Parkinson's disease (PD) is a common neurodegenerative condition marked by the gradual degeneration of dopaminergic neurons within the substantia nigra pars compacta (SNpc) [1]. This neuronal loss leads to motor dysfunction, non-motor symptoms and cognitive impairments, and non-motor symptoms, severely impacting the quality of life of the affected individuals [2,3]. The pathological hallmarks of PD include the accumulation of alpha-synuclein (α-syn) aggregates and the depletion of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis [4]. In addition to these hallmarks, a cascade of molecular and cellular dysfunctions, including oxidative stress, mitochondrial dysfunction, apoptosis, neuroinflammation, and ferroptosis have been implicated as critical mechanisms driving PD pathogenesis [5]. Despite decades of research, current treatments, such as L-dopa and carbidopa provide only symptomatic relief, failing to halt or reverse the neurodegenerative processes involved in PD [6]. Hence, there is an urgent need for newer therapeutic strategies that target the underlying pathophysiology of PD and slow PD progression.

Among the molecular mechanisms implicated in PD, ferroptosis has emerged as a significant contributor to neurodegeneration. Ferroptosis is an iron-dependent type of controlled cell death that includes lipid peroxidation, glutathione depletion, and GPX4 inactivation [5,7]. In the context of PD, excessive iron accumulation in the SNpc exacerbates oxidative stress and promotes ferroptosis, leading to dopaminergic neuronal loss [8]. Furthermore, ferroptosis is intricately linked to other pathological processes, including neuroinflammation and apoptosis [9], forming a vicious cycle that accelerates neuronal death [10]. Targeting ferroptosis and its upstream regulators, such as NRF2 and FTH1, represents a promising therapeutic approach to mitigate neurodegeneration in PD [9,11].

Apoptosis, another prominent cell death pathway in PD, is driven by oxidative stress, mitochondrial dysfunction, and pro-inflammatory signals [12]. The intrinsic apoptotic pathway, involving the dysregulation of Bcl-2 family proteins and the activation of caspase-9 and caspase-3, is particularly relevant in PD pathology [13]. The interplay between apoptosis and other mechanisms, such as ferroptosis and neuroinflammation, underscores the complexity of neuronal death in PD and highlights the need for multifaceted therapeutic interventions. Moreover, neuroinflammation, mediated by the activation of microglia and astrocytes, plays a pivotal role in exacerbating dopaminergic neuronal loss in PD [14]. Activated microglia release pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, further amplifying oxidative stress and neuronal damage [15]. Additionally, the activation of astrocytes, marked by GFAP expression, contributes to the disruption of the BBB and the perpetuation of neuroinflammatory cascades [16]. In addition, p-p38/NFκB signaling pathways are central to the regulation of these inflammatory responses, making them critical targets for therapeutic intervention [17]. Natural compounds with potent antioxidative, anti-inflammatory, anti-ferroptosis, and anti-apoptotic characteristics have emerged as potential targets for the development of neuroprotective treatments.

Betulinic acid (BA), a pentacyclic triterpenoid isolated from various plant sources typically from white birch trees [18] largely found in South Asia [19]. It has garnered significant attention due to its diverse biological activities, including anti-cancer, anti-inflammatory, and antioxidant effects. BA has been utilized therapeutically in Ayurveda, the ancient Indian system of medicine, for treating central nervous system (CNS) disorders [20]. Notably, the ability of BA to cross the blood-brain barrier (BBB) makes it a promising compound for the treatment of CNS-related conditions [21,22]. Most preliminary evidence suggests that BA may also possess neuroprotective properties [22,23]; however, its efficacy and mechanisms of action in PD remain largely unexplored. Furthermore, the oral LD50 of BA in mice is found to be 2000 mg/kg, indicating its relatively low toxicity. We used 10 mg/kg as the highest oral dose in our study, which is well below 1/10th of the reported LD50 value [24]. The various doses of BA (1, 3, and 10 mg/kg) in this study were selected based on the existing in-vivo pharmacological studies reported in literature [23]. Thus, BA presents a compelling candidate for further investigation in the context of neurodegenerative disorders. Lipopolysaccharide (LPS)-induced neuroinflammation is widely used to study PD, as it triggers microglial activation and the release of proinflammatory cytokines (TNF-α, IL-6, IL-1β), leading to dopaminergic neuronal damage [[25], [26], [27], [28], [29], [30]]. However, LPS alone does not fully replicate the progressive neurodegeneration, iron accumulation, oxidative stress, and other characteristics of PD. FeSO4, on the other hand, is a known contributor to iron dyshomeostasis, ferroptosis, and apoptosis, which plays a crucial role in dopaminergic neurodegeneration [[31], [32], [33], [34], [35], [36]]. A combination of systemic LPS and oral ferrous sulfate (FeSO4)-induced rat model of PD may offer a robust experimental platform to investigate the complex interplay of neuroinflammation, ferroptosis, oxidative stress, and dopaminergic neuronal loss. This model replicates several key features of PD pathology, including (α-syn) aggregation, TH depletion, and the activation of pro-inflammatory signaling pathways such as p-p38 and NFĸB. Additionally, this model highlights the important role of ferroptosis, an iron-dependent form of regulated cell death in mediating neurodegeneration. The multifactorial nature of this model makes it particularly well-suited for evaluating the potential neuroprotective effects of candidate compounds like BA.

Hence, we aimed to evaluate the neuroprotective potential of BA in mitigating neurobehavioral deficits, biochemical alterations, structural and histopathological changes induced by LPS + FeSO4 in a rat model of PD.