Evaluation of paeonol as a neurorestoration agent in a rotenone-induced in vitro model of Parkinson's disease: Post-injury recovery of cell viability, migration, and pCREB signaling

Parkinson's disease (PD) is defined by progressive dopaminergic neurodegeneration involving a complex interplay of mitochondrial dysfunction, impaired redox homeostasis, and diminished cellular resilience. While most experimental models evaluate candidate agents in pre-treatment paradigms, clinically meaningful intervention requires the capacity to rescue already-injured cells. This study therefore investigated whether paeonol, a natural phenolic compound, could exert post-injury restorative effects in a rotenone-induced SH-SY5Y in vitro model of PD. A reproducible sub-lethal injury window was first established using MTT assays, identifying a rotenone paradigm (50 nM, 48 h) that induced significant but non-terminal metabolic suppression. To model a clinically realistic therapeutic scenario, paeonol was administered only after rotenone injury and washout. Cell viability and functional recovery were assessed using MTT and wound-healing assays; intracellular ROS was evaluated via DCFH-DA fluorescence; pCREB signalling was examined by immunofluorescence; and exploratory transcriptional changes in TH and SNCA were analysed by qPCR. Rotenone significantly impaired both cell viability and migration. Following washout, metabolic activity recovered spontaneously; however, paeonol post-treatment enhanced wound closure and restored pCREB signalling, indicating functional and molecular recovery beyond metabolic restoration.Contrary to classical expectations, rotenone did not elevate intracellular ROS; instead, total fluorescence was markedly reduced relative to controls, likely reflecting decreased cell density and metabolic suppression. Paeonol did not substantially modify ROS, suggesting that its restorative effects are not primarily mediated by bulk antioxidant activity. At the signalling level, rotenone markedly suppressed pCREB, whereas paeonol robustly restored pCREB expression (p < 0.0001), supporting a mechanism involving reactivation of pro-survival pathways rather than simple redox scavenging. Exploratory qPCR findings were interpreted cautiously due to methodological limitations and are presented as hypothesis-generating. Overall, paeonol demonstrates meaningful post-injury rescue effects, improving cellular viability, functional migration, and survival signalling in a rotenone-based PD model. These findings highlight paeonol as a promising modulator of cellular resilience and provide a strong rationale for further mechanistic and translational investigation.