Efeito neuroprotetor da Riparina III após exposição ao lipopolissacarídeo: modulação de comportamento depressivo, reversão de danos celulares in vitro e envolvimento do óxido nítrico sintase induzível

Abstract

Depression is a disorder whose pathophysiology has not yet been fully elucidated. Conventional treatment, largely based on the monoaminergic theory, faces significant limitations, including delayed onset of therapeutic effects and the occurrence of adverse side effects. In this context, the neuroinflammation hypothesis has gained increasing attention, supported by evidence that dysregulation of inflammatory processes may be associated with the onset and persistence of depressive disorders. Within this framework, Riparin III (Rip III) emerges as a promising synthetic compound, given its previously demonstrated antidepressant and anxiolytic potential, as well as its anti-inflammatory and antioxidant properties. Thus, this study aimed to investigate the effects of Rip III in both in vivo and in vitro models following exposure to Escherichia coli lipopolysaccharide (LPS). Male C57BL/6 mice were subjected to an intraperitoneal LPS administration protocol (0.5 mg/kg, i.p.) for 10 consecutive days. From day 5 onward, animals received oral treatment with Rip III (50 mg/kg), saline (0.9%), or escitalopram (10 mg/kg). On the final day of the experiment, behavioral tests predictive of depressive-like behavior (Forced Swim Test and Sucrose Preference Test), locomotion, exploration, and anxiety-related behaviors (Open Field Test, Elevated Plus Maze, and Hole-Board Test), as well as short-term memory (Y-Maze), were performed. Subsequently, animals were euthanized, and brain tissues (prefrontal cortex and hippocampus) were collected and properly stored for the determination of nitrite levels, thiobarbituric acid reactive substances (TBARS), and the pro-inflammatory cytokines IL-6 and IL-1β. In the in vitro assays, the effects of Rip III at concentrations ranging from 1000 to 15.6 μM on the viability of PC12 cells and astrocytes were evaluated using the MTT reduction assay. Additionally, flow cytometry analyses were performed to assess cell death mechanisms through propidium iodide (PI) and Annexin V (Anx V) staining, as well as mitochondrial transmembrane potential using rhodamine staining. Morphological alterations were evaluated by scanning electron microscopy and light microscopy. To identify potential molecular targets, molecular docking analyses were conducted. The study demonstrated that Rip III reversed LPS-induced depressive-like behavior and improved anxiety-related, exploratory, and short-term memory parameters. Rip III also reduced TBARS and nitrite levels. In vitro, Riparin III reduced cell viability at 1000 μM in PC12 cells and at 500 and 1000 μM in astrocytes, and these concentrations were excluded from subsequent experimental phases. Conversely, Rip III protected both PC12 cells and astrocytes from LPS-induced structural damage, na effect supported by reduced PI/Annexin V positivity and preservation of mitochondrial transmembrane potential. Molecular docking indicated relevant interactions with inducible nitric oxide synthase (iNOS), particularly involving residues Glu371, Arg382, and the heme group, suggesting this enzyme as a potential therapeutic target. These findings reinforce the neuroprotective and anti-inflammatory potential of Rip III and encourage further studies to elucidate its mechanisms of action in neuroinflammatory conditions.