In vitro evaluation of the neuroprotective activity of Resolvin E1 and nanoencapsulated Resolvin E1 on astrocyte reactivity

Abstract

When the acute inflammatory process in the central nervous system (CNS) is uncontrolled, neuroinflammatory processes are activated, depleting the mechanisms of inflammation resolution. This initiates the production of neurotoxic signals underlying neurodegenerative diseases. Among these, multiple sclerosis (MS) is characterized by a neuroinflammatory state involving neuronal demyelination, axonal degeneration, loss of motor function, and the presence of gliosis in the CNS. Specifically, astrocytes respond to this state through a process termed "astrogliosis", becoming reactive and exacerbating the inflammatory process, which promotes a neurotoxic environment. This occurs because astrocytes secrete various cytokines, chemokines, and growth factors that recruit peripheral immune cells, generating active inflammatory infiltrates characteristic of MS progression. Additionally, neurotoxic astrocytes have been shown to be highly expressed in MS and are directly linked to synaptic disruption and loss of neural trophic support. On the other hand, the resolution of neuroinflammation is an active process regulated by specialized pro-resolving lipid mediators (SPMs), which activate transcriptional and transductional pathways that reduce inflammatory signaling associated with the recruitment of polymorphonuclear cells. Following exposure to an inflammatory stimulus, DAMPs and PAMPs are released and recognized by resident immune cells in the tissue, such as macrophages and mast cells. This recognition occurs through pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and NOD-like receptors (NLRs), which initiate the acute inflammatory response. This process is subdivided into two stages: initiation and resolution of inflammation. In particular, Resolvin E1 (RvE1), an Omega-3-derived SPM, has been proposed as a potential high-potency therapeutic agent for the resolution of inflammatory and neurodegenerative diseases due to its potential to specifically and stereoselectively bind and activate two G protein-coupled receptors that inhibit the NF-κB signaling pathway. However, due to its susceptibility to enzymatic, oxidative, and light-induced degradation, its biomedical application has been hindered. In serum samples and active brain lesions from patients with MS, as well as in the experimental autoimmune encephalomyelitis (EAE) model, a significant decrease in the expression of enzymes involved in SPM synthesis has been observed. In these cases, exogenous administration of resolvins (e.g., RvD1) improves clinical signs of EAE and suppresses the infiltration of auto reactive T cells, while also inducing an anti-inflammatory phenotype in macrophages and brain glial cells, serving as a potential treatment for neuroinflammation in vivo. This suggest the potential role of other resolvins, such as RvE1, as promising regulators of neuroinflammation. However, whether RvE1 exerts protective effects on reactive astrocytes remains unknown. Therefore, in this project, we propose the use of a lipid-based nanovehicle to enhance stability, bioavailability, and pro-resolving activity of RvE1 on astrocyte reactivity. To test this hypothesis, the DITNC1 astrocyte cell line and primary astrocyte cell cultures derived from neonatal rat cortex were used. First, a polyarginine-coated lipid nanocapsule was synthesized using a modified solvent displacement method, providing stability to the molecule and increasing its bioavailability. Subsequently, astrocyte reactivity, determined as glial fibrillary acidic protein (GFAP) expression and astrogliosis-associated morphology were analyzed using flow cytometry and fluorescence microscopy. Finally, the activation state of the proinflammatory NF-κB transcriptional pathway was studied using fluorescence microscopy and immunoblotting. The RvE1 nanoparticle, with a size of 163.9±18 nm, polydispersity index of 0.127±0.051, and positive charge (PZ: 47.3±10.3 mV), showed no cytotoxicity. Moreover, nanoencapsulated RvE1, unlike free RvE1, significantly reduced GFAP expression in astrocytes treated with IFN-γ and LPS, and delayed cellular hypertrophy associated with astrogliosis. Lastly, nanoencapsulated RvE1 inhibited NF-κB pathway activation. These data collectively indicate that the encapsulation of RvE1 exerts neuroprotective effects by modulating the reactive state of astrocytes, a key contributor to neuroinflammation in neurodegenerative diseases. Given that RvE1 has potent inhibitory activity on neutrophil migration and macrophage, lymphocyte, and microglia activation, the results of this thesis could be projected toward a potential new therapeutic agent for the treatment of MS and other neurodegenerative diseases.