# Roflumilast Elicits Therapeutic and Neuroprotective Effects in 3-Nitropropionic Acid-Induced Huntington’s Disease-Like Neurodegeneration in Rats by Mitigating NLRP3 Inflammasome-Mediated Pyroptosis, Ferroptosis, and Glial Activation

## TL;DR

Roflumilast protects against Huntington’s disease-like brain damage in rats by reducing harmful cell death and inflammation.

## Contribution

This study demonstrates roflumilast's therapeutic effects in Huntington’s disease by targeting pyroptosis, ferroptosis, and glial activation.

## Key findings

- Roflumilast improved motor and behavioral deficits in 3-NP-induced HD-like rats.
- The drug reduced NLRP3 inflammasome activation and markers of pyroptosis and ferroptosis.
- Roflumilast attenuated astrogliosis, microglial activation, and neuroinflammation in the striatum.

## Abstract

Huntington’s disease (HD) pathogenesis involves diverse cellular mechanisms, yet the contributions of pyroptosis and ferroptosis remain elusive. Roflumilast, a phosphodiesterase-4 (PDE-4) inhibitor, has shown neuroprotective effects, but its precise mechanisms are yet to be elucidated. We evaluated the potential neuroprotective and therapeutic effects of roflumilast in 3-nitropropionic acid (3-NP)-induced HD-like neurodegeneration, focusing on pyroptotic and ferroptotic cell death signaling. Adult male Wistar rats were assigned to five groups: normal control (saline + 0.5% carboxymethyl cellulose), roflumilast-control (1 mg/kg/day, p.o. for 21 days), 3-NP (20 mg/kg/day, i.p. for seven days), roflumilast-prophylactic (1 mg/kg/day, p.o. for 21 days prior to 3-NP), and roflumilast-treatment (1 mg/kg/day, p.o. for 21 days post-3-NP). Behavioral outcomes of the open-field, rotarod, and grip strength tests were assessed. Striatal PDE-4, total and p-CREB, BDNF, interleukin-1β, and markers of pyroptosis (NLRP3, caspase-1, and gasdermin D) and ferroptosis (iron, GPx4, GSH, and malondialdehyde) were measured alongside histopathological alterations and GFAP and Iba-1 immunohistochemical staining. Bioinformatics was used to visualize the target genes’ protein-protein interaction network. Behavioral assessments revealed impaired locomotion, motor coordination, and muscle strength in the 3-NP-injected rats. Biochemical analysis showed increased striatal PDE-4 expression and decreased p-CREB/BDNF axis alongside NLRP3 inflammasome/caspase-1/gasdermin D activation and elevated interleukin-1β. In parallel, ferroptosis was evidenced by increased striatal iron and malondialdehyde levels, along with reduced GPx4 and GSH. Histopathological examination revealed pronounced striatal neurodegeneration, accompanied by enhanced GFAP and Iba-1 immunostaining, indicating astrogliosis and microglial activation. Roflumilast, administered prophylactically or therapeutically, significantly improved functional and behavioral abnormalities while ameliorating biochemical, histopathological, and immunohistochemical derangements induced by 3-NP. The therapeutic regimen exhibited superior efficacy relative to prophylaxis. Conclusively, roflumilast exerts therapeutic and neuroprotective effects in HD-like neurodegeneration by mitigating pyroptosis and ferroptosis, attenuating astrogliosis, microglial activation, and neuroinflammation, and restoring synaptic plasticity.

A graphical abstract illustrating the proposed mechanistic pathway underlying the neuroprotection of the PDE-4 inhibitor roflumilast through reducing striatal pyroptosis, ferroptosis, microglial and astrocyte activation, and neuroinflammation, while restoring synaptic plasticity in experimental Huntington’s disease-like neurodegeneration induced by 3-NP.