization of the effect of sodium butyrate on short-chain fatty acid receptors in the enteric nervous system of mice submitted to experimental ulcerative colitis, BP.DR

Abstract

The enteric nervous system (ENS) is affected by inflammatory bowel diseases (IBDs). The enteric glial cells (EGC) play roles in intestinal physiology and also in the pathophysiology of IBDs. There has been much discussion about the role of the gut microbiota as well as its metabolic products such as short-chain fatty acids (SCFAS), produced by the fermentation of dietary fibers, and their effects on ENS. Among the SCFAS, butyrate stands out due to its anti-inflammatory potential. Also, butyrate can bind to receptors such as GPR41, GPR43 and GPR109A, improving gut health, reducing cytokines, inhibiting histone deacetylase 3 (HDAC3), increasing expression of tight junctions and mucus production. Knowledge about the effects of butyrate on EGC in the inflammatory context is scarce. Therefore, the aim of this project is to investigate the effects of sodium butyrate on EGC in vitro. For this purpose, a primary culture of EGC will be established from the myenteric plexus of the mouse colon. Briefly, colon tissues from adult mice will be collected and washed with ice-cold DMEM-F12 medium supplemented with L-glutamine and HEPES. After cleaning, the longitudinal muscle layer together with the myenteric plexus will be dissected, digested and the cell suspensions will be filtered and seeded in 24-well plates. The plates will be kept in an incubator and will be maintained with medium composed of DMEM-F12 supplemented every 2 days. EGC are expected to reach 50-80% confluence in 2 weeks. EGC will be inflamed with a mix of proinflammatory cytokines: IL-1B (10ng/mL); IL-6 (10ng/mL); TNF-a (10ng/mL) and IFN-Y (10ng/mL) for 24h. The cells will then receive 0.5 mM sodium butyrate for 24h. The positive control will be performed by applying only the cytokine mix for 24h followed by 24h with the common compound medium. The negative control will be performed by applying only the medium with the common compound medium without cytokines. After the treatment protocol, immunohistochemical staining for GFAP, S100B, IL-6, iNOS and IL-1B will be performed to evaluate pro-inflammatory phenotypic changes. Colocalization analyses, EGC number/field, morphology, and corrected total cell fluorescence will be assessed. PCR will be performed to analyze markers of reactive gliosis gliosis following exposure to proinflammatory cytokines and treatment or not with butyrate.