Necroptosis Regulators and Dystrophin-Deficient Muscle: Exploring Therapeutic Interventions in the mdx Mouse Model of Duchenne Muscular Dystrophy

• Grant number: 24/22008-8
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: March 01, 2025
• End date: February 28, 2026
• Field of knowledge: Biological Sciences - Morphology - Anatomy
• Principal Investigator: Renato Ferretti
• Grantee: Bruno dos Santos Ricardi
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

In the absence of dystrophin, muscle fibers undergo membrane rupture, releasing damage-associated molecular patterns (DAMPs), which leads to elevated serum creatine kinase (CK) levels, inflammation (characterized by increased TNF-¿ levels), and ultimately functional loss. The mdx mouse model, which mimics Duchenne muscular dystrophy (DMD), presents muscle fibers lacking dystrophin, resulting in elevated CK levels, activation of the innate immune system via DAMPs through TLR4 signaling, and high TNF-¿ levels, all of which play a critical role in the pathogenesis of DMD. Additionally, in the absence of dystrophin, muscle fibers activate necroinflammation through DAMPs, promoting the activation of M1 macrophages and the release of TNF¿. When TNF¿ binds to TNFR1, it recruits RIPK1 and RIPK3, forming the necrosome. The phosphorylation of MLKL by RIPK3 disrupts the plasma membrane, leading to necrosis through the necroptosis pathway. Recent advancements highlight combined therapeutic strategies, such as using prednisolone (PDZ) in conjunction with curcumin (CURC), which have shown promise in reducing inflammation, necrosis, and promoting muscle regeneration. However, it remains unclear whether the anti-inflammatory protective effects of PDZ, either alone or combined with CURC, on muscle fibers in the absence of dystrophin are linked to the modulation of the Tlr4 gene, reduction of TNF¿ production, regulation of macrophage polarization (from M1 to M2 phenotype), or inhibition of genes involved in necroptosis (Ripk1, Ripk3, and Mlkl). This study aims to investigate whether the combination of PDZ and CURC exerts a protective anti-inflammatory effect on dystrophic muscle fibers by modulating the Tlr4 gene pathway, promoting the transition of macrophages to the reparative M2 phenotype, and inhibiting the activation of necroptosis-associated genes. We will use control and dystrophic animals at 30 days of age. The mdx mice were subjected to 15 days of daily treatment by gavage with PDZ, CURC, and P+CURC. The doses will be adjusted according to body weight. A muscle strength test will be conducted, followed by the harvesting of the quadriceps muscle. Serum CK levels, histopathology, and gene expression will be analyzed using qRT-PCR. The results may provide valuable insights into the transcriptional control of inflammatory processes and cell death in muscle fibers in the absence of dystrophin during pharmacological therapies.