Anti-Neuroinflammatory Potential of Drospirenone in A TLR4-Driven In Vitro Model of Neuropathic Pain

Neuroinflammation plays a crucial role in neuropathic pain, of which toll-like receptor 4 (TLR4) is a key mediator. Virtual screening identified Drospirenone as a potential TLR4 inhibitor. This study aims to evaluate the in vitro effects of Drospirenone on TLR4 signalling in U87-MG astrocytoma cells under different inflammatory conditions.

In this experimental study, three treatment approaches with Drospirenone were employed to model various stages of neuroinflammation: co-incubation with lipopolysaccharides (LPS) to assess preventive effects; treatment after inflammation to evaluate its impact on sustained inflammation; and delayed treatment after LPS removal to investigate its role in reducing persistent inflammation. Expression levels of TLR4, myeloid differentiation primary response 88 (MyD88), nuclear factor-κB (NF-κB) p65, and interleukin-1 beta (IL-1β) were assessed by Western blotting, while nitric oxide (NO) secretion was measured by ELISA.

The potential ability of Drospirenone to inhibit early-stage inflammation was shown by significant reductions in TLR4, MyD88, NF-κBp65, IL-1β, and NO. Treatment after inflammation showed that Drospirenone reduced NO secretion but did not significantly affect TLR4 and other inflammatory markers, which could indicate its potential efficacy in controlling sustained inflammation. In the delayed treatment approach, inflammation persisted after LPS removal, and Drospirenone did not return the inflammatory state to baseline.

Drospirenone may exhibit potential as a prophylactic agent in vitro during the early phases of neuroinflammation, though its efficacy appears limited in models of chronic or prolonged inflammation. These preliminary findings require in vivo validation, and future studies could explore possible synergistic effects with other treatments or alternative dosing strategies for neuropathic pain management.