Effects of melatonin on the activation and inflammation of astrocytes in the central nervous system: an ex vivo study on primary cell cultures

• Grant number: 25/08439-9
• Support Opportunities: Scholarships abroad - Research Internship - Master's degree
• Start date: September 01, 2025
• End date: February 28, 2026
• Field of knowledge: Health Sciences - Dentistry - Endodontics
• Principal Investigator: Doris Hissako Matsushita
• Grantee: Anna Clara Cachoni
• Supervisor: Alberto Ferri
• Host Institution: Faculdade de Odontologia (FOA). Universidade Estadual Paulista (UNESP). Campus de Araçatuba. Araçatuba , SP, Brazil
• Institution abroad: Fondazione Santa Lucia Irccs, Italy
• Associated to the scholarship: 24/07729-0 - COGNITIVE EVALUATION IN RATES WITH APICAL PERIODONTITIS TREATED WITH MELATONIN, BP.MS

Abstract

This study investigates the effects of melatonin on inflammatory pathways in astrocytes subjected to lipopolysaccharide (LPS)-induced neuroinflammation, using an ex vivo model. The main objective is to evaluate how melatonin can modulate the inflammatory response in LPS-induced primary astrocytes, focusing on proteins involved in inflammatory pathways, such as NF-kB, Akt, JNK and TNF-¿, using the Western blotting technique. For this, astrocytes will be isolated from neonatal hippocampus of rat brain and cultured in culture medium supplemented with fetal bovine serum until they reach confluence. Neuroinflammation will be induced with LPS (100 ng/mL), which activates glial cells, mimicking the inflammatory process observed in the Central Nervous System (CNS) pathologies. Melatonin treatment will be performed for 24h and 48h, using two different dose of 100 ¿M and 200µM in dissolved Dulbecco's modified Eagle's medium (DMEM). As a control, DMEM alone will be used, ensuring that any observed effects are attributable to melatonin itself and not to solvent interference. After treatment, cells will be harvested, lysed in a protein extraction buffer containing protease and phosphatase inhibitors, and target proteins will be analyzed by Western blotting. The inflammatory proteins, NF-kB (p65), p-Akt, p-JNK and TNF-¿, will be detected with specific antibodies and visualized by chemiluminescence. In addition, an Seahorse XF96 technique will be performed evaluating oxygen consumption (OCR) and extracellular acidification (ECAR) to analyze mitochondrial function and glycolytic activity of cultured cells. The data obtained will be analyzed by image analysis software, ImageJ, to quantify the protein bands and compare them between the experimental, control and LPS groups. Melatonin is expected to reduce the inflammation and the release of inflammatory cytokines, suggesting its therapeutic potential in neuroinflammatory diseases such as Alzheimer's and Parkinson's. This study may provide new insights into the use of melatonin to treat neurodegenerative diseases by modulating inflammatory pathways. (AU)