Evaluation of cholinergic receptors in A549 and BEAS-2b cells stimulated with IL13 and neutrophil elastase

• Grant number: 23/09694-7
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: November 01, 2023
• End date: October 31, 2024
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Principal Investigator: Iolanda de Fátima Lopes Calvo Tibério
• Grantee: Vitória Caroline de Queiroz
• Supervisor: Ayman K. Hamouda
• Host Institution: Faculdade de Medicina (FM). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: University of Texas at Tyler (UT Tyler), United States
• Associated to the scholarship: 22/02510-5 - The role of cholinergic antiinflammatory pathway in a model of Asthma-COPD Overlap Syndrome (ACOS) modelo experimental, BP.DD

Abstract

Bronchial asthma and chronic obstructive pulmonary disease (COPD) are common respiratory disorders characterized by chronic inflammation and airway obstruction. Asthma is associated with bronchial hyperreactivity and is influenced by genetic and environmental factors, while COPD encompasses emphysema and bronchitis, resulting in small airway obstruction. The coexistence of asthma and COPD, known as asthma a COPD overlap (ACO), presents diagnostic challenges due to overlapping symptoms. Both diseases significantly contribute to global morbidity and mortality. Aim: To evaluate whether IL-13 and Neutrophilic elastase (NE) modulate the nicotinic receptors expression in pulmonary cells. Methodology: The bronchial epithelial cell line, BEAS-2B, and the human lung adenocarcinoma cell line, A549, will be employed for in vitro experiments. To mimic asthma-related injury, both cell lines will be treated with IL-13, while neutrophilicelastase (NE) will be used to simulate chronic obstructive pulmonary disease (COPD).Additionally, a combination of IL-13 and NE will be applied to mimic the overlap of asthma and COPD (ACO). Cell viability will be assessed using the MTT assay, and the levels ofIL-17, IL-8, IL-4, and IL-6 will be measured in the culture medium using ELISA kits. Furthermore, quantitative real-time PCR will be performed to evaluate the gene expression of ±7, ±4, ²2 nicotinic and muscarinic (M) receptors subtypes. Additionally, the evaluation of reactive oxygen species (ROS) will be conducted using a specific kit. Radioligand binding assays will be performed using centrifugation assays to determine the binding of non-selective and subtype-selective radio ligands to nAChR-rich membranes. The experimental methods applied in this study will provide a comprehensive analysis of the expression of nicotinic and muscarinic receptor expression in pulmonary cells submitted to different types of injury. Data analysis will be performed using the SigmaStat program. (AU)