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

• Grant number: 22/14406-8
• Support Opportunities: Scholarships in Brazil - Doctorate
• Effective date (Start): March 01, 2024
• Effective date (End): November 30, 2026
• Field of knowledge: Biological Sciences - Morphology - Anatomy
• Principal Investigator: Patricia Castelucci
• Grantee: Marcos Antônio Ferreira Caetano
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Inflammatory Bowel Diseases affect the neurons of the Enteric Nervous System (ENS). Butyrate is a short-chain fatty acid (SCFA) produced by bacteria of the intestinal microbiota, from the fermentation of dietary fibers. It binds to G protein-coupled fatty acid receptors such as GPR41, GPR43 and GPR109A and triggers anti-inflammatory effects, improves intestinal barrier and protection against pathogens. The literature reports the presence of SCFA receptors in the ENS, however it is not known in which types of neurons these receptors are present and what are their effects on these neurons. Furthermore, butyrate has been promising in the treatment of ulcerative colitis due to its anti-inflammatory effects. Therefore, this project aims to study the GPR43 and GPR109A receptors and analyze the use of Butyrate in the myenteric plexus neurons of mice submitted to experimental ulcerative colitis. For this, 100 µL of 2, 4, 6 trinitrobenzene sulfonic acid (TNBS) 1.5% will be injected intrarectally in C57BL/6 mice (TNBS Group). The Sham group will receive 35% ethanol (vehicle). A group of animals will be treated with 100mg/kg of Sodium Butyrate (BUT group), via gavage, and the TNBS and Sham groups will receive saline. Animals will be euthanized 7 days after injection of TNBS or vehicle. The following will be analyzed: A) The colocalization of GPR43, GPR43 and GPR109A receptors with neurons immunoreactive (-ir) to neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), Calretinin (Carl), Calbindin (Calb), to the pan- neuronal PGP9.5 and with glia immunoreactive to the glial marker glial fibrillary acidic protein (GFAP); B) Dual immunofluorescence staining of uncleaved Caspase-3, cleaved Caspase-3, NF-ºB and Acetylhistone 3 in nNOS-ir, ChAT-ir, Calr-ir, Calb-ir and PGP9.5-ir neurons ; C) The number of GPR43-ir, GPR109A-ir, nNOS-ir, ChAT-ir, Calr-ir, Calb-ir, PGP9.5-ir neurons/ganglion and GFAP-ir glials per ganglion; D) The area of nNOS-ir, ChAT-ir, Calr-ir, Calb-ir and PGP9.5-ir neurons; E) Corrected Total Cell Fluorescence (CTCF) of GPR43-ir, GPR109A-ir, nNOS-ir, ChAT-ir, Calr-ir, Calb-ir, PGP9.5-ir and GFAP-ir glial neurons; F) The morphology of the distal colon by histology technique, using Hematoxylin & Eosin, Periodic Acid Schiff and Picrosirius Red staining; G) The tight junction proteins ZO-1 and occludin; H) Protein expression of receptors GPR41, GPR43 and GPR109A, cleaved caspase-3, NF-º², histone deacetylase and MAPKinases (ERK1 and ERK2); I) Gene expression of receptors GPR41, GPR43 and GPR109A, cleaved caspase-3, NF-ºB, acetylated histone and MAPKinases (ERK1 and ERK2); J) Neuronal death through Apoptosis using the Tunel technique; K) Intestinal transit and intestinal motility; L) Inflammatory mediators by measuring IL-1², IL-6, IL-10 and TNF-± through enzyme-linked immunosorbent assays. Results will be expressed as mean ± standard error and statistical analysis will be performed with a significance level of p<0.05.