Chitosan-selenium nanoparticles alleviate fluoride-induced hippocampal neurotoxicity by targeting the DUSP5-ERK-CREB-BDNF signaling axis.

Introduction

Learning and memory impairment is an increasingly serious global issue that can directly lead to behavioral and mental state deviations in both humans and animals. [1] In recent years, fluoride (F) has been recognized as a neurotoxicant that contributes to learning and memory impairment [[1], [2], [3]]. F exists widely in the diet, ambient air, and drinking water in nature in both bound and ionic forms [4,5]. Most cases of endemic fluorosis are caused by high levels of F in drinking water [6]. F concentrations in groundwater in fluorosis-affected areas range from 0.1 to 22 mg/L, whereas the World Health Organization (WHO) recommends less than 1.5 mg/L for drinking water [7,8]. In animal experiments, almost all brain structures accumulate F, which is directly related to F intake [3,9]. Prolonged exposure to F at concentrations of 125–200 mg/L resulted in grooming disorders, increased spontaneous motor activity, reduced learning ability, and impaired memory acquisition [2,9,10]. Despite efforts to study the effects of F on the central nervous system (CNS), the specific mechanisms of F-induced learning and memory impairment, as well as their prevention and treatment, are still worth exploring.

The impact of F on neurological and cognitive impairment is associated with alterations in neurotransmitter transmission and synaptic receptor structure. Most studies have demonstrated that after F exposure, the acetylcholine (Ach) content in the hippocampus or brain of experimental animals and humans decreases significantly [11,12], whereas the levels of 5-hydroxytryptamine (5-HT), glutamic acid (Glu), histamine (HIS), and gamma-aminobutyric acid (GABA) increase substantially [[12], [13], [14]]. Furthermore, oxidative stress is one of the leading causes of F-induced neuronal cell damage and death. The CNS is vulnerable to oxidative stress because the cell membrane is rich in oxidative metabolism and oxidizes polyunsaturated fatty acids. When the antioxidant defense system is relatively weak, the functions of individual neurons and even the whole nervous system are impaired. F promotes the accumulation of reactive oxygen species (ROS) by inhibiting the activity of antioxidant enzymes, leading to an imbalance between oxidation and antioxidation in vivo. Oxidative stress can activate the mitogen-activated protein kinase (MAPK) cascade response, and the stimulation of extracellular signal-regulated kinase (ERK1/2) by ROS has been demonstrated in neurons [15,16]. Mounting evidence has demonstrated that the ERK1/2 signaling pathway is related to memory storage and synaptic plasticity [17]. Diverse signaling pathways, including those mediated by growth factor receptors, transmembrane receptors, and synaptic afferent neurons, can activate ERK [18]. ERK facilitates learning and memory by regulating intranuclear transcription [19]. Specifically, ERK modulates transcription by phosphorylating of cAMP response element binding protein (CREB), thereby promoting long-term potentiation (LTP) and memory formation [20,21]. Brain-derived neurotrophic factor (BDNF) is pivotal for regulating neuronal regeneration and growth and modulating neuronal plasticity [22]. The expression of BDNF is regulated by serine–threonine kinases; consequently, alterations in BDNF concentration within nerve cells may be attributable to the activation of MAPK family proteins by F [23]. However, it remains unclear whether F-induced learning and memory impairment are mediated by the ERK-CREB-BDNF signaling pathway.

Selenium is an essential component of antioxidant selenoproteins, such as glutathione peroxidase (GSH-Px). Available evidence indicates that supplementation with different forms of selenium can lead to its accumulation in the brain [24]. Selenium has been demonstrated to counteract the neurotoxicity of F, and this effect may be related to its antioxidant properties [25]. An appropriate concentration of selenium can promote the excretion of F in urine, stimulate the activation of antioxidant factors, and reduce oxidative stress, thereby alleviating fluorosis [26]. However, the range between the nutritionally active dose and the toxic dose of inorganic selenium is very narrow, making it challenging to control the dosage. Compared with inorganic selenium, selenium nanoparticles demonstrate lower toxicity, greater stability, and enhanced antioxidant properties. Moreover, they are more readily absorbed and efficiently utilized by organisms. Over the past few years, selenium nanoparticles have surfaced as a potential therapeutic approach for neurological disorders [27]. Animal experiments have demonstrated that the administration of an appropriate dose of selenium nanoparticles can effectively alleviate F-induced depressive behavior [24]. Additionally, in vitro investigations have revealed that B6-SA-SeNPs can protect PC12 cells from AB-induced toxicity [28]. However, to date, no studies have reported how selenium nanoparticles modulate neurotransmitters and their receptors to mitigate the neurotoxicity of F, and the underlying mechanism of action remains elusive.

Chitosan (CS) is a natural biopolymer obtained from the deacetylation of chitin [29]. It is not only widely sourced, nontoxic, and relatively low in cost but also, more importantly, has excellent biocompatibility, biodegradability, and biosafety [30]. CS has many amino and hydroxyl functional groups on its molecular chain, making it an ideal stabilizer and functional carrier. These characteristics make it a perfect drug delivery vehicle that ensures effectiveness while minimizing potential biotoxicity risks, which is crucial for nanopreparations applied in neuroprotection and other fields [29,30].In addition, CS has shown great potential for mucosal delivery (e.g., oral) because of its unique ability to promote adhesion and penetration. It can effectively enhance the adhesion and absorption of nanoparticles in the intestinal mucosa, thus improving oral bioavailability [30].

In this study, chitosan-selenium nanoparticles (CS-SeNPs) were synthesized via a green chemical method utilizing chitosan as a stabilizer, followed by comprehensive characterization and functional analysis. The structure of the CS-SeNPs and their good environmental stability and pH stability in gastrointestinal enzymes were revealed. The localization and accumulation of CS-SeNPs in hippocampal neurons were detected via the fluorescence labeling method to estimate the impact of CS-SeNPs on F-induced hippocampal neuron injury (Scheme 1A). In vitro and in vivo models of F and/or CS-SeNPs were established to explore the mechanisms underlying the effects of CS-SeNPs on F-induced neurotoxicity. Studies have demonstrated that CS-SeNPs can markedly alleviate F-induced neuronal damage and improve learning and memory function by regulating neurotransmitter release, modulating oxidative stress, and activating the ERK-CREB-BDNF signaling pathway. Furthermore, transcriptomic analysis revealed the regulatory role of CS-SeNPs in F-induced gene expression changes, further confirming the multitarget properties of CS-SeNPs in neuroprotection. Notably, we confirmed that CS-SeNPs can reverse fluorine neurotoxicity by upregulating DUSP5 to inhibit ERK overactivation and activate CREB-BDNF signaling, thereby promoting neuronal survival (Scheme 1B). In this study, we systematically characterized CS-SeNPs and their protective effects on F-induced neurotoxicity via the DUSP5-ERK-CREB-BDNF axis both in vitro and in vivo. This study also suggested that DUSP5 serves as a pivotal target for intervention in F-related neurodegenerative diseases, which provides theoretical support for the development of new neuroprotective agents based on CS-SeNPs.